EFFECTS OF STRESS ON TESTICULAR STEROIDOGENESIS AND ROLE OF ANTIOXIDANT SUPPLEMENTATION

By

Dr Ghulam Mustafa Lodhi

MBBS, M.Phil (2007-NUST-TfrPhD-Med-15)

A thesis submitted in partial fulfillment of the requirements for the degree of

PhD in Physiology

In

Department of Physiology Army Medical College, Rawalpindi

National University of Sciences and Technology Islamabad, Pakistan

(2012)

ii

Dedicated to my beloved parents

iii

ACKNOWLEDGEMENTS

All praises and thanks to Almighty Allah, the Most Beneficent and the Most

Merciful, Who blessed me with wisdom, expertise and knowledge to complete this

endeavor.

I express my sincere and profound gratitude to Professor Muhammad Aslam, for

his valuable guidance at every step of my post-graduation. I will never forget his

fatherly attitude, kind heartedness and care for me. Thank you sir, for all you have

done for me. May Almighty Allah give you health and prosperity in this life and

may He also shower His special blessings on you in your life hereafter.

I am grateful to Brig Muhammad Mazhar Hussain, Head of Physiology Department

Army Medical College for taking personal interest to solve my tribulations in

completing this project. I think, without his facilitation and care, it would have

been far difficult for me to accomplish this project.

I am extremely thankful to Maj Gen Abdul Khaliq Naveed, Dean and Head of

Biochemistry department for his guidance during my course work and facilitation

of research in Army Medical College.

I am thankful to my team of cell culture, Dr Waqas Hameed and Dr Rabia Latif,

who were with me during hard times of research work. Also, all faculty members,

post graduate trainees and staff of Physiology Department Army Medical College

deserve special thanks for cooperation and caring attitude towards me.

iv

I would like to offer special thanks to Mr. Tausif Ahmed, Biochemistry

Department, Army Medical College. He really provided me a lot of help in

statistical analysis and calculations. I feel indebted to him.

I am specially obliged to Dr Hussain Ali, Incharge animal house, National Institute

of Health Islamabad for providing me every possible help in animal handling and

care.

Not to be forgotten are staff of CREAM, Army Medical College for their

cooperation and help to complete this project. Also, Syed Iftekhar Gilani deserves

special thanks for his sincere efforts to facilitate me in completing this project.

I would also pay my by best regards to Prof Sheikh Abdul Saeed and Aga Khan

University, from where I learnt the Leydig cell culture technique.

I am thankful to all members of my family for their continuous support to me for

completing my Ph.D. Special regards and prayers for my parents, who supported

me in all possible ways to achieve the goals of my life.

Lastly, I express my thanks to my university (NUST) and Higher Education

Commission, Pakistan, to provide me the funding for this project.

In the end, I pray to Almighty Allah that He may guide and help me to carry on

more research work and also facilitate others in the research pursuits. Ameen

Dr Ghulam Mustafa Lodhi August 08, 2012

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TABLE OF CONTENTS

Content Page

Introduction and review of literature 01

Objectives and hypothesis 37

Materials and Methods 38

Results 94

Discussion 137

Limitations of study 163

Conclusions 164

Future directions 165

References 166

Annexures 199

vi

List of abbreviations

Abbreviation Used for

ALCs

Adult Leydig cells

3 β -HSD 3β-hydroxysteroid dehydrogenase AA arachidonic acid AA Ascorbic acid

AA-CoA Arachidonyl-CoA AAS ascorbic acid supplementation AChE acetyl cholinesterase

ARTISt Arachidonic acid -related thioesterase AT Alpha tocopherol

ATS alpha tocopherol supplementation AVP arginine – vasopressin

B0 Absorb of Blank BSA Bovine Serum Albumin

cAMP Cyclic adenosine mono phosphate COX cyclooxygenase

CREAM centre for research in experimental and applied medicine CREB cAMP response-element binding protein

DA dopamine dbcAMP Dibutryl cyclic adenosine mono phosphate DHEA dehydroepianderosterone DHT dihydrotestosterone

vii

Abbreviation Used for

DMEM Dulbecco’s modified Eagles Medium DMSO Dimethyl sulfoxide DOPA dihydroxyphenylalanine

E Epinephrine EIA Enzyme immune assay

ELISA Enzyme linked immune sorbent assay EM Eagles medium FDA Food and Drug Administration FLCs Fetal Leydig cells GnRH Gonadotrophic releasing hormone GREs glucocorticoid responsive elements hCG Human Chorionic gonadotropin HDL High density lipoprotein

HEPES Hydroxy ethyl piperazine ethane sulphonic acid. HPA axis hypothalamo-pituitary-adrenal axis

HPT Hypothalamo-pituitary testicular axis HRP Horse radish peroxidase

IBMX Iso butryl methyl Xanthine LC locus ceruleus

LDL Low density lipoprotein LH Luteinizing hormone

MDA malondialdehyde

viii

Abbreviation Used for

NE norepinephrine NIH National Institute of Health

NUST National University of Sciences and Technology PBS Phosphate Buffer Solution PKA Protein kinase A

PKC pathway Protein kinase C pathway PLA2 phospholipase A2

PUFAs polyunsaturated fatty acids PVN Para ventricular nuclei RDA Recommended daily allowance ROS reactive oxygen species

RPM Revolutions Per Minute SER Smooth endoplasmic reticulum

SHBG sex hormone-binding globulin SOD superoxide dismutase

StAR protein Steroid acute regulatory protein TBA Thio Barbituric Acid

TBARS thiobarbituric acid reacting substance TH tyrosine hydroxylase

TMB Tetra methyl benzidin

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List of figures

Figure Title Page 1.1 Overview of percentage of cell organelles and cell

composition in Leydig cells

3

1.2 Overview of intra cellular pathways of steroidogenesis

8

1.3 Steps of testosterone synthesis 11

1.4 Some of the interactions and feedback mechanisms of testosterone

15

1.5 Effects of stress on hypothalamo pituitary adrenal axis

20

2.1 Testosterone standard calibration curves (ELISA)

59

2.2 Testosterone standard calibration curves (for in vitro analysis)

65

2.3 Corticosterone standard calibration curves.

71

2.4 Nor epinephrine standard curve (ELISA) 78

2.5 Standard calibration curve for LH.

82

2.6 Malondialdehyde EIA standard calibration curve

88

2.7 Standard Calibration curve for Super oxide dismutase

93

3.1 Effect of LH on testosterone production by cultured Leydig cells

102

3.2 Effect of different concentrations of corticosterone on testosterone production by cultured Leydig cells incubated with LH

104

3.3 Effect of different concentrations of nor epinephrine on testosterone production by cultured Leydig cells incubated with LH

104

x

Figure

Title Page

3.4 Effects of different concentrations of Isobutyl methyl xanthine (IBMX) on testosterone production by cultured Leydig cells incubated with LH

105

3.5 Effect of different concentrations of pregnenolone on testosterone production by cultured Leydig cells incubated with LH

107

3.6 Effect of different concentrations of ascorbic acid (AA) in the presence of LH on testosterone production by cultured Leydig cells incubated with LH.

107

3.7 Effect of different concentrations of alpha tocopherol (AT) in the presence of LH on testosterone production by cultured Leydig cells incubated with LH

109

3.8 Effect of combination of corticosterone and nor epinephrine on testosterone production by LH stimulated cultured Leydig cells incubated with LH.

110

3.9 Effect of IBMX on testosterone production by Leydig cells incubated without LH

113

3.10 Effect of corticosterone on IBMX induced increment in testosterone production by Leydig cells incubated without LH

113

3.11 Effect of corticosterone on IBMX induced increment in testosterone production by Leydig cells incubated with LH

114

3.12 Effect of nor epinephrine on IBMX induced increment in testosterone production by Leydig cells incubated without LH

114

3.13 Effect of nor epinephrine on IBMX induced increment in testosterone production by Leydig cells incubated with LH

114

3.14 Effect of pregnenolone on testosterone production by Leydig cells incubated without LH

117

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Figure

Title Page

3.15 Effect of corticosterone on pregnenolone induced increment in testosterone production by Leydig cells incubated without LH

117

3.16 Effect of corticosterone on pregnenolone induced increment in testosterone production by Leydig cells incubated with LH

117

3.17 Effect of nor epinephrine on pregnenolone induced increment in testosterone production by Leydig cells incubated without LH

118

3.18 Effect of nor epinephrine on pregnenolone induced increment in testosterone production by Leydig cells incubated with LH

118

3.19 Effect of ascorbic acid on corticosterone induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH

120

3.20 Effect of alpha tocopherol on corticosterone induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH

120

3.21 Effect of both ascorbic acid and alpha tocopherol on corticosterone induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH.

120

3.22 Effect of ascorbic acid on nor epinephrine induced changes in testosterone production by isolated and cultured Leydig cells incubated with LH

122

3.23 Effect of alpha tocopherol on nor epinephrine induced changes in testosterone production by isolated and cultured Leydig cells incubated with LH

122

3.24 Effect of both ascorbic acid and alpha tocopherol on nor epinephrine induced changes in testosterone production by isolated and cultured Leydig cells incubated with LH

122

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Figure Title Page 3.25 Effect of ascorbic acid on corticosterone and nor

epinephrine induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH

125

3.26 Effect of alpha tocopherol on corticosterone and nor epinephrine induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH

125

3.27 Effect of ascorbic acid and alpha tocopherol on corticosterone and nor epinephrine induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH

125

3.28 Effect of ascorbic acid on stress hormones corticosterone and norepinephrine induced changes in malondialdehyde levels

131

3.29 Effect of alpha tocopherol on stress hormones corticosterone and norepinephrine induced changes in malondialdehyde levels

131

3.30 Effect of ascorbic acid and alpha tocopherol combination on stress hormones corticosterone and norepinephrine induced changes in malondialdehyde levels

132

3.31 Effect of ascorbic acid on stress hormones corticosterone and norepinephrine induced changes in super oxide dismutase levels

135

3.32 Effect of alpha tocopherol on stress hormones corticosterone and norepinephrine induced changes in super oxide dismutase levels

135

3.33 Effect of ascorbic acid and alpha tocopherol combination on stress hormones corticosterone and norepinephrine induced changes in super oxide dismutase levels

136

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List of tables

Table

Title Page

2.1 Pipetting protocol for incubation of cultured Leydig cells from rats, step 1.

53

2.2 Pipetting protocol for incubation of cultured Leydig cells from rats, step 2

54

2.3 Absorbance and B/ B0 % of standards of Testosterone

58

2.4 Concentrations for the preparation of Malondialdehyde standards

86

2.5 Absorbance and concentrations of Malondialdehyde standards

87

3.1 Effects of 06 hours restraint stress on serum Testosterone, serum Corticosterone, plasma Nor epinephrine serum LH, serum Malondialdehyde and serum Super oxide dismutase levels in male Sprague Dawley rats.

96

3.2 Effects of one month, ascorbic acid supplementation (AAS) on changes in serum Testosterone, serum Corticosterone, plasma Nor epinephrine, serum Luteinizing hormone, serum Malondialdehyde and Superoxide Dismutase levels in male Sprague Dawley rats exposed to six hours restraint stress

97

3.3 Effects of one month alpha tocopherol supplementation (ATS) on changes in serum Testosterone, Corticosterone, plasma Nor epinephrine, serum Luteinizing hormone, serum Malondialdehyde and Superoxide Dismutase levels in male Sprague Dawley rats exposed to six hours restraint stress

99

3.4 Effect of combined ascorbic acid and alpha tocopherol supplementation on changes in serum Testosterone, serum Corticosterone, plasma Nor epinephrine, serum Luteinizing hormone, serum Malondialdehyde and Superoxide Dismutase levels in male Sprague Dawley rats exposed to six hours restraint stress

100

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Table Title Page 3.5 Effect of stress hormones in the presence of LH on

lipid peroxidation in terms of Malondialdehyde (MDA) levels and Superoxide dismutase (SOD) activity at Leydig cell level:

127

3.6 Effect of antioxidants ascorbic acid (AA) and alpha tocopherol (AT) in the presence of LH on levels of Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity at Leydig cell level

128

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Summary Stress is a widespread problem nowadays. Stress of various origins suppresses

male reproductive functions through release of stress hormones. Various aspects

of hormonal mechanisms affecting testicular steroidogenesis are unclear.

Catecholamines increase steroidogenesis and corticosterone decreases it, but net

effect of stress is considered to be decreased testosterone. How these two

hormones affect the testosterone production is investigated in this study. Stress is

thought to disrupt the balance between prooxidants and antioxidants by

generating the reactive oxygen species. Thus stress damages the testicular tissue

and decreases steroidogenesis. Antioxidants, ascorbic acid and alpha tocopherol

are thought to protect the body against stress induced damages. Whether these

antioxidants confer protection against stress is investigated in this study.

The present study, comprised of in vivo and in vitro components, was designed to

investigate the effects of stress on testicular steroidogenesis, lipid peroxidation and

antioxidant enzyme activity. Also the preventive role of antioxidants, ascorbic acid

and alpha tocopherol on stress induced derangements was investigated.

The study was carried out at department of Physiology, Army Medical College

Rawalpindi, Pakistan in collaboration with National Institute of Health Islamabad.

In vivo part of study comprised 80 male Sprague Dawley rats, which were divided

into five groups with each group comprising of 16 rats. Group I was control group,

fed non supplemented normal standard diet. Group II to Group V were exposed to

acute immobilization stress in a mesh wire restrainer for 6 hours. Group II rats

were given normal standard rat diet without any supplementation. Group III was

fed normal standard diet. Ascorbic acid supplementation was given as 500 mg

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ascorbic acid per liter drinking water. Group IV was fed alpha tocopherol

supplemented diet with 300 mg alpha tocopherol/kg chow + 2% soyabean oil.

Group V was fed alpha tocopherol supplemented diet as well as ascorbic acid

supplementation in drinking water. The levels of testosterone, corticosterone,

norepinephrine, luteinizing hormone, malondialdehyde and superoxide dismutase

were measured.

Also the in vitro model of isolated and cultured Leydig cells of male Sprague

Dawley rats was set to investigate the effects and mechanism of action of stress

hormones corticosterone and nor epinephrine and the effects of antioxidants,

ascorbic acid and alpha tocopherol separately and combined in the presence of

stress hormones at Leydig cell level. Purified Leydig cells were incubated with

corticosterone, nor epinephrine and antioxidants ascorbic acid and alpha tocopherol

in the presence of LH. The testosterone production and extent of lipid peroxidation

and superoxide dismutase activity were evaluated at cell level. Data were analyzed

by SPSS version 17.

The data of this study indicate that acute restraint stress decreased testosterone and

increased corticosterone and norepinephrine levels. LH remained unchanged in all

the study groups. Lipid peroxidation increased while super oxide dismutase

decreased by stress.

In vitro findings suggest that corticosterone in acute stress decreases the Leydig

cell steroidogenesis through non genomic rapid pathways. It may decrease the

production of cAMP or may inhibit the enzymes like 17 α hydroxylase or C-17-20

lyase. However, nor epinephrine increases the testosterone synthesis at Leydig cell

xvii

level and it may affect the cAMP or the enzymes involved in testosterone

synthesis.

In vivo, the stress induced increase in nor epinephrine may contribute towards

decline in testosterone synthesis probably by causing vasoconstriction and

inhibiting the LH access to Leydig cell or by activating the direct hypothalamo-

testicular axis which upon activation is thought to inhibit testosterone synthesis by

Leydig cells.

Among anti-oxidants, ascorbic acid has shown favorable preventive effects on

stress only in in vitro part of our studies, while alpha tocopherol prevented fall in

testosterone and rise in malondialdehyde levels, both in vivo and in vitro.

Combination of antioxidants, ascorbic acid and alpha tocopherol shown synergistic

effect in reducing oxidative stress and prevented stress-induced derangements in

testicular steroidogenesis both in vivo and in vitro.

Therefore, it is concluded from the findings of this study that the antioxidants

ascorbic acid and alpha tocopherol decrease the stress induced lipid peroxidation

and the resultant membrane stabilization prevented stress induced decline in

testicular steroidogenesis.

Key words: Acute restraint stress, testosterone, corticosterone, nor epinephrine,

ascorbic acid, alpha tocopherol.

1

.0]INTRODUCTION AND REVIEW OF LITERATURE

>In order to maintain homeostasis, along with other systems of body, the

reproductive system also contributes by generating new individuals to replace the

dying individuals and thus ensures continuity of life, as narrated by great

physiologist Arthur C Guyton (Guyton and Hall, 2011). It is known centuries ago

that fertility and sexuality are related to the functions of gonads. Significant

knowledge about anatomy and physiology of gonads was established about 1500

years ago while about 100 years ago the relationship between gonads and pituitary

was recognized (Lindholm and Nielsen, 2009).

1.1 Testis and Leydig cells

The word testis means, ‘witness’ and it was named by De Graaf, because

testis provide evidence of virility, as men with testis are thought to be capable of

generating offspring’s

Two important cells of testis are Leydig cells and Sertoli cells. In 1850

Franz Leydig described that cells are present in testicular interstitium. Those cells

were named as Leydig cells while Enrico Sertoli found cells in the tubules which

were named after his name (Lindholm and Nielsen, 2009).

In males, the interstitial cells of Leydig, which are the major sources of

sexual steroid hormone testosterone (Wang et al., 2009). Leydig cells arise from

interstitial mesenchymal tissue between the tubules during the eighth week of intra

uterine life. They are located in the connective tissue between the seminiferous

tubules (Sikka and Wang, 2008).

In rat’s testis, two different types of Leydig cells have been differentiated,

which are fetal Leydig cells (FLCs), and adult Leydig cells (ALCs). ALCs are

Chapter 1

2

abundant in smooth endoplasmic reticulum and have few rough endoplasmic

reticulum. Mitochondria are present as tubulovesicular cristae, while Golgi

apparatus is large and well differentiated. Also there are present few lipid droplets

with a diameter of about 0.5 um. Nuclei are elliptical or round and cell surface has

few small protrusions, while basal laminae usually absent in ALCs (Haider et al

1995; Kuopio et al 1989 a, b).

In Leydig cells of rat, the typical mitochondrion is 0.35 um in diameter

while length is 2.4 um. The average Leydig cell has about 622 mitochondria. The

inner mitochondrial membrane which is the site of enzymes that regulate the

testicular steroid synthesis has a surface area 1.8 times larger than the outer

membrane of the mitochondrion, but its surface area is less than smooth

endoplasmic reticulum (SER) that has the majority of steroidogenic enzymes by a

factor of 3.6 (Mori and Christensen, 1980). At least 30 or more enzymes that may

control the cholesterol synthesis are located on the SER of Leydig cells

(Christensen, 1975).

An important organelle in Leydig cells is the smooth endoplasmic reticulum

(SER), because most of the steriodogenic enzymes in Leydig cells are bound to

membranes of the SER. It has a surface area, which is about 6.9 times the plasma

membrane which is about 60% of the total membrane area of the cell (Mori and

Christensen, 1980).

Majority of the enzymes that regulate the testicular steroidogenesis are in

bound form with SER of Leydig cells. Some enzymes are also positioned on the

inner membrane and cristae of mitochondria. Acetyl-CoA, an enzyme present

within mitochondria, testosterone synthesis is catalyzed by those enzymes which

3

Figure 1.1: Overview of percentage of cell organelles and cell composition in Leydig cells. (Adopted from Mori and Christensen, 1980)

4

are bound to SER. Therefore SER and inner mitochondrial membranes are of

primary importance (Mori and Christensen, 1980). Important requirements for

adequate steroid biosynthesis include adequate membrane potential within the

mitochondria, sustained ATP production by mitochondria and maintenance of

suitable pH within mitochondria. It means optimally functioning mitochondria are

required for acute Leydig cell steroidogenesis (Allen et al., 2006).

The ability of testosterone production by Leydig cells is proportionate to

amount of the smooth endoplasmic reticulum within these cells (Zirkin et al.,

1982). Blockage of hypothalamo-pituitary testicular axis (HPT) axis with

gonadotropin releasing hormone (GnRH) antagonist not only results in Leydig cells

atrophy, but also decline in quantity and distribution of SER in Leydig cells along

with reduction in number of mitochondria and lipid droplets (Prince et al., 1998).

Leydig cells are present in interstitium of testis in such a way that they are usually

widely separated from blood vessels, therefore the steroids secreted from Leydig

cells diffuse through surrounding loose connective tissue before reaching the blood

vessels (De Kretser and Kerr, 1994).

Regulation of Leydig cells functional activity is accomplished by

luteinizing hormone (LH), which binds to LH receptors which are located on the

Leydig cell membrane (Catt and Dufau, 1976). It is reported that amount of LH

reaching Leydig cells is about one tenth that of circulation. This reduction is due to

impediment of hormone by endothelial cells. It means Leydig cells are extremely

sensitive to actions of LH or endothelial cells might modulate the actions of LH

(Satchell, 2003). It has been estimated that there are about 20,000 LH receptors on

each Leydig cell (Conn et al., 1977).

5

1.2 Steroidogenesis:

In order to maintain homeostasis and continue reproductive function in

males, steroid hormones are synthesized and released by steroid producing cells of

testis, adrenals and brain. Trophic hormones control the regulation of

steroidogenesis in minutes to hours (Stocco et al., 2005).

Leydig cells are stimulated by luteinizing hormone that is secreted in a

pulsatile fashion into circulation by pituitary gland in response to gonadotropin

releasing hormone from hypothalamus. A negative feedback mechanism operates

here in hypothalamo pituitary gonadal axis, by which increased levels of

testosterone in circulation inhibit release of GnRH from hypothalamus and LH

from pituitary (Bremner et al., 1993; Ellis and Desjardins, 1983). The process of

steroidogenesis in Leydig cells is regulated by various endocrine, autocrine and

paracrine factors (Rommerts et al., 2001). Along with above factors,

microcirculation in the testis is an important determinant for the regulation of both

steroidogenesis and gametogenesis. The microvasculature permits exchange of

oxygen, nourishment and steroid hormones between the steroidogenic and

gametogenic sections of the testis freely (Ergun et al., 1994).

1.2.1 Role of cholesterol in steroid synthesis

Cholesterol is principal precursor for synthesis of all steroid hormones. The

serum is the chief source of cellular cholesterol. Transport of cholesterol into cell

occurs by protein carriers, which may include high or low-density lipoprotein

(HDL or LDL). On entry into the cell, cholesterol is either utilized or it is stored in

the form of lipid droplets. Upon LH-induced stimulation, mobilization of newly

synthesized and stored cholesterol occurs in lipid droplets. Cholesterol is

6

transported out of the cytoplasm into the mitochondria. It is transported from the

outer to the inner membrane. This cholesterol transfer is considered as rate limiting

step of steroidogenesis (Stocco, 1999).

Cholesterol entry from the outer to the inner mitochondrial membrane

needs a transport protein. Stimulation of Leydig cells by LH stimulates the

synthesis of the cholesterol transport protein. As this protein is essential for

synthesis of steroid hormones and it regulates the rate limiting step of steroid

hormone production, it is named as the steroid acute regulatory (StAR) protein

(Stocco, 1999). Another protein is involved in cholesterol transport is peripheral

benzodiazepine receptor and may play role in cholesterol transport from outer to

inner mitochondrial membrane. It is suggested that it may act as a channel for

cholesterol entry (Haider, 2004).

1.2.2 Role of cAMP in steroidogenesis

Leydig cells stimulation results in activation of G proteins which in turn

stimulate the adenylate cyclase with consequent increase in cAMP inside Leydig

cell. Also there is activation of protein kinase A (PKA) (Selstam, Rosberg, 1976;

Zhu and Birnbaumer, 1996). Action of PKA results in the phosphorylation of

various proteins like cholesteryl ester hydrolase and also there is phosphorylation

of transcription factors that may include cAMP response-element binding protein

(CREB) and the cAMP response element modulator. This may result in activation

of genetic machinery involved in steriodogenesis, like StAR protein synthesis and

activation (Stocco et al., 2001; Manna et al., 2003; Tremblay et al., 2002). Various

factors that do not need cAMP and/or protein synthesis are also thought to potently

stimulate steroidogenesis (Cooke, 1999).

7

1.2.3 Role of calcium in steroidogenesis

The increase in intracellular calcium, whether it is released from

intracellular stores or mobilized from extracellular spaces, is believed to have

considerable role in steroidogenesis. As a result of hormonal stimulation, there is

change in intracellular cAMP and calcium levels. All stimulants of steroidogenesis

like LH, hCG and GnRH rapidly increase intracellular calcium within 1-5 minutes

(Sullivan and Cooke, 1986; Manna et al., 1999).

Mitochondria in the Leydig cells store calcium and play important role in

regulation of calcium levels within the Leydig cell (Nicholls, 2005). If there is

inhibition of the electron transport chain, it results in intracellular hypercalcemia.

The process of steroidogenesis is stimulated by increased calcium levels, but if

intracellular calcium in Leydig cells falls, as induced through chelation in

experimental models, it will lead to suppression of steroidogenesis (Sullivan and

Cooke, 1986; Kumar et al., 1994, Tomic et al., 1995).

Calcium is known to potentiate the process of steroidogenesis. Literature shows

that in calcium free media, despite stimulation by LH/hCG (100ng/ml), synthesis of

steroids was 50% after 06 hours (Ramnath et al., 1997; Sullivan and Cooke, 1986;

Manna et al., 1999). Also, there is experimental evidence that removal of calcium

decrease steroid synthesis and StAR protein expression while addition of calcium

to calcium free media increases steroidogenic capacity at cell level. Also role of

calcium after hCG stimulation were studied with calcium channels blockers, which

decreased steroidogenic capacity of steroid producing cells. It has been narrated

that if calmodulin, (a protein which binds calcium) is blocked, steroidogenesis

declines (Capponi et al., 1988; Irusta et al., 2003).

8

Figure 1.2: Overview of intracellular pathways of steroidogenesis.

(Adopted from Stocco et al., 2005)

9

1.2.4 Protein Kinase pathway in steroidogenesis

After binding of LH to Leydig cells and formation of cAMP, there is

activation of phospholipase, which activates the protein kinase C (PKC) pathway.

Also cAMP can activate the protein kinase A (PKA) pathway (Nikula and

Huhtaniemi, 1989; Kuhn and Gudermann, 1999; Nishimura et al., 2004).

Stimulation of Protein kinase C pathway results in signal transduction that can lead

to transcription and translation of StAR and phosphorylation of StAR protein must

occur by the activation of PKA, which is a mandatory requirement for its

cholesterol-transferring function (Stocco et al., 2005).

1.2.5 Role of arachidonic acid in steroidogenesis

Stimulation of Leydig cells causes the release of arachidonic acid (AA)

from intracellular stores. It is also proposed that cAMP also activates arachidonic

acid -related thioesterase (ARTISt), an enzyme that catalyzes AA release from

arachidonyl-CoA (AA-CoA). Release of AA occurs within one min of LH

stimulation (Cooke et al., 1991; Stocco et al., 2005). It is also advocated that,

hormone-receptor interaction at Leydig cell level cause the activation of G proteins

that will result in activation of phospholipase A2 (PLA2) which as a result

catalyzes the release of AA from phospholipids (Ronco et al., 2002). Various other

pathways for arachidonic acid release are also cited in literature.

AA is thought to act at the rate limiting step of steroidogenesis, which is the

transfer of the substrate cholesterol from outer to the inner mitochondrial

membrane (Abayasekara et al., 1990). AA after release may be metabolized

through one of three enzymatic pathways, the cyclooxygenase (COX), the

lipoxygenase, or the epoxygenase pathways. Inhibition of either lipoxygenase or

10

epoxygenase activity as shown in some experiments inhibits StAR protein

expression and steroid synthesis. It is also shown via experimental work that if AA

release is blocked, it will result in inhibition of LH- or dbcAMP-stimulated StAR

expression and steroidogenesis. However, the level of PKA activity remained high

in above experimental models of Leydig cells, but such high levels of PKA were

unable to affect the steroidogenesis significantly (Wang et al., 2000).

Only one pathway is not working for steroidogenesis at Leydig cell level,

but various signaling pathways are operating which results in the stimulation of

steroid biosynthesis and StAR protein expression (Stocco et al., 2005).

1.2.6 Overview of enzymatic steps of testosterone synthesis

LH binds to receptors on plasma membrane of Leydig cells. This results in

various events which include activation of adenylate cyclase and increased

formation of cyclic adenosine monophosphate (cAMP) inside the cell. There is

translocation of cholesterol into the mitochondria, connection of cholesterol with

the P450 side-chain cleavage enzyme (P450scc) and formation of pregnenolone

from cholesterol in the mitochondria. From mitochondria, pregnenolone is

translocated to the smooth endoplasmic reticulum. Cholesterol transfer from the

outer to the inner mitochondrial membrane is considered as rate limiting step in the

process of steriodogenesis. The exact process of translocation is unclear but the

steroidogenic acute regulatory protein (StAR protein), a cycloheximide-sensitive

30-kDa mitochondrial protein which is synthesized in response to LH or cAMP, is

involved (Stocco and Clark, 1996; Stocco, 1996; Stocco, 1999).

The next step is the transformation of pregnenolone to progesterone by the

enzyme 3β-hydroxysteroid dehydrogenase (3 β -HSD). Now the steroidogenesis

11

Figure 1.3 Steps of testosterone synthesis

(Adopted from: Zirkin and Chen, 2000)

12

operates via either hydroxysteroid or ketosteroid pathways. In hydroxy steroid

pathway, pregnenolone is converted to 17 α hydroxy pregnenolone, which is

converted to dehydroepianderosterone (DHEA), and then androstenedione is

formed. In ketosteroid pathway, progesterone is converted to 17 α

hydroxyprogesterone and then androstenedione that will lead to formation of

testosterone. Testosterone is converted to dihydrotestosterone (DHT) by 5 alpha

reductase which is present in cell membrane, nuclear envelope and endoplasmic

reticulum. DHT is the end product hormone and more potent androgen than

testosterone (Stocco and Clark, 1996; Stocco, 1996; Stocco, 1999).

1.2.7: Testosterone release and transport in circulation

Testosterone is the principal androgen in males. It was first isolated and

discovered in 1935 (Ruzicka and Wettstein, 1935). Testosterone levels in testicular

lymph and testicular venous blood are same, but the main route for steroid

secretion into circulation is through venous blood (Morse et al., 1973).

Testosterone in bound form with plasma proteins is incapable to produce the

androgenic effects. More than 50% of testosterone circulating in blood is in state of

binding with sex hormone-binding globulin (SHBG) while main part of remaining

testosterone is in albumin bound state (Wheeler, 1995). The binding affinity of

albumin for testosterone is low but it binds a substantial percentage of the total

testosterone because the plasma concentration of albumin is much higher than

SHBG. The free and albumin-bound testosterone togetherly is called bioavailable

testosterone (Manni et al., 1985). It is also documented that diminished serum

testosterone levels are responsible for stimulation of the SHBG production by the

liver (Petak et al., 1996).

13

1.2.8: Regulation of testosterone production

Regulation of testosterone production occurs via a negative feedback loop

system involving the anterior pituitary, hypothalamus and testis called the

hypothalamo-pituitary-testicular axis. The hypothalamus releases pulses of

gonadotropin-releasing hormone (GnRH) into the hypophyseal circulation. The

GnRH stimulates the release of luteinizing hormone (LH) by anterior pituitary. The

pulsatile release of GnRH results in LH release in a analogous pulsatile manner

(Ismail et al., 1986). Circulating testosterone then feedback to hypothalamus and

pituitary to suppress LH and consequently testosterone production. This negative

feedback cycle results in pulsatile secretion of LH followed by pulsatile production

of testosterone (Ellis et al., 1983). Besides luteinizing hormone, the follicle

stimulating hormone and growth hormone play roles in the regulation of

testosterone (Celec and Ostatnikova, 2003).

There are also various paracrine factors that may control the testosterone

production. These include

a) Testicular peptides e.g., Inhibin and Activin.

b) Growth factors like Transforming growth factor α and ß, Fibroblast

growth factor and Insulin like growth factor I.

c) Cytokines that may include interleukin and tumor necrosis factor α.

d) Vasoactive peptide like angiotensin II, atrial natriuretic peptide and

endothelin. (Saez, 1994; Gnessi et al., 1997; Schlatt et al., 1997).

1.2.9: Mechanism of action of testosterone. Testosterone acts via cytoplasmic

receptors within the cells of the target tissues. The steroid-receptor complex then

14

reach nucleus where it enhances the gene transcription and may result in formation

of new proteins.

Experimental evidence has also displayed a membrane-bound testosterone

channel. Testosterone is also thought to change the functional status of ionic

channels. In prostate gland the predominant receptor type is a Na+-K+-ATPase

(Leung et al., 2001). In neurons and macrophages, a G protein- linked androgen

receptor increases cAMP or inositol triphosphate (IP3) and diacyglycerol (DAG)

through the activation of either adenylate cyclase or phospholipase C. In case of red

blood cells, even the cytoskeleton functions as a steroid receptor and effector

(Ramirez and Zheng, 1996). Androgen receptors are present on platelets, which are

thought to play some role in the gender differences in platelet function and the

associated risks of thrombotic diseases (Khetawat et al., 2000).

1.2.10: Physiological actions of testosterone

In tissues, testosterone is converted to dihydrotestosterone (DHT) in the

presence of enzyme 5α-reductase. The physiological actions of testosterone on

scalp, prostate and beard are performed by dihydrotestosterone. Testosterone plays

a key role in the growth and development of the male genitalia as well as secondary

sex characteristics (Gronowski and Landau-Levine, 1999). Testosterone causes

increase in the growth of muscle and larynx that may lead to physical maturation as

well as change in voice that occurs in boys at puberty. Development of acne is also

related with testosterone levels. DHT not only stimulates the growth of axillary and

pubic hair, but may also be responsible for their maintenance. Increased levels of

DHT have been associated with the loss of scalp hair with resultant male pattern

balding which is also called as androgenic alopecia (Randall., 2001). Testosterone

15

Fig. 1.4. Some of the interactions and feedback mechanisms of testosterone (Adopted from Zitzmann and Nieschlag, 2001).

16

causes increase in red blood cell count and hemoglobin concentration by increasing

body metabolism and stimulating production of erythropoietin (Gooren, 2003). It is

due to fact that testosterone increases erythropoiesis and also has important effects

on behavior. Testosterone enhances the libido, competitiveness, and aggression

(Ismail et al., 1986).

Testosterone is an anabolic hormone and it has important effects on

metabolism in body (Mauras et al., 2000). Many studies indicate the relationship

between levels of testosterone and blood pressure. Various authors are in favor of

the opinion that testosterone has blood pressure lowering effects, which are

probably mediated through atrial natriuretic peptide, vascular endothelial growth

factor or could be due to the induction of nitric oxide synthase (Pandey et al., 1999;

Sordello et al., 1998).

1.2.11: Testosterone levels in different phases of life in males

Testosterone levels are different in different during different phases of the

the life cycle (Gronowski and Landau-Levine, 1999). At puberty, the levels of

testosterone are increased with resultant changes in male secondary sexual

characteristics (Harries et al., 1997).

Significant physiological changes occur in male as age advances, most of

them are due to decline in testosterone levels. The prime cause of this decrease in

testosterone production is testicular failure, although reduced gonadotropin release

can also be a contributing factor (Basaria and Dobs, 2001).

1.2.12 Factors affecting testosterone levels

Restraint stress in rats leads to decrease in testosterone levels (Dong et al.,

2004). The levels of testosterone are affected by various endogenous as well as

17

environmental factors (Zitzmann and Nieschlag, 2001), as shown in figure 1.4.

The Leydig cells activity is suppressed by stress and accordingly the levels of

testosterone decrease (Haddy and Pankhurst, 1999). Similarly in humans, the

stressful conditions may range form exams to sports and result in decline in

testosterone levels. On the other hand stress release can result in rise of androgen

levels (MacLean et al., 1997). The same effect may appear after loss of jobs or

even change in work places (Grossi et al., 1999). The increased glucocorticoid

secretion is observed in stressful situations that are due to increased corticotropin-

releasing hormone production which may be responsible for down regulated

testosterone biosynthesis in the Leydig cell (Hardy and Ganjam, 1997). There is

feedback mechanism between testosterone and aggression, which is further

influenced by various other factors like education, culture and socio economic

status (Adler et al., 2000).

Supraphysiological levels of testosterone are suspected as a cause for

carcinoma prostate (Shaneyfelt et al., 2000). Life threatening illnesses and traumas

may also cause decline in testosterone levels which may also be accompanied by

decrease in LH (Dong et al.., 1992). Hyperthyroidism, autoimmune diseases

(Handelsman, 2000), and various systemic diseases like Hodgkin’s disease and

malignancies may also cause low testosterone levels thereby affecting testes as well

as the hypothalamo pituitary axis (Baker, 1998). Vegetarians have significantly

higher levels of SHBG, therefore the bioavailable testosterone levels are reduced in

them (Belanger, et al., 1989).

Various factors affect reproductive system in males also include

environmental disrupters like organic chemicals and pesticides, heavy metals like

18

lead, mercury, cadmium and ionizing radiations. Pharmacological threats include

various agents that range from X rays to drugs like steroids and nicotine like

agents. Among biological threats to reproductive system, oxidative stress is one of

important factors (Sikka and Wang, 2008).

1.2.13 Effects of testosterone deficiency

Diminished testosterone in adult males may result in loss of libido and that

may or may not be accompanied by erectile dysfunction (Petak et al., 2002). Older

men with decreased plasma testosterone may show irritability, loss of

concentration, lessened libido, erectile dysfunction, and may experience decreased

sense of well-being (Tenover, 1998). Depression and behavioral symptoms like

irritability and fatigue can also occur. Some secondary sexual characteristics may

show regression such as reduced axillary and pubic hair, but quality of voice,

length of penis and prostate size often remain unchanged (Petak et al., 2002).

Decreased in testosterone production is an important cause of infertility

among men (Gronowski and Landau-Levine., 1999). Decrease in testosterone

concentrations can result by a problem at Leydig cell level called testicular failure

which may lead to primary hypogonadism. It can also occur when gonadotropins

are insufficient to stimulate testis which is termed as secondary hypogonadism

(Petak et al., 1996., Gronowski and Landau-Levine., 1999).

Measurement of testosterone and gonadotropin levels are useful parameters

that may help in the differential diagnosis of cases of infertility (Petak et al., 1996).

Therefore, we used testosterone levels as hormonal marker for well-being of male

reproductive system in our study species, i.e., male Sprague Dawley rats. Our

19

research is focused on effects of stress on testicular steroidogenesis, which will be

investigated.

1.3: Stress

Rapid industrialization, environmental contamination, changed life styles

and stressful working conditions may result in stress with resultant adverse effects

(Turek, 2000). Stress is imbalance between production of reactive oxygen species

and antioxidant defense (Halliwell and Whiteman, 2004). Cannon was one of the

first investigators to recognize the role of emotional reaction in the development of

diseases (Cannon, 1909).

. Homeostasis is facing continuous threats from various internal and external

challenges which are called stressors (Miller and O’Callaghan, 2002). Selye was

among the initial researchers to identify that stressors may disrupt homeostasis and

have pathophysiological consequences (Selye, 1936). Stress is defined as “a

general body response to initially threatening external or internal demands,

involving the mobilization of physiological and psychological resources to deal

with them” (Montoro et al., 2009). A large variety of homeostatic challenges can

activate the hypothalamo-pituitary-adrenal (HPA) axis, including cold, infection,

emotional distress, electric shock, sleep deprivation, social stress (Blanchard et al.,

2001; Taylor et al., 1997). To overcome these stressors, hormonal, autonomic and

behavioral adjustments are required. Activation of HPA and sympathetic nervous

system with resultant release of glucocorticoids and catecholamines play important

roles in stress induced diseases (Miller and O’Callaghan, 2002). Stress hormones

are adaptive if their action remains for shorter durations, but if these hormones are

released in excess or they remain in circulation for a longer time when they are no

20

Figure 1.5: Effects of stress on hypothalamo pituitary adrenal axis. (Modified from Miller and O’Callaghan, 2002)

21

more required, may become harmful. Consequently, they may contribute to

disturbances in homeostasis (McEwen, 2000). Better understanding of

pathophysiology of stress and the specific adjustments associated with an

anomalous handling of glucocorticoids can help in better elucidation of the

mechanisms by which stress may generate a disease (Bjorntorp, 1991).

1.3.1: Generation of reactive oxygen species

In order to maintain life, oxygen is mandatory requirement because the

physiological levels of reactive oxygen species (ROS) are essential for the

preservation of normal functions of the cell. However, breakdown products of

oxygen with resultant excessive liberation of ROS can be injurious to cell function

and survival (De Lamirande and Gagnon, 1995). Reactive oxygen species as

reviewed by Makker et al., are present in the form of free radicals. ROS include a

extensive range of molecules, including collection of radical (hydroxyl ion,

peroxyl, superoxide) and non-radical (lipid peroxide, hydrogen peroxide) oxygen

derivatives (Makker et al., 2009; Agarwal and Prabakaran, 2005). Environmental

pollution and radiation exposure can also generate various ROS such as hydrogen

peroxide, super oxides and hydroxyl radicals (Gate et al., 1999). ROS take part in

various chemical reactions that liberate free radicals which can damage organic

substrates (Agarwal and Prabakaran, 2005)

1.3.2: Stress: Imbalance between pro oxidants and anti-oxidants: Oxidative

stress may occur due to imbalance between prooxidants and antioxidants. This

imbalance may result from either a decrease in level of antioxidants or an increased

production of prooxidants.

22

1. Decline in the levels of antioxidants that may occur due to mutations

affecting the activities of antioxidant defense enzymes like glutathione peroxidase.

Also deficiency of some minerals like zinc, magnesium, copper and selenium and

various other antioxidants can also result in oxidative stress.

2. Increased production of reactive species that may result from exposure of

cells or organisms to high levels of oxidative stress or excessive activation of

intrinsic ‘natural’ systems that can produce such reactive oxygen species e.g.,

excessive phagocytic activity in chronic inflammation (Halliwell and Whiteman,

2004).

1.3.3: Effects of stress at cell level

Oxidative stress affects various components of cell that may be the lipids or

the protein components of cell or the nucleic acids. The magnitude of the

detrimental effects is affected by duration of ROS exposure, amount of ROS

present in vicinity of that cell and some extracellular factors are also important like

oxygen tension, temperature and amount of antioxidants present in area

surrounding the cell (Makker et al., 2009; Agarwal and Prabakaran, 2005).

ROS most commonly target polyunsaturated fatty acids of cell membrane

with resultant oxidation of lipids called Lipid peroxidation. ROS generate chain

reactions and steps of a chain reaction include initiation, propagation and

termination (Makker et al., 2009). Initiation involves reaction of free radicals with

fatty acids and result in release of lipid free radicals. Lipid free radicals on reaction

with the molecular oxygen may result in generation of lipid peroxyl radical, which

again reacts with fatty acids to propagate the reaction with resultant more free

radicals production (Makker et al., 2009). To assess the degree of per oxidative

23

damage, one of the byproducts of lipid peroxidation i.e., malondialdehyde is

estimated, the levels of which are indicative of the extent of damage by oxidative

stress (Aitken and Fisher, 1994).

1.3.4: Consequences of stress at cell level

Oxidative damage is the bimolecular damage that can be caused by direct

attack of reactive species during oxidative stress. Consequences of oxidative stress

can include:

1.3.4.1. Adaptation of the cell: can occur by up regulation of defense systems.

This could have various effects:

(a) Cell is completely protected against damage.

(b) There is protection of cell against oxidative damage to some extent but

complete protection is not there.

(c) Overprotection of cell to the extent that the cell may become resistant to

oxidative stress.

1.3.4.2. Cell injury: Various components of cell are damaged as a result of

oxidative stress. Consequently, cellular proteins, lipids and even DNA can be

damaged. Oxidative stress may change the various ion levels like that of calcium,

with drastic effects on cell functions. Similarly activation of proteases can result in

cell injury.

1.4.3.3. Cell death: Oxidative stress induced injury to cell will end up in three

ways

(a) Cell may repair or replace the damaged molecules and recuperate, or

(b) Oxidative damage persists and cell may stay alive with that damage, or

24

(c) Oxidative injury involving genetic machinery of cell may not be repaired and it

can result in cell death, by apoptosis or necrosis (Halliwell and Whiteman, 2004).

1.3.5: Effects of stress on organs and systems:

Oxidative stress is thought to be involved in the pathogenesis of many

diseases such as malignancies, Parkinson’s disease, diabetes mellitus,

atherosclerosis, AIDS, inflammatory bowel disease, nervous system disorders,

motor neuron disease and even related with premature child birth (Agarwal and

Prabakaran, 2005), therefore stress may affect multiple organs and systems of

body.

The stressors involving psychological factors stimulate various neural

circuits mediating neuroendocrine responses involving cortical activation of the

basolateral amygdala, which in turn activates the central nucleus of the amygdala.

The central amygdala then activates hypothalamic neurons, either directly by

central amygdala or indirectly through the stria terminalis. The stimulation of

hypothalamus can also result via circuits involving brainstem serotonergic and

catecholaminergic neurons (Van de Kar and Blair, 1999).

In order to control the stress response, neurons are located in the

hypothalamus and brain stem. These include the parvocellular corticotropin

releasing hormone (CRH), arginine – vasopressin (AVP) neurons of the

paraventricular nuclei (PVN) of the hypothalamus, and the locus ceruleus (LC) –

nor epinephrine system which is also called the central sympathetic system

(Chrousos, 1992; Tsigos and Chrousos, 1994). The hypothalamo pituitary adrenal

(HPA) axis, along with the sympathetic/adreno medullary systems are the effectors

which are activated in response to stress with resultant effects of stress on different

25

organs of body (Gold et al., 1988a; Gold et al., 1988b). Enteric nervous system of

gastrointestinal tract may also respond to stress via activation of Vagus nerve and

sacral parasympathetic outflow is enhanced (Habib et al., 2001).

Neurons of the hypothalamic Para ventricular nucleus secrete the

corticotropic releasing hormone (CRH) into the portal circulation of the pituitary

gland (Van de Kar and Blair, 1999). CRH is a peptide containing 41 amino acids

and was first isolated in 1981 by W. Vale (Vale et al., 1981). CRH reaches the

anterior pituitary, where it stimulates adreno corticotrophic hormone (ACTH)

secreting cells causing the release ACTH into the blood (Van de Kar and Blair,

1999). AVP stimulate the ACTH release in synergism with CRH, however AVP

alone do not increase the release of ACTH significantly (Lamberts et al., 1984).

ACTH acts on the adrenal cortex, and result in amplified release of cortisol into the

blood. Also there is enhanced release of prolactin from the anterior pituitary in

stress, that’s why prolactin is also considered as a stress hormone (Van de Kar and

Blair, 1999).

When there is no stress, both CRH and AVP are secreted in a pulsatile

manner with a circadian rhythm (Engler et al., 1989). Under resting conditions,

early morning hours are associated with increased pulses of CRH and AVP as a

result of which ACTH and Cortisol levels increase in circulation in early morning

hours (Horrocks et al., 1990; Chrousos and Gold, 1998). The diurnal patterns of

variations can be disturbed by changes in the duration of dark and light cycles and

are disrupted by stress. Number of pulsations in the hypophyseal portal system

relevant to CRH increase during acute stressful conditions with consequent

increase in ACTH and cortisol secretory episodes (Tsigos and Chrousos, 1994).

26

The high levels of cortisol as well as nor epinephrine and gamma-amino butyric

acid inhibit the release of CRH (Miller and O’Callaghan, 2002).

Repeated stress influences the functions of various parts of brain.

Hippocampus is affected significantly due to the presence of receptors for stress

hormones like cortisol (McEwen et al., 1986). It is also thought that hippocampus

is also important in regulation of stress response by inhibiting the stress induced

hyper activity of HPA axis (Jacobson and Sapolsky, 1991).

1.3.6: Stress hormones

1.3.6.1: Glucocorticoids

Glucocorticoids are produced by adrenals from cholesterol in response to

adrenocorticotrophic hormones (ACTH) (Pizarro and Troster, 2007). They are

produced with a circadian rhythm, but stress disrupts their circadian rhythm.

Glucocorticoids inhibit the hypothalamo-pituitary-adrenal axis via negative

feedback mechanism (Pizarro and Troster, 2007).

The ultimate effectors of the HPA axis are glucocorticoids and they play

role in the control of stress response and maintenance of homeostasis. They not

only regulate basal activity of HPA axis but also control the termination of stress

response (de Kloet, 1991). Negative feedback inhibition by glucocorticoids

decreases the exposure time of body tissues and cells to glucocorticoids and in this

way decreases the catabolic, immunosuppressive and anti-reproductive effects of

stress hormones. The mechanism of action of glucocorticoids involves cytoplasmic

receptors which are present everywhere on cell (Pratt, 1990; Smith and Toft, 1993).

On ligand binding, the glucocorticoid receptors translocate into the nucleus, where

specific glucocorticoid responsive elements (GREs) are activated within the DNA

27

to activate appropriate hormone-responsive genes. Glucocorticoids thus induce

translation of messenger RNAs with resultant activity of several glucocorticoid-

responsive proteins (Pratt, 1990).

If the HPA axis remains hyper active for prolonged periods, the resultant

increase in the glucocorticoids may suppress the secretion of growth hormone.

Glucocorticoids also inhibit the action of Somatomedin C with resultant decrement

in effects of Growth hormone on target tissues (Burguera et al., 1990). Similarly, if

there is hyperactive HPA axis, it may lead to decrease in production of thyroid-

stimulating hormone and it may also block the conversion of thyroxine to tri-

iodothyronine with decline in thyroid function (Benker et al., 1990).

1.3.6.1.1: Corticosterone: Major glucocorticoid in rats

Predominant glucocorticoid in rodents like rats is corticosterone (Miller and

O’Callaghan, 2002). Therefore, plasma corticosterone level is an indicator of

activity of HPA axis in rats. Corticosterone levels are increased in psychological

and physical stress, e.g., experimentally induced restraint stress in rats will increase

the plasma levels of corticosterone (Bauer et al., 2001), and so the levels of

corticosterone may serve as a good indicator of stress in rats.

Corticosterone has many metabolic effects that may include the glucose

production from protein and diverse effects on fat metabolism. Various other

important effects include inflammatory responses, vascular responsiveness and the

effects on the central nervous and immune systems.

1.3.6.2: Catecholamines

Stress activates sympathetic adrenal system with release of catecholamines

like epinephrine and nor epinephrine. Usually cortisol release is also in

28

proportionate to release of catecholamines in response to stress. Therefore, greater

the cortisol, more will be the norepinephrine (Cacioppo et al., 1995). As narrated

by Fernstrom & Fernstrom, norepinephrine (NE) is synthesized in sympathetic

neurons and in the adrenal glands. The initial step involves hydroxylation of

tyrosine to dihydroxyphenylalanine (DOPA) which is catalyzed by the enzyme

tyrosine hydroxylase (TH), DOPA is promptly decarboxylated to dopamine (DA)

by aromatic L-amino acid decarboxylase. In neurons that use DA as a transmitter,

no further change occurs and dopamine is used as such. Neurons that use NE as a

transmitter, NE is produced from the dopamine by the action of the enzyme

dopamine β -hydroxylase. Neurons using epinephrine as a transmitter contain

another enzyme, phenylethanolamine-N-methyl transferase, which catalyze the

conversion of NE to epinephrine. The rate limiting is the initial step in the pathway

i.e., Tyrosine hydroxylation, which controls the rate of synthesis of both nor

epinephrine and epinephrine (Fernstrom and Fernstrom, 2007; Nemeroff, 1998).

The sympathoadrenal system activation is dependent not only on the

impulse activity in nerves or amounts of NE and E released, but the sensitivity and

responsiveness of the tissues to catecholamines is also very important, which is

changed in various physiological and pathophysiological states by up regulation or

down regulation of receptors and change in affinity of receptors (Christensen and

Jensen, 1994).

Norepinephrine (NE) as summarized by Weinshenker and Schroeder plays

an important role in attention, arousal, and stress reactions in challenging

environments. NE is also thought to play role in learning and memory, neuronal

excitability, pain and affective disorders (Weinshenker and Schroeder, 2007). The

29

levels of epinephrine and nor epinephrine rise in males during physical and mental

stress (Gustafson and Kalkhoff, 1982; Barnes et al., 1982), and in rats during

immobilization stress (Kvetnansky et al., 1979). Catecholamines are shown to

decrease the plasma testosterone levels in human and animal studies (Damber and

Janson, 1978).

As mentioned before, CRF is increased in stress and if CRF is injected into the

locus ceruleus of rats, it stimulates tyrosine hydroxylase (TH), an enzyme which

regulates the NE synthesis, which results in increased synthesis of NE. Similarly

activation of TH is seen in experimentally induced stress, in animals (Melia and

Duman, 1991), therefore to assess the stress; it is worth to measure the levels of

norepinephrine.

Role of catecholamines during stress at Leydig cell level is less known

(Hardy et al., 2005). Catecholamines are generally thought to increase

steroidogenesis in vitro (Mayerhofer et al., 1993). Beta blockers which decrease

sympathetic activity decrease testicular steroidogenesis (Khan et al., 2004). So nor

epinephrine increase levels of testosterone, while stress decreases the testicular

steroidogenesis. But in stress, along with other hormones like corticosterone, the

levels of nor epinephrine increased. How stress induced increase in nor epinephrine

affect Leydig steroidogenesis, will be evaluated in vivo as well as in vitro on

isolated and cultured Leydig cells.

1.3.7: Malondialdehyde and stress

Reactive oxygen species are known to cause oxidative damage to

macromolecules (Cross et al., 1987). Reactive oxygen species usually cause lipid

peroxidation which is a multifaceted phenomenon that occurs in biological

30

membranes made up of polyunsaturated fatty acids and may lead to the formation

of lipid hydroperoxides and their metabolites. Most cases concerning lipid

peroxidation start from a chain reaction which is initiated by free radicals. Lipid

hydroperoxides accumulate in the membrane which may cause its receptors and

enzymes to become inactive thereby affecting its functions, causing instability and

making the membranes permeable to ions (Kazez et al., 1997). In order to

determine the levels of ROS, we can use the levels of lipid peroxidation products in

the body fluids as a diagnostic tool (Kiarostami et al., 2006).

The degradation of lipid peroxides of the biological membranes may result

in formation of a number of aldehydes such as malondialdehyde (MDA), which

may cause damage to cells by reacting with proteins, lipids and nucleic acids (Sim

et al., 2003). A simple method of high sensitivity, frequently used as a lipid

peroxidation marker, involves thiobarbituric acid-reactive substances. Therefore,

MDA which is a thiobarbituric acid reacting substance (TBARS) is considered as a

suitable indicator of lipid peroxidation triggered by free radicals (Kazez et al.,

1997). Polyunsaturated fatty acids after oxidation may result in increased levels of

MDA, which can be used as an in vivo marker to assess lipid peroxidation in

diseases like atherosclerosis and diabetes (Haberland et al., 1990; Slatter et al.,

2000). Therefore in order to assess the amount of lipid peroxidation and to estimate

whether the antioxidants confer production against stress induced lipid

peroxidation, we estimated the levels of MDA in our study.

1.3.8: Role of superoxide dismutase as natural antioxidant in stress

Halliwell & Gutteridge defined an antioxidant as “any substance that when

present at low concentrations compared with those of an oxidizable substrate

31

significantly delays or prevents oxidation of that substrate” (Halliwell and

Whiteman, 2004).

Antioxidants are the most important defense mechanism against oxidative

stress that results from generation of free radicals. Antioxidants may play a role to

prevent oxidants induced injury and may also act as free radicals scavenger.

Prevention antioxidants include the metal chelators and metal-binding proteins

which play a role in blocking the formation of new ROS. On the other hand,

scavenger antioxidants eradicate the ROS that have previously been generated as a

result of oxidative stress process (Agarwal and Prabakaran, 2005).

Endogenous antioxidant systems that may act as free radical scavengers

may have important role to combat against stress induced lipid peroxidation. One

of important endogenous antioxidant defense system is an enzyme, superoxide

dismutase (Ferrari et al., 1991). Superoxide dismutase was discovered by Irwin

Fridovich and Joe McCord and initially it was considered that these metalloproteins

have no function (McCord and Fridovich, 1988).

The importance of superoxide dismutase (SOD) was investigated in

genetically engineered mice lacking these enzymes. Various diseases were found in

these mice and excessive oxidative stress caused even death of these SOD deficient

mice (Li et al., 1995). SOD deficient mice are more prone to develop diseases like

hepatocellular carcinoma (Elchuri et al., 2005). If SOD is given, it will decrease

reactive oxygen species generation and oxidative stress in clinical conditions like

inflammatory bowel disease (Segui et al., 2004). The mechanism by which SOD

exerts its effect is that, it prevents oxidative stress induced damage to cells caused

by reactive oxygen species. SOD converts superoxide to H2O2 and oxygen.

32

Consecutively, H2O2 is reduced by CAT and GPx to H2O and oxygen. SOD thus

helps the body to combat with the deleterious effects of reactive oxygen species

(Jones et al., 1981).

In mammalian cells, including humans and rats, super oxide dismutase is

present in three forms, which include the Mn SOD, Cu-Zn SOD, and extracellular

Cu-Zn SOD. Different genes encode each of these super oxide dismutases and these

differ in amino acid sequence and localization (Keller et al., 1991). In humans

mental stress is reported to increase antioxidant enzymes, super oxide dismutase

(SOD) of seminal plasma with abnormal sperms (Eskiocak et al., 2005). Restraint

stress on rats caused increased circulating levels of SOD, catalase. Testicular tissue

is highly susceptible to stress because testicular membranes are rich in

polyunsaturated fatty acids (Chainy et al., 1997).

Therefore, lipid peroxidation and spontaneous oxygen toxicity is prevented

by Superoxide dismutase (Fridovich, 1985). The levels of SOD may thus indicate

the activity of natural intrinsic anti-oxidant defense system of body. Therefore in

order to assess the natural antioxidant activity in acute restraint stress to rats and to

estimate whether the antioxidants confer production against stress induced

derangements in antioxidant activity, we estimated the levels of SOD in our study.

1.4: Antioxidant supplements and stress

Antioxidants obtained from dietary sources constitute an essential

component of human antioxidant defense system. Various fruits and vegetables and

diet supplements are the possible sources of various antioxidants (Agarwal and

Prabakaran, 2005). Oxidative stress can be controlled by use of chain breaking

antioxidants like ascorbic acid and alpha tocopherol (Agarwal et al., 2005). Many

33

epidemiologic and clinical trials are in favor that if the diet is supplemented with

antioxidant vitamins it may result in a decrease in the occurrence of chronic disease

morbidity and mortality (Weber et al., 1996; Enstrom, 1997; Gey, 1998). Among

various dietary antioxidants, ascorbic acid and alpha tocopherol are antioxidants

which are recognized by US Food and Drug Administration (FDA) as dietary

antioxidants (Monsen, 1996).

1.4.1: Ascorbic acid

Ascorbic acid is one of the important and essential micronutrients and it is

required for various metabolic functions of the body (Jaffe, 1984). It is thought

that human beings have lost the capability to produce ascorbic acid within their

body due to mutation in gene coding for L-gulonolactone oxidase, which is an

enzyme required for the biosynthesis of ascorbic acid via the glucuronic acid

pathway (Woodall and Ames, 1997). So, ascorbic acid forms an essential

constituent of diet. Ascorbic acid is found abundantly in fresh citrus fruit and in

various vegetables (Bendich, 1997). Deficiency of ascorbic acid in the diet results

in the disease called Scurvy (Levine, 1986). Consumption of 10 mg ascorbic acid

per day is sufficient to prevent scurvy (Weber et al., 1996), and it can be gained

through intake of fresh fruit and vegetables.

Ascorbic acid is a cofactor for numerous enzymes concerned with

biosynthesis of carnitine, collagen, and various neurotransmitters (Burri and Jacob,

1997; Tsao, 1997). Procollagen-proline dioxygenase (proline hydroxylase) and

procollagen-lysine 5-dioxygenase (lysine hydroxylase), which are the two enzymes

concerned with procollagen biosynthesis, need ascorbic acid for optimum activity

(Phillips and Yeowell, 1997).

34

The deficiency of ascorbic acid may lead to the weakening of collagenous

structures, the consequences of which may include the tooth loss, joint pains, bone

and connective tissue disorders and poor wound healing which are important

features of scurvy (Burri and Jacob, 1997). Hypochondria, depression, and mood

variations frequently take place during scurvy and these could be correlated with

incomplete dopamine hydroxylation. The actions of a variety of other enzymes may

also need ascorbic acid. These enzymes include mono and dioxygenases, involved

in peptide amidation and tyrosine metabolism (Burri and Jacob, 1997; Tsao, 1997)

Data from many various studies have narrated that ascorbic acid helps to

reduce the incidence of various diseases which may include cancer, cataract and

cardiovascular disease (Weber et al., 1996; Enstrom, 1997; Gey, 1998). It is also

proposed that endogenous and exogenous ascorbic acid decreased lipid oxidation

(Carr and Frei, 1999). Generally ascorbic acid has beneficial effects but it is also

claimed to play prooxidant role in some situations (Podmore et al., 1998).

1.4.2: Alpha tocopherol

Alpha tocopherol was discovered in 1922 by Evans and Bishop as an

essential dietary factor for reproduction in rats (Evans and Bishop, 1922). It was

later named as ‘factor 2’ in 1950’s by Klaus Schwarz and placed in the group of

cellular antioxidant systems, along with sulfur amino acids and selenium (Schwarz,

1965).

Like ascorbic acid, alpha tocopherol must be obtained from the diet

(Bertinato et al., 2007). Recommended daily allowance (RDA) for alpha

tocopherol is 8 mg (12 IU) for females and 10 mg (15 IU) for males. Alpha

tocopherol is a vital nutrient which is required for normal growth and development.

35

It is considered as a major lipid-soluble antioxidant in animals (Burton, 1994).

Major sources of alpha tocopherol include whole grains, nuts and vegetable oils.

Dietary vitamin present in the form of tocopherol esters are hydrolyzed in the

intestinal lumen by pancreatic esterases (Debier and Larondelle. 2005; Hacquebard,

and Carpentier, 2005; Rigotti, 2007).

Free radical chain reactions are broken up by alpha tocopherol molecules by

capturing the free radicals. This action is responsible to make alpha tocopherol an

ideal antioxidant. The antioxidant properties are due to free hydroxyl group on the

aromatic ring. The hydrogen of the hydroxyl group is given to the free radical,

which may result in formation of relatively stable free radical form of alpha

tocopherol (Sies and Murphy, 1991). Chain reaction of lipid peroxidation in

biomembranes and lipoproteins is blocked by alpha tocopherol (Dieber-Rotheneder

et al., 1991).

The antioxidant activity of alpha tocopherol has been proven by various

studies and its ability to prevent chronic diseases, especially caused by oxidative

stress component such as cardiovascular diseases, atherosclerosis, and cancers have

been extensively investigated. Epidemiological studies have reported that high

alpha tocopherol intakes are correlated with a decreased risk of coronary heart

disease (Rimm et al., 1993). Diabetes mellitus also induces stress and alpha

tocopherol prevents diabetic rat testis against oxidative stress (Naziroglu, 2003).

Therefore, alpha tocopherol has antioxidant properties and it is useful in

preventing various diseases. We can say that alpha tocopherol protects various

biological systems of our body (Packer, 1991), by preventing lipid peroxidation

(Evstigneeva et al., 1998). Alpha tocopherol, if present in an adequate amount at

36

site of oxidative stress where there is free radical generation, it may reduce the

toxic effects of ROS (John et al., 2001). Due to these antioxidant properties of

alpha tocopherol, we used this antioxidant in our research to evaluate its preventive

role as an antioxidant against stress.

The rationale of this study was to investigate the effects of acute restraint stress on

testicular steroidogenesis. Catecholamines are thought to increase steroidogenesis

and corticosteroids decrease steroidogenesis, but net effect of stress is considered to

be decreased testosterone. So the separate and combined effects of these hormones

on testicular steroidogenesis will be evaluated at the level of Leydig cell. Also the

mechanism of action of stress hormones, corticosterone and nor epinephrine on

cultured Leydig cells is also investigated.

Testicular tissue is highly susceptible to stress because testicular membranes are

rich in polyunsaturated fatty acids (Chainy et al., 1997). So by giving acute

restraint immobilization stress to rats, oxidant and antioxidant status will be seen

by MDA and SOD respectively. As explained earlier in detail that antioxidant

ascorbic acid and alpha tocopherol are thought to protect the body against stress

induced damages (Zaidi et al., 2003). So the role of these antioxidants on stress

induced decline of testosterone and the effects of these antioxidant vitamins on

stress induced lipid peroxidation and SOD was evaluated, first in vivo study on rats

and then in vitro on isolated and cultured Leydig cells of rats.

37

OBJECTIVES OF STUDY In vivo studies

1 To determine the effects of six hours acute restraint stress on male

Sprague Dawley rats on the levels of serum testosterone, serum

corticosterone, plasma nor epinephrine and serum LH.

2 To determine the effects of six hours acute restraint stress on male

Sprague Dawley rats on lipid peroxidation (in terms of

malondialdehyde) and antioxidant enzyme, superoxide dismutase.

3 To determine the preventive effects of antioxidants, ascorbic acid and

alpha tocopherol separately and combined on derangements induced by

stress hormones on testicular steroidogenesis in male Sprague Dawley

rats.

In vitro studies

1 To develop an in vitro model of isolated and cultured Leydig cells of

male Sprague Dawley rats to study the effects of stress hormones at

Leydig cell level.

2 To explore the mechanism of action of corticosterone and nor

epinephrine on testicular steroidogenesis at Leydig cell level.

3 To ascertain the role of antioxidants like ascorbic acid and alpha

tocopherol to overcome the stress hormones induced effect on testicular

steroidogenesis.

Hypothesis Stress hormones adversely affect testicular steroidogenesis which can be prevented

by the use of antioxidants like ascorbic acid and alpha tocopherol.

38

MATERIALS AND METHODS 2.1 In vivo study

2.1.1 Setting

The study was conducted in the department of Physiology, Army Medical

College, Rawalpindi, Pakistan, in collaboration with National Institute of Health

Islamabad. Laboratory work was done primarily in Centre for Research in

Experimental and Applied Medicine, (CREAM) which is a multi-disciplinary

research laboratory at Army Medical College, established under auspices of

National University of Sciences and Technology (NUST), Islamabad, Pakistan.

2.1.2 Duration of study

Two years as full time resident student.

2.1.3 Study design

Quasi experimental studies.

2.1.4 Chemicals used in in vivo part of study

Following chemicals were purchased from local supplier:

2.1.4.1 Ascorbic acid.

MERCK, Research grade. Cat No: 500074.

2.1.4.2 Alpha tocopherol

MERCK, Research grade. Cat No: 500854.

Chemicals were weighed with the help of Sartorius Balance. After cleaning

balance top, leveling was done by adjusting the bubble in the centre of ring. First of

all, balance was zeroed by pushing the tare button. Then a clean weighing boat was

placed on the balance and again tarred to account for its weight. Then chemical was

placed into weighing boat and adding continued with the help of spatula till the

Chapter 2

39

desired weight achieved. When the displayed reading for desired weight steadied, it

was then shifted to storage container which was then sealed and labeled.

2.1.5 Animals of study: Rats

Male Sprague Dawley healthy rats 90 days old, bred at National Institute of

Health, Islamabad were used in this experiment. Average weight of rats was

275±50 grams. Animals were handled two to three days prior to start of

experiments to make them acclimatized to new handler.

2.1.6 Animal house

Animal house facility of National Institute of Health (NIH), Islamabad was

used. This animal house has a setup according to international standards for

breeding and housing of research animals. A separate room was allocated for study

groups of this research. Metallic rack was used to place different cages of the rats.

Room was well ventilated and 12 hour light and 12 hour dark cycle was

maintained. Temperature of that room was maintained at 22 ± 3 ° C, with the help

of central temperature regulating systems with ducts opening in each room (Dong

et al, 2004).

2.1.6.1 Cages and Water Bottles

Animals were placed in cages of 2 x 3 feet size, ten animals per cage. Clean

water bottles specific to fit over these cages for continuous supply of water were

used.

2.1.6.2 Water for rats

Rats were provided with water in clean bottles that are specific for the rat

cages and have valve at their opening from which water comes out when the rat

sucks it. Bottle was fitted inverted over the cages with one bottle per cage. Bottles

40

were cleaned every alternate day as per protocol of the animal house. Water bottles

were filled at least twice a day.

2.1.6.3 Rats feed.

It was provided by animal house of NIH. Food was supplemented with

antioxidants as described in grouping. Food composition given to rats is attached as

annexure A and B.

2.1.7 Sample size: Eighty male Sprague Dawley rats.

2.1.8 Sample selection

2.1.8.1 Inclusion criteria

1 Male Sprague Dawley rats

2 Healthy rats with weight 275 ± 50 grams.

3 90 days old

2.1.8.2 Exclusion criteria

1 Rats with disease

2 Rats who develop disease during course of study.

2.1.9 Grouping (N=80)

16 rats were taken per group and they were fed on standard and

supplemented diets for one month. Each cage labeled both for group numbers and

type of diet.

Group I (n=16): This was control group. Rats were fed normal standard rat diet

(see Annex A & B) without any supplementation. These rats were given plain tap

water.

Group II (n=16): Rats were fed normal standard rat diet (see Annex A and B)

without any supplementation. These rats were given plain tap water. These rats

41

were exposed to acute immobilization stress in a mesh wire restrainer for 6 hours.

(Dong et al, 2004)

Group III (n=16):

This group was fed normal standard diet. Ascorbic acid supplementation

was given as 500 mg ascorbic acid/ L drinking water (Hsu et al., 1998). These rats

were exposed to acute immobilization stress in a mesh wire restrainer for 6 hours,

after one month of antioxidant supplemented diet (Dong et al, 2004).

Group IV (n=16)

This group was fed alpha tocopherol supplemented diet with 300 mg alpha

tocopherol /kg chow + 2% soyabean oil (Hsu et al., 1998). These rats were given

plain tap water. These rats were exposed to acute immobilization stress in a mesh

wire restrainer for 6 hours, after one month of antioxidant supplemented diet

(Dong et al, 2004).

Group V (n=16)

This group was fed alpha tocopherol supplemented diet with 300 mg alpha

tocopherol/kg chow + 2% soyabean oil. Also, ascorbic acid supplementation was

given as 500 mg ascorbic acid/ L drinking water. These rats were also exposed to

acute immobilization stress in a mesh wire restrainer for 6 hours, after one month

of antioxidant supplemented diet (Dong et al, 2004).

2.1.10 SAMPLING TECHNIQUE

To avoid bias among different values among cortisol and testosterone because of

diurnal variations, all samples were taken at same timings of the day between 11

am and 12 noon (Debigare et al., 2003).

42

2.1.10.1 Procedure for sampling

First of all, rats were placed one by one in a closed chamber with ether

soaked in cotton in that chamber in the main laboratory of animal house. Rats were

thus anesthetized (which took approximately 3-5 minutes per rat). After this, rat

was placed on dissection board and after ensuring proper anesthesia, intracardiac

blood sample was drawn with the help of BD 5cc disposable syringe (Hsu et al.,

1998).

2.1.10.2 Intra cardiac sampling:

For intra cardiac sampling, each rat was placed at its back. After palpation

of lower rib cage and sternal margin, syringe needle was inserted into heart taking

care that it may not pierce the heart up to its posterior wall. Then blood was

withdrawn in syringe to ensure that needle is in heart. If no blood came in syringe,

then needle was gently pulled out but not to the extent that it comes out of body of

rat. After this, needle of syringe was reinserted into the heart and checked for

proper insertion.

2.1.10.3 Sampling tubes- 5 ml CP tubes

After this, blood was drawn slowly up to 5 ml in the syringe. Then 3.5 ml

from this blood was immediately shifted into BD vacutainer. Blood was allowed to

clot. Another 1.5 ml was placed in BD vacutainer containing potassium oxalate for

obtaining plasma

2.1.11 Processing of samples:

2.1.11.1 Cold centrifugation.

Samples were first centrifuged at 4000 rpm at 4ºC in the cold centrifuge.

Here eppendorf centrifuge was used. First of all, samples were adjusted in the

43

centrifuge machine taking care of balance on opposite site. First temperature was

set (to ensure that temperature inside eppendorf chamber is 4ºC) and then speed

adjusted to 4000 rpm for 15 minutes.

2.1.11.2 Storage of samples

After cold centrifugation, serum was pipetted out and 1.5 ml of serum was

taken per sample and placed in 1.5 ml eppendorf storing tubes (which were labeled

according to grouping protocol).

Also about 0.5 ml of plasma was obtained from all samples in which

preservative (EDTA) added and were placed in separate labeled eppendorf storage

tube.

Samples were stored at – 80 ° C temperature refrigerator till analysis.

2.2 IN VITRO EXPERIMENTATION

2.2.1 Chemicals used for in vitro experimentation

• Eagles medium M 199 (sigma-Aldrich)

• Hydroxy ethyl piperazine ethane sulphonic acid. (HEPES) (sigma-

aldrich)

• Theophylline (sigma-Aldrich)

• Isobutryl methyl Xanthine (IBMX)

• Bovine serum albumin (sigma-Aldrich)

• Gentamycin (sigma-Aldrich)

• Collagenase (sigma-Aldrich)

• Bovine serum Albumin (sigma-aldrich)

• Dulbecco’s modified Eagles Medium (DMEM) (sigma-aldrich)

• Percoll (sigma-Aldrich)

44

• Phosphate Buffer Saline (sigma-Aldrich)

• Dimethyl sulfoxide (DMSO) • Trypan blue

• Sodium Hydoxide 1 M (NaOH-Sigma-Aldrich)

• Hydrochloric acid 37 % (HCl-Merck)

• Ethanol

• Distilled water (Rock land) • Ether

2.2.2 Chemicals used for staining

a. Trypan-blue stain 0.4% (sigma-aldrich)

b. 3-beta-HSD stain (5 ml)

Nitro-blue-tetrazolium (Sigma-Aldrich) = 1 mg

Beta-NAD (Sigma-Aldrich) = 5 mg

Dehydroandrosterone (Sigma-Aldrich) = 0.2 ml

PBS = 4.8 ml

2.2.3 Equipment used

• Analytical balance (Sartorius)

• Parafilm (Chicago, USA)

• Micropipettes

• Stirring plate Thermix® -310T (Allied Fisher Scientific, USA)

• pH meter (Selectra)

• Ice flaker (Ilshin)

• Centrifuge (temperature controlled), Eppendorff-5810 R (Hamburg, Germany)

45

• Shaking Incubator JSSI-100 C (JS Research Inc. Korea)

• Shaking water bath JSSB-30 T (JS Research Inc. Korea)

• Neubauer Chamber 0.100 mm, 0.0025 mm2 (Bright Line, Assistant,

Germany)

• Microscope (Nikon)

• Glassware Pyrex® Corning (USA)

• Culture tubes 12 x 75 mm, Pyrex ® (USA)

• Conical centrifuge tubes 15 and 50 ml (Falcon)

• Timer (Sanyo)

• Racks

• Glass beakers and bottles (Autoclaved)

• Nylon mesh

• Funnel

• Spinal needle

• Pipettes (all sizes)

• Ethanol washed magnets as stirrers

• Eppendorf tubes and Pasteur pipettes

• Discarding beaker

• Surgical set, kidney tray10 cc syringes

• Ice bucket

46

2.2.4 Reconstitution of Solutions

2.2.4.1 Eagle’s Medium

In 100 ml M-199, HEPES (0.595 g), Theophylline (0.009 g) and

Gentamycin (100 µl) were mixed. Later 0.1 g of BSA was added and

allowed to mix without stirring. The pH was maintained at 7.4.

2.2.4.2 Biophosphate Buffer

100 ml bio phosphate buffer was prepared by adding 0.001g of BSA and

100 ul of gentamycin in 100 ml PBS.

2.2.4.3 Discontinuous Percoll gradient (Four gradients for 4 rats)

Stock: 10X M 199 + 1.0% BSA (30 ml M199 + 0.3 g BSA)

Solution A (Percoll 0%)

Stock 12.5 ml

Water 112.5 ml

Total 125 ml

Solution B (Percoll 90%)

Stock 15.5 ml

Water 139.5 ml

Total 155 ml

pH of solution A and B was maintained at 7.4

2.2.4.4 Gradient Steps

For four gradients (4 rats) % Percoll Solution A (ml) Solution B (ml) 68 8.8 27.2

47

43 18.8 17.2 35 22 14 20 28 8 Preparation of Percoll Gradient

Percoll gradient was layered starting from bottom of 50 ml centrifuge tube, using a long spinal needle in the following sequence;

8 ml each of solution A, Percoll 20 %, 35 %, 43 %, 68 % and 90 %.

2.2.5 Reconstitution of Chemicals

Following chemicals were reconstituted as their molar solution and

concentration constituted are shown in pipetting protocol:-

Corticosterone (soluble in DMSO)

Cortisol (soluble in DMSO)

Nor epinephrine (soluble in DMSO)

Alpha tocopherol (soluble in DMSO)

Ascorbic acid (soluble in ultra pure water)

Cortisol (soluble in DMSO)

Pregnenolone (soluble in DMSO)

2.2.6 Procedure - Rat Leydig cells isolation and culture

Rats:

The rats were of same specifications as mentioned in 2.1.5 above.

48

2.2.6.1 Orchidectomy

We took four male Sprague Dawley rats per experiment from NIH and kept

them in our research lab for two days for acclimatization. The animals were fed

on standard diet and water ad libitum. Dark and light cycles were maintained as

per standards as mentioned before.

After anesthetizing the rats with ether, they were decapitated. The abdomen

was opened by a midline incision. The testes were brought out and examined, if

they were of same size and shape, only then the testes were dissected out and

decapsulated. Then the testis were placed in eagles medium on ice, in a tray

with ice crystals. (Testis were washed with Eagles Medium and exposed media

was removed)

2.2.6.2 Collagenase dispersion of testicular interstitial cells

The testis after decapsulation were placed in eagles medium (EM)

containing 0.25 mg/ml (10 ml) Collagenase in two 50 ml falcons tube. This

falcon tube was then sealed with parafilm.

Then we incubated these falcon tubes at 37°C in shaking water bath at a

speed of 75 cycles per min for 25 minutes. To stop the incubation process, 20

ml of EM was added and the contents were transferred to a beaker. Beaker was

placed in kidney tray with ice, smallest magnet placed in beaker; beaker was

covered with parafilm and then stirred with slow speed for 10 minutes.

Later, the contents were filtered through Nylon mesh. For that, we took

funnel, mesh and falcon tube. Pasteur pipette was used and contents were

shaken before filtering. Also the beaker was washed with medium at end to

49

remove the left over cells. After this, 15 ml falcon tube was centrifuged at a

speed of 800 rpm for 10 minutes at 4°C to remove collagenase.

2.2.6.3 Purification of Leydig cells by density gradient Percoll

centrifugation

The supernatant was discarded and pellet was resuspended in 24 ml Eagles

medium. Preformed Percoll gradient (as described in 2.2.4.4) was there, with us

at this stage. Then we layered the 6 ml cell suspension on the top of pre formed

gradient (20, 35, 43, 68 and 90 % Percoll in M199 with BSA) with Pasteur

pipette, taking care not to disturb gradient steps/layers for which we poured

along walls with Pasteur pipette by rotating the falcon tube.

Gradient was then centrifuged at 800 g for 25 minutes at 27°C in eppendorf

swinging rotor. Initially, needle and 10 ml syringe were cleaned with solution

A. Then, the third fraction from the top of each gradient was collected through

long needle (about 20 to 24 ml). Caution was exercised to prevent collection of

erythrocytes from fourth layer. (We first removed last layer and then removed

Leydig cell layer). That fraction was diluted in 2 volumes of M-199 with BSA

and centrifuged at 800 rpm, at room temperature for 20 min. For one gradient,

we resuspended cells in 32 ml M 199 with BSA

2.2.6.4 Leydig cell staining for 3-β-HSD

The presence of Leydig cells in the isolated cell fraction was confirmed by

3-β-HSD staining (Sharpe and Fraser, 1983). For that, 50 uL of cell suspension

was mixed with 150 ul staining solution, incubated at 37°C for 60 minutes in

incubator. To checked purity we put 50 µl of above mixture on

haemocytometer and purity of the cells was checked by counting total number

50

of blue stained cells and unstained cells in WBCs counting chamber (16 small

squares). The blue stained and the unstained cells represent presence and

absence of 3-beta HSD, respectively. Percentage of the Leydig cells was

calculated by following formula:

% age purity of LC = # of blue stained cells/total # of cells x 100

Total number of blue stained cells = 131

Total number of unstained cells = 12

Total number of cells = 148

% age purity of LC = 131/148 x 100 = 88.51 %

The purity of Leydig cells was 88.51 %.

2.2.6.5 Preincubation:

Cell suspension was incubated at 34°C in shaking water bath in a conical

flask at a speed of 75 cycles per min for 60 minutes to remove endogenous

testosterone. After incubation, cell suspension was diluted in triple volume of

PBS and then centrifugation was done at 900 rpm for 20 min, at room

temperature. Then, we discarded the supernatant and resuspended the pellet in

10 ml EM. For this, the pellet was dispersed in 1 ml EM and then added to

complete 10 ml EM.

2.2.6.6 Cell viability and cell count

Cell viability was checked by trypan blue exclusion method (Aldred and

Cooke, 1983). To check viability of cells, 50 ul of CS and 50 ul of Trypan blue

were mixed properly (Abdul-Saeed et al., 1995).

After charging the Neubauer Chamber, the cells were viewed under

microscope. The number of unstained and stained cells represents viable and

51

damaged cells, respectively. The cells were counted, and the percentage of viable

cells was calculated by following formula

% age of viable cells = # of unstained cells/total # of cells x 100

Cells were counted under microscope (40 x) as follows.

Total number of cells counted in four WBCs chamber = x

Cells/ml = x x 2 x 10,000

Total cells = cells/ml x total volume

In our experiment x = 31

So cells/ml = 31 x 2 x 10,000 = 580000

Total volume with us was 10 ml

So total cells = 620000 x 10 = 6200000 or 62 x 105 or 6.2 x 106

According to the rule of 85,000 cells/200 ul ((Abdul-Saeed et al., 1995),

85,000 cells are present in 200 µl or 0.2 ml

Therefore 01 cell was present in 0.2/85000

6.2 x 106 cells were present in 0.2/85000 x 6.2 x 106 = 14.588 ml

So we added more Eagle’s medium in cell suspension till total volume

became 14.588 ml to get 85000 cells/200 µl.

2.2.6.7 Incubation

All culture tubes were numbered in triplicates. In all the tubes, 200 ul of cell

suspension was pipetted to get 85,000 Leydig cells per tube

Cell sample were placed in culture tubes and different chemicals added as

shown in pipetting protocol and then we incubated at 34°C in shaking water

bath at 75 RPM. The reaction was then stopped after 03 hours by placing tubes

52

in 60°C water bath. After stopping reaction, the supernatant was pipetted in 1.5

ml eppendorf storing tubes and stored at -80°C till analysis.

Cell culture experiment was run in two steps. In first step we determined

appropriate doses of each chemical/ antioxidant. Then cells were exposed to

most appropriate dose of chemical. All the combinations were run with and

without LH, but here only that components of pipetting protocol are shown

whose results are narrated in this study i.e., with LH only (except with Isobutyl

methyl xanthine and pregnenolone containing wells, where their mechanism of

action is explored).

53

Table 2.1: Pipetting protocol for incubation of cultured Leydig cells from rats, step 1. (All were run in triplicate).

Tube No

Cell suspension 200 µl -

Sample (S) Stimulation

Chemical Bio

phosphate 1 S

-

- 100 µl

2 S LH 50 µl -

50 µl

3 S LH 50 µl Corticosterone 10 nM: 15 µl 35 µl

4 S LH 50 µl Corticosterone 100 nM: 15 µl 35 µl

5 S LH 50 µl NE 10 µM: 15 µl 35 µl

6 S LH 50 µl NE 100 µM: 15 µl 35 µl

7 S LH 50 µl NE 1000 µM: 15 µl 35 µl

8 S LH 50 µl IBMX 0.1 mM: 15 µl 35 µl

9 S LH 50 µl IBMX 1 mM: 15 µl 35 µl

10 S LH 50 µl IBMX 10 mM: 15 µl 35 µl

11 S LH 50 µl Pregnenolone 1 µM: 15 µl 35 µl

12 S LH 50 µl Pregnenolone 10 µM: 15 µl 35 µl

13 S LH 50 µl Pregnenolone 100 µM: 15 µl 35 µl

14 S LH 50 µl AA 10 µM: 15 µl 35 µl

15 S LH 50 µl AA 100 µM: 15 µl 35 µl

16 S LH 50 µl AA 200 µM: 15 µl 35 µl

17 S LH 50 µl AT 10 µg/ml: 15 µl 35 µl

18 S LH 50 µl AT µg/ml: 15 µl 35 µl

19 S LH 50 µl AT µg/ml: 15 µl 35 µl

LH: Luteinizing hormone; NE: Nor epinephrine; IBMX: Isobutyl Methyl

Xanthine; AA: Ascorbic acid; AT: Alpha tocopherol.

54

Table 2.2: Pipetting protocol for incubation of cultured Leydig cells from rats, step 2. (All were run in triplicate).

LH: Luteinizing hormone; NE: Nor epinephrine; IBMX: Isobutyl Methyl

Xanthine; AA: Ascorbic acid; AT: Alpha tocopherol.

Tube No Cell suspension 200 µl -

Sample (S)

Chemical

Chemical

Bio phosphate

1 S LH 50 µl VIT C 15 µl + VIT E 15 µl - 20 µl

2a S LH 50 µl Corticosterone 15 µl + NE 15 µl -

20 µl

2b S - Corticosterone 15 µl IBMX 15 µl 70 µl

3a S LH 50 µl Corticosterone 15 µl IBMX 15 µl 20 µl

3b S - NE 15 µl IBMX 15 µl 70 µl

4a S LH 50 µl NE 15 µl IBMX 15 µl 20 µl

4b S - Corticosterone 15 µl + NE 15 µl IBMX 15 µl 55 µl

5a S LH 50 µl Corticosterone 15 µl + NE 15 µl IBMX 15 µl 05 µl

5b S - Corticosterone 15 µl Pregnenolone 15 µl 70 µl

6a S LH 50 µl Corticosterone 15 µl Pregnenolone 15 µl 20 µl

6b S - NE 15 µl Pregnenolone 15 µl 70 µl

7a S LH 50 µl NE 15 µl Pregnenolone 15 µl 20 µl

7b S - Corticosterone 15 µl + NE 15 µl

Pregnenolone15 µl 55 µl

8 S LH 50 µl Corticosterone 15 µl + NE 15 µl

Pregnenolone15 µl 05 µl

9 S LH 50 µl Corticosterone 15 µl AA 15 µl 20 µl

10 S LH 50 µl Corticosterone 15 µl AT 15 µl 20 µl

11 S LH 50 µl Corticosterone 15 µl AA 15 µl + AT 15 µl 05 µl

12 S LH 50 µl N E 15 µl AA 15 µl 20 µl

13 S LH 50 µl N E 15 µl AT 15 µl 20 µl

14 S LH 50 µl N E 15 µl AA 15 µl + AA 15 µl 05 µl

15 S LH 50 µl Corticosterone 15 µl + NE 15 µl

AA 15 µl 05 µl

16 S LH 50 µl Corticosterone 15 µl + NE 15 µl

AT 15 µl 05 µl

17 S LH 50 µl Corticosterone 15 µl + NE 15 µl

AA 15 µl + AT 15 µl -

55

2.3 ESTIMATION OF HORMONES

2.3.1 TESTOSTERONE (for In vivo samples) KIT: ADALTIS- EIAgen Testosterone- LI 4011 K. It is an enzyme immune assay kit for measurement of Testosterone in the serum or

plasma quantitatively.

2.3.1.1 Assay Principle

It is based on solid phase competitive enzyme immuno assay. Horseradish

peroxidase labeled Testosterone (HRP-testosterone) competes with the testosterone

present in sample for a fixed and limited number of antibody sites immobilized on

the walls of microstrips. After completion of competitive immuno reaction, wells

of microplate are washed and the amount of HRP testosterone that is in bound state

with antibody in the solid phase is measured by adding a chromogen/ substrate

solution that which converts it to blue compound by the bound conjugate.

After 15 minutes of incubation, sulphuric acid is added to stop the enzymatic

reaction and as a result the color of solution is changed to yellow color. At 450 nm,

the absorbance of solution is measured photometrically. The value of absorbance

which we obtain is related inversely to the concentration of testosterone which is

present in the sample. A calibration curve is drawn to find out the final values of

testosterone in our study sample.

2.3.1.2 REAGENTS

Testosterone Microplate

The microplate which is provided has 12 strips x 8 wells. Each well is

coated with anti testosterone obtained from rabbit.

56

Testosterone Calibrators The vials contain standard Testosterone

concentration and 0.09 % Sodium Azide. Testosterone concentrations are as

follows: 0 – 0.2 – 1 – 4 – 8 – 16 ng/ml. regarding calibrators volume, the volume of

zero calibrator was to be kept 2 ml and volume of other calibrators was maintained

at 0.5 ml.

HRP Testosterone Conjugate

The bottle has 22 ml horseradish peroxidase labeled testosterone in buffer

which is supplemented by the bovine serum albumin (0.5 %) and the testosterone

binding protein displacers.

Washing solution. (10 x concentrated)

A bottle of the washing solution 10 x has Tween 20 (0.1 %) and

Amphotericin B (2.5 ug/ml) which is present in the citrate- borate buffer, 50 ml.

Contents of this vial are to be diluted 500 ml distilled water and thorough mixing is

also required.

Substrate HS

Another bottle of substrate HS containing 0.26 mg/ml of 3,3' ,5,5' Tetra

methyl benzidin (TMB) along with 0.01 w/v Hydrogen peroxide, in citrate buffer.

There is 13 ml volume of this container.

2.3.1.3 TESTOSTERONE ASSAY PROCEDURE

o All reagents and samples were brought to the room temperature.

o Enough strips in strip holder were placed so that all tests can be run

adequately.

o For photometer blank, 100 μl of substrate along with 100 μl stop

solution were pipetted into 1st well.

57

o Then we prepared the washing solution by mixing the contents of bottle

with 450 ml of distilled water.

o Fifty μl of calibrators and samples were pipetted into appropriate wells

of the strips.

o Then, 200 µl of HRP testosterone conjugate was added to each well in

sequence. Then incubated for 120 minutes at 37°C without covering the

plate.

o After incubation, the incubation solution was discarded, wells were

rinsed with washing solution three times with automatic washer and

residual fluid was removed.

o Then immediately 100 µl of chromogen/ substrate mixture was pipetted

into the rinsed wells.

o It was then incubated for 15 minutes at room temperature.

o The reaction was stopped by pipetting 100 µl of stop solution into the

wells in the same sequence adopted to dispense the chromogen/substrate

mixture.

o Microplate was then shaken gently to avoid splashing.

o It was then read at 450 nm within one hour. (Ballester et al., 2005)

2.3.1.4 CALCULATION OF RESULTS

For all standards as well as samples, B/ B0 % was calculated.

B/ B0 % = Absorb of Sample - Absorb of Blank /Absorb of C0 – Absorb of Blank Then the standard curve was plotted on graph paper with calibrators on y axis and

standard values of Testosterone on x axis, to find out the Testosterone

concentrations, as shown in figure 2.1. (Ballester et al., 2005)

58

Table 2.3: Absorbance and B/ B0 % of standards of Testosterone.

Name

Absorbance

B/ B0 %

Blank

0.053

-

C0

2.098

100%

C1

2.045

97%

C2

1.693

80%

C3

1.423

67%

C4

1.078

50%

C5

0.861

39%

59

testosterone Invivo

1.562, 76.11.398, 68.1

1.056, 51.5

0.74, 36.1

2.052, 1001.994, 97.2

0

20

40

60

80

100

120

0 0.5 1 1.5 2 2.5

Testosterone Abs.

B /

Bo %

testosterone Invivo

testosterone invivo

y = -0.0769x + 1.8414R2 = 0.8666

0

0.5

1

1.5

2

2.5

0 5 10 15 20

Calibrators Conc.

Abso

rban

ce

testosterone Invivo

Linear (testosterone Invivo)

Figure 2.1: Testosterone standard calibration curves for in vivo analysis (ELISA)

Testosterone In vivo

Testosterone In vivo

60

2.3.2 TESTOSTERONE (For In vitro analysis)

Testosterone EIA kit - Cayman chemicals

Catalogue No: 582701.

Lot No: 181722

2.3.2.1 Principle of the assay

Competition between testosterone and a testosterone acetyl cholinesterase

conjugate (testosterone tracer) for a restricted amount of testosterone anti serum is

the basis of this test. As the concentration of testosterone is fixed, so the amount of

testosterone tracer that has ability to bind with the testosterone serum will be

inversely proportional to the concentration of that testosterone which is present in

the well. This antiserum-testosterone complex is in bound state to the anti rabbit

IgG obtained from mouse that has been previously attached to the well. After that,

the plate is washed any unbound reagents are removed after which the Ellman’s

Reagent (which contains substrate to acetyl cholinesterase) is added to the well.

The product which is obtained from this enzymatic reaction has a distinctive

yellow color and absorbs strongly at 412 nm. The amount of testosterone tracer

bound to well is narrated by intensity of this color on spectrophotometer. This

amount of testosterone tracer is inversely related to the amount of free testosterone

that is present in the well during the incubation.

2.3.2.2 Buffer preparation

1 EIA Buffer preparation

We diluted the contents of one vial of EIA Buffer concentrate (10x)

(Cat No: 400060) with 90 ml of ultra pure water. Caution was taken

to rinse the bottle to remove any precipitated salts.

61

2 Wash Buffer preparation

We diluted 5 ml wash buffer concentrate (400 X) (cat No: 400062)

to total volume of 2 liters ultra pure water and then added 1 ml

Tween 20 (Cat No: 400035)

2.3.2.3 Preparation of Assay specific reagents

Testosterone standard

We equilibrated the pipette tip for which we used ethanol by repetitively

filling and expelling the tip with ethanol. Then we transferred 100 ul of

testosterone EIA standard (Cat No 482704) in a test tube that was clean and then

we diluted with 900 ul ultra pure water to achieve a final conc of 5 ng/ml.

To prepare the standard, we took eight test tubes and labeled them from 1 to 8.

Then we aliquoted 900 ul of EIA buffer to tube no 1, and 500 ul of EIA buffer into

tubes no 2 to 8. Then 100 ul of the bulk standard was transferred to tube no 1 and

mixed thoroughly. The serial dilution was done by pipetting 500 ul from tube 1 and

transferring it to tube no 2. After thorough mixing, 500 ul removed from tube no 2

and placed in tube no 3. This process was repeated for tubes no 4 to 8.

Testosterone AChE Tracer

100 dtn Testosterone AChE Tracer (Cat No 482700) was reconstituted with 6 ml

EIA Buffer.

Testosterone EIA Antiserum

100 dtn Testosterone EIA Antiserum (Cat No 482702) was reconstituted with 6 ml

EIA Buffer.

2.3.2.4 ASSAY PROCEDURE

Reagents addition: Following reagents were added:

62

1. EIA Buffer

We added 100 ul of EIA Buffer to those wells labeled as the Non

Specific Binding (NSB) wells. Then, 50 ul of EIA Buffer was added to

Maximum binding (B0) well. In case of assay of cell culture wells,

culture medium was substituted for EIA buffer both in the NSB and B0

wells.

2. Testosterone EIA Standard

From tube no 8 we added 50 micro liters to both of lowest standard

wells. Then added fifty micro liters from tube no 7 to succeeding

standard wells till all standards were aliquoted. We used the same

pipette tip for all standards and pipette tip was equilibrated before

pipetting.

3 Samples:

Then 50 ul of sample added to each well.

4 Testosterone Ache Tracer

Then we added 50 ul of tracer to each well. However total activity and

blank wells were spared from this step.

5 Testosterone EIA Antiserum

Then we added 50 ul of serum to each well. Here we spared the total

activity well, NSB and Blank well.

Incubation of plate

Plate was then incubated for 2 hours at room temperature by covering it with

plastic film

63

Development of plate

1. Ellman’s reagent was reconstituted immediately before use. To develop

100 wells, 100 dtn vial of Ellman’s reagent was reconstituted with 20

ml ultra pure water.

2. Then wells were emptied with rinsed five times with wash buffer.

3. After this, 200 ul of Ellman’s reagent was added to each well.

4. Then 5 ul of tracer was added to total activity wells.

5. Then plate was covered with plastic film. Plates were placed in dark for

90 then minutes.

Reading the plate

1 Bottom of plate was cleaned to remove dirt and finger prints.

2 Plate cover was removed carefully to prevent splashing of Ellman’s

reagent.

3 Plate was read at 405 to 420 nm.

Performance characteristics:

The assay range to determine the testosterone concentration with this kit was 3.9 to

500 pg/ml. The intra assay coefficient of variance was 9%. The inter assay

coefficient of variance was 8%.

2.3.2.5 Calculations for testosterone estimation

1. Absorbance readings of blank wells were abstracted from all wells

2. Then the absorbance readings from NSB wells were averaged

3. After this, absorbance from B0 wells were averaged

4. NSB average was subtracted from B0 average. This is labeled as the

corrected B0 or the corrected maximum binding.

64

5. Then % B/ B0 was calculated for remaining wells. To do this, average NSB

absorbance was subtracted from S1 absorbance and then divided by B0.

6. Then it was multiplied by 100 which will give us the value of % B/ B0.

Repeat for S2-S8 and all sample wells.

Plotting the Standard curve.

Standard curve was plotted for standards S1-S8 versus testosterone concentration

using linear (y) along with the log (x) axis.

65

Testosterone Invitro

0.039, 12.30

0.091, 28.70

0.124, 39.11

0.206, 64.98

0.251, 79.17 0.301, 94.95

0.323, 101.890.358, 112.93

0

20

40

60

80

100

120

0 0.1 0.2 0.3 0.4

Testosterone Abs.

B /

Bo %

Testosterone Invitro

Figure 2.2 Testosterone standard calibration curves (for in vitro analysis)

Testosterone Invitro

y = -0.0006x + 0.2852R2 = 0.7662

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 200 400 600

Calibrators Conc.

Abso

rban

ce

Testosterone Invitro

Linear (Testosterone Invitro)

Testosterone In vitro

66

2.3.3 Corticosterone EIA Kit

Catalogue No: 500651

Lot number: 0404965

2.3.3.1 Principle

This assay is based on competition between corticosterone and a

corticosterone acetyl cholinesterase (AChE) conjugate (Corticosterone tracer) for a

limited number of corticosterone specific rabbit anti serum binding sites. Because

the concentration of the corticosterone tracer is held constant while the

concentration of corticosterone varies, the amount of corticosterone tracer which is

able to bind to rabbit anti serum is considered to be inversely proportional to the

concentration of corticosterone in the well. This rabbit anti serum corticosterone

(either free or tracer) complex binds to the mouse monoclonal anti rabbit IgG that

was previously attached to the well. The plate is washed to remove any unbound

agents and then Ellman’s Reagent (which contains the substrate to AChE) is added

to the well. The product of this enzymic reaction has discrete yellow color and

absorbs strongly at 412 nm. The intensity of this color determined

spectrophotometrically is in proportion to amount of corticosterone tracer bound to

the well which is inversely proportional to the amount of free corticosterone

present in well during incubation.

2.3.3.2 Buffer preparation

EIA Buffer preparation

We diluted the contents of one vial of EIA Buffer concentrate with 90 ml of ultra

pure water. Caution was taken to rinse the bottle to remove any precipitated salts.

67

Wash Buffer preparation

We diluted 5 ml wash buffer concentrate to total volume of 2 liters ultra pure water

and then added 1 ml Tween 20.

2.3.3.3 Preparation of Assay specific reagents

Corticosterone standard

We equilibrated the pipette tip using ethanol by repeatedly filling and expelling the

tip with ethanol. Then we transferred 100 ul of corticosterone EIA standard into a

clean test tube and diluted with 900 ul ultra pure water to gain conc of 100 ng/ml.

To prepare the standard, we took eight test tubes and labeled them from 1 to 8. then

we aliquoted 900 ul of EIA buffer to tube no 1, and 750 ul of EIA buffer into tubes

no 2 to 8. Then 100 ul of the bulk standard was transferred to tube no 1 and mixed

thoroughly. The serial dilution was done by removing 500 ul from tube 1 and

placing in tube no 2. After thorough mixing, 500 ul removed from tube no 2 and

placed in tube no 3. This process was repeated for tubes no 4 to 8.

Corticosterone AChE Tracer

100 dtn corticosterone AChE Tracer (vial no 2) was reconstituted with 6 ml EIA

Buffer.

Corticosterone EIA Antiserum

100 dtn corticosterone EIA Antiserum (vial no 1) was reconstituted with 6 ml EIA

Buffer.

2.3.3.4 ASSAY PROCEDURE:

Addition of reagents:

The addition of reagents is explained below:

68

EIA Buffer

We added 100 ul of EIA Buffer to Non Specific Binding (NSB) wells. Then, 50 ul

of EIA Buffer was added to Maximum binding (B0) wells. In case of assay of cell

culture wells, culture medium was substituted for EIA buffer in NSB and B0 wells.

Corticosterone EIA Standard

We added 50 ul from tube no 8 to both of lowest standard wells. Then added 50 ul

from tube no 7 to next standard wells until all standards aliquoted. Same pipette tip

was used for all standards and pipette tip was equilibrated before pipetting.

Samples:

Then 50 ul of sample was added to each well.

Corticosterone AchE Tracer

Then we added 50 ul of tracer to each well except total activity and blank wells

Corticosterone EIA Antiserum

Then we added 50 ul of serum to each well, except the total activity well, NSB and

Blank well.

Incubation of plate

Plate was then incubated for 2 hours at room temperature by covering it with

plastic film

Development of plate

• Ellman’s reagent was reconstituted immediately before use. To develop 100

wells, 100 dtn vial Ellman’s reagent was reconstituted with 20 ml of ultra

pure water.

• Then wells were emptied and then rinsed five times with wash buffer.

• After this, 200 ul of Ellman’s reagent was added to each well.

69

• Then 5 ul of tracer was added to total activity wells.

Then plate was covered with plastic film. Plates were placed in dark for 90

minutes.

Reading the plate

Bottom of plate was cleaned to remove dirt and finger prints.

Plate cover was removed carefully to prevent splashing of Ellman’s reagent.

Plate was read between 405 and 420 nm.

Precision:

The intra assay coefficient of variance and inter assay coefficient of variance for

the concentration of 10,000 pg/ml was 18.6% and 17.1 % respectively. The intra

assay coefficient of variance and inter assay coefficient of variance for the

concentration of 1600 pg/ml was 18.1% and 7.3 % respectively. The intra assay

coefficient of variance and inter assay coefficient of variance for the concentration

of 41 pg/ml was 16% and 12 % respectively.

Assay range:

Assay range for this kit to determine the corticosterone is 16.4 pg/ml to 10000

pg/ml

2.3.3.5 Calculations for corticosterone estimation

Absorbance readings of blank wells were abstracted from all wells

Then the absorbance readings from NSB wells were averaged

After this, absorbance from B0 wells were averaged

NSB average was subtracted from B0 average. This is the corrected B0 or corrected

maximum binding.

70

Then % B/ B0 was calculated for remaining wells. To do this, average NSB

absorbance was subtracted from S1 absorbance and then divided by B0.

Then it was multiplied by 100 to obtain % B/ B0. Repeat for S2-S8 and all sample

wells.

Plotting the Standard curve.

Standard curve was plotted % B/ B0 for standards S1-S8 versus corticosterone

concentration (usually in pg/ml) using linear (y) and log (x) axis.

71

Corticosterone

0.071, 22.4

0.108, 34.1

0.163, 51.4

0.216, 68.10.24, 75.7

0.272, 85.8

0.341, 107.6

0.111, 35.0

0.0

20.0

40.0

60.0

80.0

100.0

120.0

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Corticosterone Abs.

B /

Bo %

Corticosterone

Corticosterone

y = -2E-05x + 0.2309R2 = 0.5317

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 2000 4000 6000 8000 10000 12000

Calibrators Conc.

Abso

rban

ce

Corticosterone

Linear (Corticosterone)

Figure 2.3 Corticosterone standard calibration curves.

72

2.3.4 Nor adrenaline ELISA

Catalogue number: BA-E 5200 Labor Diagnostica Nord (LDN)

2.3.4.1 Principle

Enzyme immunoassay used for quantitative determination of nor adrenaline. Nor

adrenaline is detected by using a cis-diol specific affinity gel, acylated and then

derivatized enzymatically.

The competitive ELISA kit uses the microtiter plate format. The antigen is bound

to solid phase of the micro titer plate. The derivatized standards, controls and

samples and solid phase bound analyte compete for a fixed number of anti serum

binding sites. After the system is in equilibrium, free antigen and free antigen –

antiserum complexes are removed by washing. The antibody bound to solid phase

is detected by an anti rabbit IgG peroxidase conjugate using TMB as a substrate.

The reaction is monitored at 450 nm.

Quantification of unknown samples is achieved by comparing their absorbance

with a reference curve prepared with known standard concentrations.

Kit contents

BA- D- 0032 Microtitre plate

BA- E – 0090 Adhesive foil

BA- E – 0030 Wash buffer concentrate

BA- E - 0040 Enzyme conjugate

BA- E - 0055 Substrate

BA- E - 0080 Stop solution

BA- E - 0231 Nor adrenaline micro titer strips

BA- E - 5210 Nor adrenaline antiserum

73

BA- E - 5601 Standard A

BA- E - 5602 Standard B

BA- E - 5603 Standard C

BA- E - 5604 Standard D

BA- E - 5605 Standard E

BA- E - 5606 Standard F

BA- E - 5619 Hydrochloric acid

BA- E - 5651 Control 1

BA- E - 5652 Control 2

BA- R- 0012 Acylation concentrate

BA- R - 0050 Adjustment buffer

BA- R - 0075 Acylation diluent

BA- R - 6611 Acylation buffer

BA- R - 6613 Assay buffer

BA- R - 6614 Coenzyme

BA- R - 6615 Enzyme

BA- R - 6617 Extraction buffer

BA- R - 6618 Extraction plate

2.3.4.2 Preparation of reagents

Wash Buffer

We diluted 20 ml of wash buffer concentrate with distilled water to make a final

volume of 1000 ml.

Acylation solution:

We diluted 20 ul of Acylation concentrate in 1.2 ml of acylation diluent.

74

2.3.4.3 PROCEDURE

Step 1: Extraction and acylation:

Respective wells of extraction plate were filled with sample

volume of 100 ul, 10 ul standard and 10 ul control. Distilled

water was added to each well if final volume was less than 100

ul.

Assay buffer 50 ul was pipetted into all wells

Extraction buffer 50 ul was then pipetted into all wells

Plate was the covered with adhesive foil. Then it was shaked at

600 rpm (25 °C) for 60 minutes.

Foil was removed, plate emptied and dried by tapping over

adsorbent material.

Wash buffer was then pipetted 1 ml in all wells. Plate was

covered with adhesive foil.

Then it was shaked at 600 rpm (25 °C) for 5 minutes.

Foil was removed, plate emptied and dried by tapping over

adsorbent material

Washing repeated as in steps 6, 7, 8.

Acylation buffer 150 ul was pipetted into all wells

Acylation solution 25 ul was then pipetted into all wells

Then it was shaked at 600 rpm (25 °C) for 20 minutes.

Foil was removed, plate emptied and dried by tapping over

adsorbent material

75

Wash buffer was then pipetted 1 ml in all wells. Plate was

covered with adhesive foil

Then it was shaked at 600 rpm (25 °C) for 5 minutes.

Foil was removed, plate emptied and dried by tapping over

adsorbent material

Washing repeated as in steps 14, 15, 16.

Then 100 ul Hydrochloric acid was pipetted into all wells.

Plate was the covered with adhesive foil. Then it was shaked at

600 rpm (25 °C) for 10 minutes.

Step 2: Enzymatic Conversion

Micro titer plate was used into which 90 ul of extracted standards,

controls and samples were pipetted.

Then enzyme solution was prepared by reconstituting the contents

of vial labeled enzyme with 1 ml distilled water which were mixed

thoroughly. Then we added 0.3 ml of coenzyme after which we

added 0.7 ml of adjustment buffer. Thus a total volume of 2.0 ml

was obtained.

Then, 25 ul of enzymatic solution was added to all wells.

Plate was the covered with adhesive foil. Then it was shaked at 600

rpm (25 °C) for 1 minute.

Then it was incubated for 2 hours at 37°C.

Step 3: Noradrenaline ELISA

Enzyme plate was then used to pipette 100 ul of standards, controls

and samples, into precoated Microtitre strips.

76

Then, 50 ul of nor adrenaline antiserum was pipetted into all wells.

Plate was then covered with adhesive foil. Then it was incubated for

1 min on Room temperature on a shaker.

After this, 15 hours (over night) incubation was done at 2-8 °C.

Foil was then removed, contents aspirated and each well was

washed four times with 300 ul of wash buffer. The plate was dried

by tapping it over dry tissue paper.

Then 100 ul of enzyme conjugate was pipetted into all wells.

Plate was then covered with adhesive foil and incubated for 30 min

at room temperature on a shaker with rpm of 600.

Foil was then removed, contents aspirated and each well was

washed four times with 300 ul of wash buffer. The plate was dried

by tapping it over dry tissue paper

Then 100 ul of substrate was pipetted into all wells.

Then plate was incubated for 30 min at room temperature on a

shaker with rpm of 600.

In the end, 100 ul of stop solution was pipetted into all wells

Then the absorbance was measured by ELISA reader at wavelength

of 450 nm.

Assay characteristics:

Analytic specificity for nor epinephrine was 100%, and for epinephrine

was 0.14%. the analytical sensitivity was 0.2 ng/ml x correction factor

(0.013). Intra assay coefficient of variance was 11.7% at 1.3 ng/ml and

8.4% for a concentration of 0.5 ng/ml .

77

2.3.4.3 Results calculation

Calibration curve was obtained by plotting the absorbance readings,

measured for the standards (linear y axis) against standard concentrations

(x axis). The standards which were referred were as follows:

Standard A 0 ng/ml

Standard B 0.45 ng/ml

Standard C 1.5 ng/ml

Standard D 4.5 ng/ml

Standard E 15 ng/ml

Standard F 45 ng/ml

Concentration of samples obtained form standard curve was multiplied

with correction factor.

Correction factor = 10 ul (volume of standards extracted) / sample volume

extracted.

78

NE (Calibration Curve)

y = -0.0153x + 0.9R2 = 0.5332

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 10 20 30 40 50

NE Conc. (ng/ml)

Absorbance

NE Conc. (ng/ml)

Linear (NE Conc.

Figure 2.4 Nor epinephrine standard curve (ELISA)

79

2.3.5 LH ESTIMATION

Kit: LH EIA.

Catalogue number: 11-LUTHU-E01

Manufacturer: ALPCO Diagnostics

2.3.5.1 Principle of the method

It is a two step capture or sandwich type assay. This assay makes use of two highly

specific monoclonal antibodies. A monoclonal antibody specific for LH is

immobilized on micro well plate and another monoclonal antibody specific for a

different region of LH is conjugated to the horse radish peroxidase (HRP). LH

from sample and standards are permitted to bind to plate, washed afterwards and

incubated with HRP conjugate. After a second washing step, the enzyme substrate

is added. The enzymatic reaction is terminated by addition of stopping solution.

The absorbance is measured on Microtitre plate reader. The intensity of color

formed is in direct proportion to the concentration of LH in the sample. A set of

standards is used to plot a standard curve from which the amount of LH in sample

and control is measured.

2.3.5.2 REAGENTS

Mouse anti LH antibody coated micro well plate – Break Apart Wells

96 wells

Mouse anti LH antibody - Horse radish peroxidase (HRP) conjugate concentrate

240 ul of HRP in 12 ml assay buffer

LH CALIBRATORS

Calibrator A 0 IU/L 2.0 ml

Calibrator B 1 IU/L 0.5 ml

80

Calibrator C 4 IU/L 0.5 ml

Calibrator D 10 IU/L 0.5 ml

Calibrator E 40 IU/L 0.5 ml

Calibrator F 100 IU/L 0.5 ml

Control

Ready to use

Wash buffer concentrate

50 ml of wash buffer was diluted with 450 ml of distilled water

Assay buffer

Ready to use

TMB substrate

Ready to use, bottle contains tetra methyl Benzidine and hydrogen

peroxide.

Stopping solution

One vial containing 1 M sulfuric acid, ready to use

Performance characteristics:

Sensitivity of the kit for LH was 0.2 IU/L. The intra assay coefficient of variance

for the concentration of 4.8 IU/L was 4.5 % and for a concentration of 53.28 IU/L

was 2.9%. The inter assay coefficient of variance for the concentration of 5.15 IU/L

was 5.1 % and for a concentration of 51.50 IU/L was 9.2%.

2.3.5.3 ASSAY PROCEDURE

After bringing all samples and reagents to room temperature, working

solutions for cortisol-HRP conjugate and wash buffer were prepared.

Micro well strips were taken out of seal

81

25 ul of calibrator, control and samples were pipetted into respective wells

Then 100 ul of assay buffer was pipetted into all wells

Incubation was done for 30 minutes on plate shaker at 200 rpm.

Wells were washed three times with 300 ul diluted wash buffer per well and

plate was tapped firmly against absorbent paper to make it dry

Then, 100 ul of conjugate working solution was put into all wells.

Incubation was done for 30 minutes on plate shaker at 200 rpm at room

temperature

Wells were washed three times with 300 ul diluted wash buffer per well and

plate was tapped firmly against absorbent paper to make it dry

The 100 ul of TMB substrate was pipetted into all wells

Then it was incubated on plate shaker for 15 to 20 minutes at room

temperature.

Stop solution 50 ul was then pipetted in to all wells

Then absorbance was read on micro plate reader at 450 nm

2.3.5.4 CALCULATIONS

Mean optical density for each calibrator was calculated in duplicate

Then mean optical density of each unknown was calculated

Then we subtracted the mean absorbance value of ‘0’calibrator from the mean

absorbance values of calibrators, control and serum samples.

Calibration curve was drawn on semi Log paper with mean optical densities on y

axis and calibrator concentrations on x axis.

Values were read directly from calibration curve

82

LH (Calibration Curve)

y = 0.0186x + 0.1898R2 = 0.9703

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60 70 80 90 100 110

LH Conc.

Absorbance

LH Conc.

Linear (LH Conc.)

Figure 2.5 Standard calibration curve for LH.

IU/L

83

2.3.6 MALONDIALDEHYDE ESTIMATION

TBARS Essay kit- Cayman chemical company

Catalogue No: 10009055

Lot No: 181722

2.3.6.1 Principle of the assay

Thio Barbituric Acid Reactive Substances-(TBARS) assay provides a simple and

standard method for assaying lipid peroxidation in various body fluids including

plasma, serum, urine, tissue homogenates, and cell lysates. The chemical reaction is

MDA + TBA MDA-TBA Adduct

The MDA-TBA adduct formed by above reaction under high temperature (90-100°

C) and acidic conditions is measured colorimetrically at 530- 540 nm.(Huang et al.,

2002)

2.3.6.2 Preparation of Reagents

Thio BarbituricAcid (TBA.)

Each vial contains 2 g of TBA. It is ready to use to prepare the color

reagent. One vial is sufficient to evaluate 24 wells.

TBA –Acetic acid-

The vial contains concentrated acetic acid. To prepare the solution, 40 ml of

TBA Acetic acid was added slowly to 160 ml of HPLC grade water. This solution

was used to prepare the color reagent.

TBA-Sodium Hydroxide (10 X)

20 ml of TBA Sodium Hydroxide was diluted with 180 ml of HPLC grade

water. This solution will be used to prepare the color reagent.

84

TBA- Malondialdehyde standard.

This vial contains 500 μM MDA in water. It was ready to use

TBA SDS Solution.

This vial contains sodium dodecyl sulfate (SDS). It is ready to use

preparation.

Preparation of color reagent

We weighed 530 mg of TBA and it was added to 50 ml of diluted TBA

Acetic acid solution in 500 ml beaker. Then 50 ml of diluted TBA Sodium

Hydroxide solution was added and mixed until TBA completely dissolved. Then

other three vial were also processed in a similar way.

2.3.6.3 PERFORMING THE ASSAY

In order to prepare MDA Standards, 250 μl of MDA standard

was diluted with 750 μl of water to obtain a stock solution of

125 μM. Glass tubes, 8 in number were taken and labeled from

A to H. Water and 125 μM MDA was added to tubes as

described in table 3.3.

Vial caps were labeled with identification numbers.

Then, 100 μl of sample or standard was added to labeled vials.

After this, 100 μl of SDS solution was added to vials and mixed

on maxi mixer.

In each vial, 4 ml of color reagent was added

Vials were caped and placed in holder to keep them upright.

Vials were then placed in vigorously boiling water and allowed

to boil for 1 hour.

85

After one hour, vials were removed and placed in ice bath for 10

minutes to stop the reaction.

After 10 minutes vials were centrifuged for 10 minutes at 1600 x

g at 4°C.

Vials were brought to room temperature and 150 μl from each

vial was taken to plate

Absorbance was read at 545 nm using a plate reader ( Huang et

al., 2002).

Precision:

The intra assay coefficient of variation was 5.5% and interassay coefficient of

variation was 5.9%

Assay range:

Assay range of this kit for determining MDA is 0 - 50 uM. (uM = umole/L =

nmol/ml)

2.3.6.3 CALCULATION OF RESULTS

Average absorbance of each standard and sample was calculated.

Standard A absorbance was subtracted from all to get the value of corrected

absorbance as shown in table 2.5

3. Standard curve was plotted by plotting corrected absorbance value and

MDA concentration as shown in figure 2.6.

4. From standard curve, results were drawn.

86

Table 2.4: Concentrations for the preparation of Malondialdehyde standards.

Test tube

MDA (μl)

Water (μl)

MDA concentration (μM)

A 0 1000 0

B 5 995 0.625

C 10 990 1.25

D 20 980 2.5

E 40 960 5

F 80 920 10

G 200 800 25

H 400 600 50

87

Table 2.5: Absorbance and concentrations of Malondialdehyde standards.

Name of

standard

Absorbance

MDA concentration

(μM)

A

0.001

0

B

0.009

0.625

C

0.011

1.25

D

0.014

2.5

E

0.020

5

F

0.028

10

G

0.057

25

H

0.103

50

88

MDA (Calibration Curve)

y = 0.0021x + 0.0028R2 = 0.9791

0

0.02

0.04

0.06

0.08

0.1

0.12

0 10 20 30 40 50 60

MDA Conc. (uM)

Absorbance

MDA Conc. (uM)

Linear (MDA Conc. (uM))

Figure 2.6 Malondialdehyde EIA standard calibration curve.

89

2.3.7 Super oxide dismutase:

Superoxide dismutase assay kit- Cayman chemical company

Catalogue No: 706002

2.3.7.1 Principle:

This kit utilizes a tetrazolium salt for detection of superoxide radicals generated by

Xanthine oxidase and hypo xanthine. One unit of SOD is defined as the amount of

enzyme needed to exhibit 50% dismutation of the superoxide radical. The SOD

assay measures all three types of SOD, ( Cu/Zn, Mn, and Fe-SOD). This assay

provides a simple, reproducible and fast tool for assaying SOD activity in serum,

plasma etc.

2.3.7.2 PRE ASSAY PREPARATION

Preparation of reagents

1 Assay buffer

We diluted 2 vials of assay buffer concentrate in 54 ml of HPLC

grade water. This preparation was used to dilute radical detector.

2 Sample buffer

Two vials of sample buffer were diluted in 36 ml of HPLC grade

water. It was used to prepare

3 Radical detector

The vial contains tetrazolium salt. Transfer 100 ul of solution in a

vial and add 39.90 ml of diluted assay buffer. Then it was covered with tin foil.

90

SOD Standard:

The vial contains a solution of bovine erythrocyte SOD. It is ready to use

preparation.

6 Xanthine Oxidase

These vials contain a solution of Xanthine Oxidase. We diluted 100

ul of enzyme with 3.90 ml of sample buffer. It was stored on ice.

2.3.7.3 Assay procedure

We diluted 20 ul of SOD standard with 1.8 ml of sample buffer to obtain SOD

stock solution. Then seven glass tubes were taken and marked from A to G. Then

we added SOD and sample buffer to each tube as described in table SOD

Tube SOD Stock ul Sample buffer ul Final SOD activity

U/ml

A 0 1000 0

B 20 980 0.025

C 40 960 0.05

D 80 920 0.1

E 120 880 0.15

F 160 840 0.2

G 200 800 0.25

SOD Standard wells. We added 200 ul of diluted radical detector and 10 ul of

standard tubes per well in designated wells on plate.

91

Sample wells: we added 200 ul of diluted radical detector and 10 ul of samples to

the wells

The reaction was initiated by adding 20 ul of diluted xanthine oxidase to all wells,

and this step was performed quickly.

Plate was shaken carefully for few seconds to mix it well

Plate was then incubated at room temperature for 20 minutes and absorbance was

read at 450 nm using a plate reader.

Assay range:

Dynamic range of this kit is .025 -0.25 units/ml super oxide dismutase.

Precision:

The intra assay coefficient of variance was 3.2 % and the inter assay coefficient of

variance was 3.7%.

2.3.7.4 CALCULATION OF RESULTS

We calculated the average absorbance of each standard and sample.

Then we divided the standard A’s absorbance by itself and standard A’s absorbance

by all the other standards and samples absorbances to yield the linearized rate.

Then we plotted the linearized SOD standard rate as a function of final SOD

activity (U/ml).

Then we calculated the SOD activity of samples using the equation obtained from

linear regression of standard curve substituting the linearized rate for each sample.

STATISTICAL ANALYSIS

Data were analyzed on SPSS version 13. The arithmetic mean and standard error of

mean of all samples was calculated. One way ANOVA was applied to observe the

level of significance among groups followed by Post Hoc Tukey’s test for multiple

92

comparisons. Difference in mean among the control and treated groups was

calculated by ‘Independent sample t test’ for 2 group comparisons. The difference

was considered significant if p value was found less than 0.05. (Sengupta et al.,

2004)

93

SOD Linerized Rate (u/ml)

y = 11.689x + 1.0116R2 = 0.9915

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0 0.05 0.1 0.15 0.2 0.25 0.3

SOD Linerized Rate (u/ml)

Absorbance

SOD Linerized Rate

Linear (SOD Linerized

Figure 2.7 Standard Calibration curve for Super oxide dismutase

SOD Linearized rate (u/ml)

94

RESULTS In vivo results

The in vivo component of this project was done on eighty male Sprague

Dawley rats by dividing them into five groups with 16 rats in each group. All rats

remained alive and healthy for whole period of study and took their feed properly.

Acute stress for six hours in mesh wire restrainer resulted in a significant

decrease in levels of serum testosterone (p value < 0.01), while serum

corticosterone (p value < 0.01) levels significantly increased. The levels of plasma

nor- epinephrine were also increased in significant amount (p value < 0.001) after

exposure to restraint stress.

There was statistically insignificant (p value > 0.05) change in the levels of serum

LH. The lipid peroxidation increased after stress exposure quite significantly (p

value < 0.01) which is expressed in our study in terms of serum malondialdehyde

levels. Stress exposure also resulted in a significant fall in superoxide dismutase

activity (p value < 0.01) as shown in table 3.1.

Effects of one month supplementation with ascorbic acid on stress induced

changes:

The results shown in table 3.2 reveal that one month supplementation with

ascorbic acid was unable to prevent the acute restraint stress induced decline in

serum testosterone levels (p value > 0.05). Serum corticosterone (p value > 0.05)

and plasma nor epinephrine (p value > 0.05) showed insignificant change as

compared to non-supplemented group. The levels of serum LH were also

unchanged (p value > 0.05) after ascorbic acid supplementation.

Chapter 3

95

The lipid peroxidation which is expressed in our study in terms of serum

malondialdehyde levels remained high after stress exposure quite significantly (p

value > 0.05) despite ascorbic acid supplementation. However ascorbic acid

supplementation resulted in a significant rise in superoxide dismutase activity

despite the exposure to stress (p value < 0.05).

Effects of one month supplementation of alpha tocopherol on stress induced

changes

The results shown in table 3.3 reveal that one month supplementation with

alpha tocopherol significantly prevented the acute restraint stress induced decline in

serum testosterone levels (p value < 0.01). The serum corticosterone (p value <

0.01) decreased in the group of rats supplemented with alpha tocopherol. The

plasma nor epinephrine (p value > 0.05) showed insignificant change as compared

to non-supplemented group. The levels of serum LH were also unchanged (p value

> 0.05) after alpha tocopherol supplementation.

The lipid peroxidation in terms of serum malondialdehyde levels, decreased

significantly (p value < 0.05) in alpha tocopherol supplemented group after stress

exposure Also in alpha tocopherol supplemented group, there was a significant rise

in superoxide dismutase activity despite the exposure to stress (p value < 0.05).

96

Table 3.1: Effects of 06 hours restraint stress on serum Testosterone, serum Corticosterone, plasma Nor epinephrine serum LH, serum Malondialdehyde and serum Super oxide dismutase levels in male Sprague Dawley rats.

Parameter

Group I Control (n=16)

Mean ± SEM

Group II Acute restraint

stress (n=16)

Mean ± SEM

p value*

Serum Testosterone (ng/ml)

6.09 ± 0.15

5.18 ± 0.19

< 0.01

Serum Corticosterone (pg/ml)

8870.00 ± 175.35

9688.75 ± 219.18

< 0.01

Plasma Nor epinephrine (ng/ml)

2.59 ± 0.18

5.06 ± 0.31

< 0.001

Serum LH (IU/L)

9.10 ± 0.08

9.17 ± 0.09

> 0.05

Serum Malondialdehyde (nmol/ml)

9.71 ± 0.59

14.77 ± 1.18

< 0.01

Serum Super oxide dismutase (U/ml)

0.14 ± 0.01

0.08 ± 0.01

< 0.01

*p value less than 0.05 is taken as significant.

97

Table 3.2 : Effects of one month, ascorbic acid supplementation (AAS) on changes in serum Testosterone, serum Corticosterone, plasma Nor epinephrine, serum Luteinizing hormone, serum Malondialdehyde and Superoxide Dismutase levels in male Sprague Dawley rats exposed to six hours restraint stress

Parameter

Group II Acute restraint

stress (n=16)

Mean ± SEM

Group III AAS and

restraint stress (n=16)

Mean ± SEM

p value*

Serum Testosterone (ng/ml)

5.18 ± 0.19

5.29 ± 0.20

> 0.05

Serum Corticosterone (pg/ml)

9688.75 ± 219.18

9229.38 ± 240.95

> 0.05

Plasma Nor epinephrine (ng/ml)

5.06 ± 0.31

5.45 ± 0.21

> 0.05

Serum LH (IU/L)

9.17 ± 0.09

9.26 ± 0.11

> 0.05

Serum Malondialdehyde (nmol/ml)

14.77 ± 1.18

13.88 ± 0.96

> 0.05

Serum Super oxide dismutase (U/ml)

0.08 ± 0.01

0.12 ± 0.01

< 0.05

*p value less than 0.05 is taken as significant.

98

Effects of one month supplementation of ascorbic acid and alpha tocopherol

on stress induced changes:

The results shown in table 3.4 reveal that one month supplementation with

combination of ascorbic acid and alpha tocopherol significantly prevented the acute

restraint stress induced decline in serum testosterone levels (p value < 0.01). the

serum corticosterone (p value < 0.01) were decreased in the group of rats

supplemented with combination of antioxidants. The plasma nor epinephrine (p

value > 0.05) showed the insignificant change even in this group also. The levels of

serum LH were also unchanged (p value > 0.05) after combined antioxidants

supplementation.

The lipid peroxidation in terms of serum malondialdehyde levels decreased

significantly (p value < 0.01) in ascorbic acid and alpha tocopherol supplemented

group after stress exposure There was also a significant rise in superoxide

dismutase activity of this combination supplementation group rats despite the

exposure to restraint stress for six hours (p value < 0.01).

99

Table 3.3: Effects of one month alpha tocopherol supplementation (ATS) on changes in serum Testosterone, serum Corticosterone, plasma Nor epinephrine, serum Luteinizing hormone, serum Malondialdehyde and Superoxide Dismutase levels in male Sprague Dawley rats exposed to six hours restraint stress

Parameter

Group II Acute restraint

stress (n=16)

Mean ± SEM

Group IV ATS and restraint

stress (n=16)

Mean ± SEM

p value*

Serum Testosterone (ng/ml)

5.18 ± 0.19

6.80 ± 0.23

< 0.01

Serum Corticosterone (pg/ml)

9688.75 ± 219.18

8710.63 ± 149.15

< 0.01

Plasma Nor epinephrine (ng/ml)

5.06 ± 0.31

5.28 ± .17

> 0.05

Serum LH (IU/L)

9.17 ± 0.09

9.18 ± 0.08

> 0.05

Serum Malondialdehyde (nmol/ml)

14.77 ± 1.18

10.84 ± 1.12

< 0.05

Serum Super oxide dismutase (U/ml)

0.08 ± 0.01

0.13 ± 0.07

< 0.05

*p value less than 0.05 is taken as significant.

100

Table 3.4: Effect of combined ascorbic acid and alpha tocopherol supplementation on changes in serum Testosterone, serum Corticosterone, plasma Nor epinephrine, serum Luteinizing hormone, serum Malondialdehyde and Superoxide Dismutase levels in male Sprague Dawley rats exposed to six hours restraint stress

Parameter

Group II Acute restraint

stress (n=16)

Mean ± SEM

Group V AAS + ATS and restraint stress

(n=16) Mean ± SEM

p value*

Serum Testosterone (ng/ml)

5.18 ± 0.19

7.06 ± 0.15

< 0.01

Serum Corticosterone (pg/ml)

9688.75 ± 219.18

8776.25 ± 157.71

< 0.01

Plasma Nor epinephrine (ng/ml)

5.06 ± 0.31

5.29 ± 0.32

> 0.05

Serum LH (IU/L)

9.17 ± 0.09

9.15 ± 0.08

> 0.05

Serum Malondialdehyde (nmol/ml)

14.77 ± 1.18

10.27 ± 0.99

< 0.01

Serum Super oxide dismutase (U/ml)

0.08 ± 0.01

0.13 ± 0.007

< 0.01

*p value less than 0.05 is taken as significant.

101

IN VITRO FINDINGS:

All samples were run in triplicate. Therefore, all findings are based on mean

of three results.

Effect of LH on testosterone release by Leydig cells

Basal testosterone production was 48.67 ± 3.85 pg/well and with LH

stimulation raised to 189.78 ± 1.47 pg/ well (p value < 0.001) as shown in figure

3.1. Therefore, LH significantly increased testosterone production by isolated

Leydig cells placed in well.

Effects of different concentrations of corticosterone in the presence of LH on

testosterone production by Leydig cells:

The mean value of the testosterone produced under the effect of LH alone

was 189.78 ± 1.47 pg/ well. The mean value of testosterone production was 190.89

± 4.34 pg/ well when exposed to 10 nM concentration of corticosterone, while

addition of corticosterone in concentration of 100 nM resulted in mean testosterone

production of 112.56 ± 2.94 pg/ well as shown in figure 3.2. The production of

testosterone in the presence of LH was significantly reduced by 100 nM

concentration of corticosterone with a p value < 0.05.

Effects of different concentrations of nor epinephrine in the presence of LH,

on testosterone production by Leydig cells:

The mean value of the testosterone produced under the effect of LH alone was

189.78 ± 1.47 pg/well. The mean value of testosterone production was 220.89 ±

6.76 pg/ well when exposed to 10 µM concentration of nor epinephrine, while

addition of nor epinephrine in concentration of 100 µM resulted in mean

102

Figure 3.1: Effect of LH stimulation on testosterone production by cultured Leydig cells (*p value < 0.001 as compared to basal, without LH)

103

testosterone production of 205.33 ± 6.94 pg/ well. When we added 1000 µM

concentration of nor epinephrine, the mean value of testosterone was 196.44 ± 4.84

pg/ well, as shown in figure 3.3. The production of testosterone in the presence of

LH was significantly increased by 10 µM concentration of nor epinephrine with a p

value< 0.05.

Effects of different concentrations of Iso butryl methyl xanthine (IBMX) in the presence of LH, on testosterone production by Leydig cells:

The mean value of the testosterone produced under the effect of LH alone

was 189.78 ± 1.47 pg / well. The mean value of testosterone production was 197.55

± 4.34 pg/ well when exposed to 0.1 mM concentration of IBMX, while addition of

IBMX in concentration of 1 mM resulted in mean testosterone production of

229.78 ± 8.73 pg/ well. When we added 10 mM concentration of IBMX, the mean

value of testosterone was 191.44 ± 3.09 pg/ well. The production of testosterone in

the presence of LH was significantly increased by 1 mM of IBMX with a p value <

0.05 as shown in figure 3.4.

104

Figure 3.2: Effect of different concentrations of corticosterone on testosterone production by cultured Leydig cells incubated with LH (* p value < 0.05 as compared with the well incubated with LH alone)

Figure 3.3: Effect of different concentrations of nor epinephrine on testosterone production by cultured Leydig cells incubated with LH (*p value< 0.05 as compared with the well incubated with LH alone)

105

Figure 1.4: Effects of different concentrations of Isobutyl methyl xanthine (IBMX) on testosterone production by cultured Leydig cells incubated with LH (* p value< 0.05 as compared with the well incubated with LH alone)

106

Effects of different concentrations of pregnenolone in the presence of LH, on testosterone production by Leydig cells:

The mean value of the testosterone produced under the effect of LH alone

was 189.78 ± 1.47 pg/well. The mean value of testosterone production was 192 ±

7.70 pg/ well when exposed to 0.1 µM concentration of pregnenolone, while

addition of pregnenolone in concentration of 1 µM resulted in mean testosterone

production of 241.44 ± 8.18 pg/ well. When we added 10 µM concentration of

pregnenolone, the mean value of testosterone was 199.22 ± 6.41 pg/ well, as shown

figure 3.5. The production of testosterone in the presence of LH was significantly

increased by 1 µM of pregnenolone with a p value< 0.05.

Effects of different concentrations of ascorbic acid in the presence of LH, on testosterone production by Leydig cells:

The mean value of the testosterone produced under the effect of LH alone

was 189.78 ± 1.47 pg/tube. The mean value of testosterone production was 194.22

± 4.84 pg/tube when exposed to 10 µM concentration of ascorbic acid, while

addition of ascorbic acid in concentration of 100 µM resulted in mean testosterone

production of 207.00 ± 6.31 pg/tube. When we added 200 µM concentration of

ascorbic acid, the mean value of testosterone was 153.67 ± 5.36 pg/tube, as shown

in figure 3.6. The production of testosterone in the presence of LH was

insignificantly changed by low doses of ascorbic acid, while high dose has

inhibitory effect (p value< 0.01).

107

Figure 3.5 Effect of different concentrations of pregnenolone on testosterone production by cultured Leydig cells incubated with LH (* p value< 0.05 as compared with the well incubated with LH alone)

Figure 3.6 Effect of different concentrations of ascorbic acid (AA) in the presence of LH on testosterone production by cultured Leydig cells incubated with LH. (p value< 0.01 as compared with the well incubated with LH alone)

108

Effects of different concentrations of alpha tocopherol in the presence of LH,

on testosterone production by Leydig cells:

The mean value of the testosterone produced under the effect of LH alone

was 189.78 ± 1.47 pg/ well. The mean value of testosterone production was 256.45

± 7.22 pg/ well when exposed to 10 µg/ml concentration of alpha tocopherol, while

addition of alpha tocopherol in concentration of 100 µM resulted in mean

testosterone production of 201.45 ± 7.22 pg/well. When we added 1000 µg/ml

concentration of alpha tocopherol, the mean value of testosterone was 158.11 ±

6.26 pg/well, as shown in figure 3.7. The production of testosterone in the presence

of LH was significantly increased by 10 µg/ml of alpha tocopherol with a p value<

0.01.

Effects of combination of corticosterone and nor epinephrine in the presence

of LH, on testosterone production by Leydig cells:

The mean value of the testosterone produced under the effect of LH alone

was 189.78 ± 1.47 pg/well. However, exposure of Leydig cells in the experimental

well containing LH along with corticosterone in concentration of 100 nM and nor

epinephrine 10 µM resulted in significant reduction (p value< 0.05) in mean

testosterone production to 136.45 ± 6.83 pg/well. These results are shown in figure

3.8.

109

Figure 3.7: Effect of different concentrations of alpha tocopherol (AT) in the presence of LH on testosterone production by cultured Leydig cells incubated with LH. (*p value< 0.01 as compared with the well incubated with LH alone)

110

Figure 3.8: Effect of combination of corticosterone and nor epinephrine on testosterone production by LH stimulated cultured Leydig cells incubated with LH. (*p value< 0.05 as compared with the well incubated with LH alone)

111

Effect of IBMX on basal testosterone production by cultured Leydig cells:

The mean value of the testosterone produced without LH stimulation was

48.67 ± 3.85 pg/well. However, addition of IBMX to the well containing Leydig

cells without LH resulted in significant increase (p value< 0.001) in mean

testosterone production to 140.89 ± 6.76 pg/well as shown in figure 3.9.

Effect of corticosterone on IBMX induced increase in basal testosterone

production by cultured Leydig cells:

The mean value of the testosterone produced under the effect of IBMX was

140.89 ± 6.76 pg/well. The corticosterone in conc of 100 nM resulted in a

significant decrease (p value< 0.05) in mean testosterone production to 113.67 ±

6.01 pg/well as shown in figure 3.10.

Effect of corticosterone on IBMX induced increase in testosterone production

by cultured Leydig cells in the presence of LH:

The mean value of the testosterone produced under the effect of IBMX was

229.78 ± 8.73 pg/well. The corticosterone in conc of 100 nM resulted in a

significant decrease (p value< 0.01) in mean testosterone production to 161.47 ±

6.19 pg/well as shown in figure 3.11.

Effect of nor epinephrine on IBMX induced increase in basal testosterone

production by cultured Leydig cells:

The mean value of the testosterone produced under the effect of IBMX was

140.89 ± 6.76 pg/well. The nor epinephrine in conc of 10 µM, as shown in figure

3.12 resulted in a significant increase (p value˂0.05) in mean testosterone

production to 169.78 ± 8.01 pg/well.

112

Effect of nor epinephrine on IBMX induced increase in testosterone

production by cultured Leydig cells in the presence of LH:

The mean value of the testosterone produced under the effect of IBMX in

the presence of LH was 229.78 ± 8.73 pg/well. The nor epinephrine in conc of 10

µM resulted in an insignificant change (p value> 0.05) in mean testosterone

production with value of 221.45 ± 12.56 pg/well as shown in figure 3.13.

113

Fig 3.9: Effect of IBMX on testosterone production by Leydig cells incubated without LH. (*p value< 0.001 as compared with the well incubated without LH with basal testosterone production)

Fig 3.10: Effect of corticosterone on IBMX induced increment in testosterone production by Leydig cells incubated without LH. (*p value< 0.05 as compared with the well incubated with IBMX alone)

114

Fig 3.11: Effect of corticosterone on IBMX induced increment in testosterone production by Leydig cells incubated with LH. (*p value< 0.01 as compared with the well incubated with IBMX and LH)

Fig 3.12: Effect of nor epinephrine on IBMX induced increment in testosterone production by Leydig cells incubated without LH. (*p value< 0.05 as compared with the well incubated with IBMX)

Fig 3.13: Effect of nor epinephrine on IBMX induced increment in testosterone production by Leydig cells incubated with LH (*p value > 0.05 as compared with well containing IBMX and LH)

115

Effect of pregnenolone on basal testosterone production by cultured Leydig cells:

The mean value of the testosterone produced without LH stimulation was

48.67 ± 3.85 pg/well. However, addition of pregnenolone to the well containing

Leydig cells without LH resulted in significant increase (p value< 0.01) in mean

testosterone production to 95.89 ± 5.80 pg/well as shown in figure 3.14.

Effect of corticosterone on pregnenolone induced increase in basal

testosterone production by cultured Leydig cells:

The mean value of the testosterone produced under the effect of

pregnenolone was 95.89 ± 5.80 pg/well. The mean testosterone produced in the

experimental well where we added corticosterone in conc of 100 nM to

pregnenolone was 100.89 ± 6.01 pg/well, which showed an insignificant change (p

value> 0.05). These results are shown in figure 3.15.

Effect of corticosterone on pregnenolone induced increase in testosterone

production by cultured Leydig cells in the presence of LH:

The mean value of the testosterone produced under the effect of

pregnenolone was 241.44 ± 8.18 pg/well. The effect of corticosterone as shown in

figure 3.16, in conc of 100 nM which resulted in a significant decrease (p value<

0.01) in mean testosterone production to 160.89 ± 6.26 pg/well.

116

Effect of nor epinephrine on pregnenolone induced increase in basal

testosterone production by cultured Leydig cells:

The mean value of the testosterone produced under the effect of

pregnenolone was 95.89 ± 5.80 pg/well. The nor epinephrine in conc of 10 µM

resulted in an insignificant change (p value> 0.05) and value of mean testosterone

production is 98.11 ± 3.05 pg/well as shown in figure 3.17.

Effect of nor epinephrine on pregnenolone induced increase in basal

testosterone production by cultured Leydig cells in the presence of LH:

The mean value of the testosterone produced under the effect of

pregnenolone in the presence of LH was 241.44 ± 8.18 pg/well. The nor

epinephrine in conc of 10 µM resulted in an insignificant change (p value> 0.05) in

mean testosterone production with value of 271.45 ± 7.22 pg/well as shown in

figure 3.18

117

Fig 3.14: Effect of pregnenolone on testosterone production by Leydig cells incubated without LH. (*p value< 0.01 as compared with the well incubated without LH with basal testosterone production)

Fig 3.15: Effect of corticosterone on pregnenolone induced increment in testosterone production by Leydig cells incubated without LH. (*p value > 0.05 as compared with pregnenolone alone).

Fig 3.16: Effect of corticosterone on pregnenolone induced increment in testosterone production by Leydig cells incubated with LH. (*p value< 0.01 as compared to pregnenolone with LH)

118

Fig 3.17: Effect of nor epinephrine on pregnenolone induced increment in testosterone production by Leydig cells incubated without LH. (*p value > 0.05 as compared to pregnenolone alone).

Fig 3.18: Effect of nor epinephrine on pregnenolone induced increment in testosterone production by Leydig cells incubated with LH. (*p value > 0.05 as compared to pregnenolone and LH).

119

Effect of ascorbic acid on corticosterone induced changes in testosterone

production by cultured Leydig cells in the presence of LH.

Corticosterone, as mentioned before in a conc of 100 nM resulted in a fall

of testosterone synthesis by cultured Leydig cells. The addition of ascorbic acid in

100 µM conc to our experimental well containing cultured Leydig cells exposed to

corticosterone resulted in a significant rise (p value< 0.01) of testosterone

production to a value of 198.11 ± 9.35 pg/well as shown in figure 3.19.

Effect of alpha tocopherol on corticosterone induced decline in testosterone

production by cultured Leydig cells in the presence of LH.

The addition of alpha tocopherol in 10 µg/ml conc to our experimental well

containing cultured Leydig cells exposed to corticosterone resulted in a significant

rise (p value< 0.001) of testosterone production to a value of 197.00 ± 4.19 pg/well

as shown in figure 3.20.

Effect of addition of both ascorbic acid and alpha tocopherol on corticosterone

induced changes in testosterone production by cultured Leydig cells in the

presence of LH.

The addition of ascorbic acid in 100 µM conc and alpha tocopherol in 10

µg/ml conc in the presence of LH to our experimental well containing cultured

Leydig cells exposed to corticosterone resulted in a significant rise (p value<

0.001) of testosterone production to a value of 209.22 ± 7.78 pg/well as shown in

figure 3.21.

120

Fig 3.19: Effect of ascorbic acid on corticosterone induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH. (*p value< 0.01 as compared with corticosterone in presence of LH)

Fig 3.20: Effect of alpha tocopherol on corticosterone induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH. (*p value< 0.001 as compared with corticosterone in the presence of LH)

Fig 3.21: Effect of both ascorbic acid and alpha tocopherol on corticosterone induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH. (*p value< 0.001 as compared with corticosterone in the presence of LH)

121

Effect of ascorbic acid on nor epinephrine induced changes in testosterone

production by cultured Leydig cells in the presence of LH.

Nor epinephrine, as mentioned before in a conc of 10 µM resulted in

increase in testosterone synthesis by cultured Leydig cells. The addition of ascorbic

acid in 100 µM conc to our experimental well containing cultured Leydig cells

exposed to nor epinephrine resulted in insignificant change (p value> 0.05) of

testosterone production to a value of 220.33 ± 3.46 pg/well as shown in figure 3.22.

Effect of alpha tocopherol on nor epinephrine induced changes in testosterone

production by cultured Leydig cells in the presence of LH.

The addition of alpha tocopherol in 10 µg/ml conc to our experimental well

containing cultured Leydig cells exposed to nor epinephrine resulted in a

significant rise (p value< 0.01) of testosterone production to a value of 258.67 ±

4.41 pg/well as shown in figure 3.23.

Effect of addition of both ascorbic acid and alpha tocopherol on nor

epinephrine induced changes in testosterone production by cultured Leydig

cells in the presence of LH.

The addition of ascorbic acid in 100 µM conc and alpha tocopherol in 10

µg/ml conc in the presence of LH to our experimental well containing cultured

Leydig cells exposed to nor epinephrine resulted in a significant rise (p value<

0.05) of testosterone production to a value of 243.11 ± 3.64 pg/well. These results

are presented in figure 3.24.

122

Fig 3.22: Effect of ascorbic acid on nor epinephrine induced changes in testosterone production by isolated and cultured Leydig cells incubated with LH (*p value > 0.05 as compared with nor epinephrine in the presence of LH).

Fig 3.23: Effect of alpha tocopherol on nor epinephrine induced changes in testosterone production by isolated and cultured Leydig cells incubated with LH. (*p value< 0.01 compared with nor epinephrine in the presence of LH)

Fig 3.24: Effect of both ascorbic acid and alpha tocopherol on nor epinephrine induced changes in testosterone production by isolated and cultured Leydig cells incubated with LH. (*p value< 0.05 compared with nor epinephrine in the presence of LH)

123

Effect of ascorbic acid on corticosterone and nor epinephrine induced changes

in testosterone production by cultured Leydig cells in the presence of LH.

Addition of both corticosterone, in a conc of 100 nM and nor epinephrine in

a conc of 10 µM resulted in decrease in testosterone synthesis by cultured Leydig

cells to a value of 136.45 ± 6.83 pg/well. The addition of ascorbic acid in 100 µM

conc to our experimental well containing cultured Leydig cells exposed to both

corticosterone and nor epinephrine resulted in testosterone production to a value of

197.55 ± 4.94 pg/well (p value< 0.01) as shown in figure 3.25.

Effect of alpha tocopherol on corticosterone and nor epinephrine induced

changes in testosterone production by cultured Leydig cells in the presence of

LH.

Addition of both corticosterone, in a conc of 100 nM and nor epinephrine in

a conc of 10 µM resulted in decrease in testosterone synthesis by cultured Leydig

cells to a value of 136.45 ± 6.83 pg/well. The addition of alpha tocopherol in 10

µg/ml conc to our experimental well containing cultured Leydig cells exposed to

both corticosterone and nor epinephrine resulted in testosterone production to a

value of 198.11 ± 3.89 pg/well (p value< 0.01) as shown in figure 3.26.

Effect of addition of both ascorbic acid and alpha tocopherol on nor

epinephrine induced changes in testosterone production by cultured Leydig

cells in the presence of LH.

Addition of both corticosterone, in a conc of 100 nM and nor epinephrine in

a conc of 10 µM resulted in decrease in testosterone synthesis by cultured Leydig

cells to a value of 136.45 ± 6.83 pg/well. The addition of both ascorbic acid in 100

124

µM conc and alpha tocopherol in 10 µg/ml conc to our experimental well

containing cultured Leydig cells exposed to both corticosterone and nor

epinephrine resulted in testosterone production to a value of 200.33 ± 3.47 pg/well

(p value< 0.01) as shown in figure 3.27.

125

Fig 3.25: Effect of ascorbic acid on corticosterone and nor epinephrine induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH. (*p value< 0.01 compared with nor epinephrine and corticosterone in the presence of LH)

Fig 3.26: Effect of alpha tocopherol on corticosterone and nor epinephrine induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH. (*p value< 0.01 compared with nor epinephrine and corticosterone in the presence of LH)

Fig 3.27: Effect of ascorbic acid and alpha tocopherol on corticosterone and nor epinephrine induced decline in testosterone production by isolated and cultured Leydig cells incubated with LH. (*p value< 0.01 compared with nor epinephrine and corticosterone in the presence of LH)

126

Effect of stress hormones in the presence of LH on lipid peroxidation in terms

of Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity at

Leydig cell level:

There was insignificant effect of LH alone on MDA as well as SOD activity

(p value > 0.05) in cultured Leydig cells. The predominant steroid in rat, i.e.,

corticosterone significantly increased the lipid peroxidation (p value < 0.05) in

terms of MDA and caused the SOD activity to decline significantly (p value <

0.05) as shown in table. However, the other important hormone nor epinephrine has

shown no effect on MDA as well as SOD activity of cultured Leydig cells. But the

combination of corticosterone and nor epinephrine has resulted in a significant rise

of MDA (p value < 0.05) and also a statistically significant fall in SOD (p value <

0.01) activity in cultured Leydig cells of our study wells as shown in table 3.5.

Effect of antioxidants ascorbic acid (AA) and alpha tocopherol (AT) in the

presence of LH on levels of Malondialdehyde (MDA) and superoxide

dismutase (SOD) at Leydig cell level:

There was no significant effect (p value > 0.05) of ascorbic acid and alpha

tocopherol separately on lipid peroxidation in terms of MDA activity of Leydig

cells. However combination of AA and AT resulted in a significant (p value <

0.05) reduction in levels of MDA.

Ascorbic acid (p value < 0.05) and alpha tocopherol (p value < 0.05) has caused a

significant rise in superoxide dismutase activity. This was also significantly raised

(p value < 0.05) by combination of AA and AT, as shown in table 3.6.

127

Table 3.5: Effect of stress hormones in the presence of LH on lipid peroxidation in terms of Malondialdehyde (MDA) levels and Superoxide dismutase (SOD) activity at Leydig cell level:

Parameter Well Contents Mean ± SEM p value*

Malondialdehyde (nmol/well)

LH only

6.52 ± 0.63 > 0.05

LH + Corticosterone 100 nM

9.14 ± 0.28 < 0.05

LH + Nor epinephrine NE 10 µM

5.33 ± 0.28 > 0.05

LH + Corticosterone 100 nM + Nor epinephrine NE 10 µM

9.46 ± 0.42 < 0.05

Serum Super oxide dismutase (U/well)

LH only

0.10 ± 0.004 > 0.05

LH + Corticosterone 100 nM

0.07 ± 0.006 < 0.05

LH + Nor epinephrine NE 10 µM

0.09 ± 0.008 > 0.05

LH + Corticosterone 100 nM + Nor epinephrine NE 10 µM

0.06 ± 0.005 < 0.01

*p value less than 0.05 is taken as significant.

128

Table 3.6: Effect of antioxidants ascorbic acid (AA) and alpha tocopherol (AT) in the presence of LH on levels of Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity at Leydig cell level:

Parameter Well Contents Mean ± SEM *p value

Malondialdehyde (nmol/Well)

LH only

6.52 ± 0.63 > 0.05

LH + AA 100 µM

5.01 ± 0.69 > 0.05

LH + AT 10 µg/ml

4.70 ± 0.57 > 0.05

LH + AA 100 µM + AT 10 µg/ml

3.74 ± 0.42

< 0.05

Serum Super oxide dismutase (U/well)

LH only

0.10 ± 0.004 > 0.05

LH + AA 100 µM

0.13 ± 0.008 < 0.05

LH + AT 10 µg/ml

0.15 ± 0.006 < 0.05

LH + AA 100 µM + AT 10 µg/ml

0.15 ± 0.009

< 0.05

*p value less than 0.05 is taken as significant.

129

Effect of antioxidants on stress hormone induced changes in Malondialdehyde

(MDA) at Leydig cell level:

Effect of ascorbic acid on stress hormones induced changes in MDA levels

The levels of MDA in corticosterone added well was 9.14 ± 0.28 nmol /well

and addition of ascorbic acid to this well containing LH and corticosterone

resulted in significant reduction of MDA to 6.76 ± 0.27 nmol /well (p value <

0.01). Addition of ascorbic acid to NE exposed cells resulted in MDA levels of

5.08 ± 0.31 nmol /well, which is an insignificant change as compared to NE

addition alone (p value > 0.05). These results are shown in figure 3.28. However

ascorbic acid significantly reduced lipid peroxidation to 5.33 ± 0.73 nmol /well (p

value < 0.01) in the corticosterone and NE added well. These results are shown in

figure 3.28.

Effect of alpha tocopherol on stress hormones induced changes in MDA levels

The levels of MDA in corticosterone added well was 9.14 ± 0.28 nmol /well

and addition of alpha tocopherol to this well containing LH and corticosterone

resulted in significant reduction of MDA to 6.29 ± 0.56 nmol /well (p value <

0.05). Addition of alpha tocopherol to NE exposed cells resulted in decline in

MDA levels to 3.75 ± 0.42 nmol /well, (p value < 0.05). Also, alpha tocopherol

significantly reduced MDA to 6.29 ± 0.56 nmol /well (p value < 0.05) in the

corticosterone and NE added well. These results are shown in figure 3.29.

130

Effect of ascorbic acid and alpha tocopherol combination on stress hormones

induced changes in MDA levels

The levels of MDA in corticosterone added well was 9.14 ± 0.28 nmol /well

and ascorbic acid and alpha tocopherol addition to this well containing LH and

corticosterone resulted in significant reduction of MDA to 6.12 ± 0.42 nmol /well

(p value < 0.01). Addition of ascorbic acid and alpha tocopherol to NE exposed

cells resulted in fall in MDA levels to 3.75 ± 0.32 nmol /well, (p value < 0.05).

Similarly, ascorbic acid and alpha tocopherol significantly reduced MDA levels to

7.24 ± 0.55 nmol /well (p value < 0.01) in the corticosterone and NE added well as

shown in figure 3.30.

131

Figure 3.28: Effect of ascorbic acid (AA) on stress hormones corticosterone and norepinephrine (NE) induced changes in MDA levels *p value < 0.01 as compared with corticosterone with LH ** p value > 0.05 as compared with nor epinephrine with LH *** p value < 0.01 as compared with corticosterone and nor epinephrine with LH

Figure 3.29: Effect of alpha tocopherol (AT) on stress hormones corticosterone and norepinephrine (NE) induced changes in MDA levels

*p value < 0.05 as compared with corticosterone with LH, ** p value < 0.05 as compared with nor epinephrine with LH *** p value < 0.05 as compared with corticosterone and nor epinephrine with LH

132

Figure 3.30: Effect of ascorbic acid (AA) and alpha tocopherol (AT) combination on stress hormones corticosterone and norepinephrine (NE) induced changes in MDA levels

*p value < 0.01 as compared with corticosterone with LH ** p value < 0.05 as compared with nor epinephrine with LH *** p value < 0.01 as compared with corticosterone and nor epinephrine with LH

133

Effect of antioxidants on stress hormone induced changes in superoxide

dismutase (SOD) at Leydig cell level:

Effect of ascorbic acid on stress hormones induced changes in SOD levels

The levels of SOD in corticosterone added well was 0.07 ± 0.005 U/well

and addition of ascorbic acid to this well containing LH and corticosterone

resulted in significant rise of SOD to 0.11 ± 0.01 U/well (p value < 0.05).

Addition of ascorbic acid to NE exposed cells resulted in SOD levels of 0.14 ±

0.006 U/well, which is a significant rise as compared to NE addition alone (p value

< 0.05). Also, ascorbic acid significantly increased SOD to 0.11 ± 0.008 U/well (p

value < 0.05) in the corticosterone and NE added well as shown in figure 3.31.

Effect of alpha tocopherol on stress hormones induced changes in SOD levels

The levels of SOD in corticosterone added well was 0.07 ± 0.006 U/well

and addition of alpha tocopherol to this well containing LH and corticosterone

resulted in significant rise of SOD to 0.15 ± 0.008 U/well (p value < 0.05).

Addition of alpha tocopherol to NE exposed cells resulted a significant rise in SOD

levels to 0.13 ± 0.005 U/well, (p value < 0.05). Also, alpha tocopherol cause a

significant rise in SOD to 0.12 ± 0.01 U /well (p value < 0.05) in the corticosterone

and NE added well. These results are shown in figure 3.32.

Effect of ascorbic acid and alpha tocopherol combination on stress hormones

induced changes in SOD levels

The ascorbic acid and alpha tocopherol addition to the well containing LH

and corticosterone resulted in significant rise of SOD to 0.07 ± 0.006 U/well (p

value < 0.01). Addition of ascorbic acid and alpha tocopherol to NE exposed cells

134

resulted in a significant rise in SOD levels to 0.15 ± 0.01 U/well (p value < 0.05).

Similarly, ascorbic acid and alpha tocopherol significantly increased SOD levels to

0.14 ± 0.01 U/well (p value < 0.05) in the corticosterone and NE added well as

shown in figure 3.33.

135

Figure 3.31: Effect of ascorbic acid (AA) on stress hormones corticosterone and norepinephrine (NE) induced changes in SOD levels *p value < 0.05 as compared with corticosterone with LH ** p value < 0.05 as compared with nor epinephrine with LH *** p value < 0.05 as compared with corticosterone and nor epinephrine with LH

Figure 3.32: Effect of alpha tocopherol (AT) on stress hormones corticosterone and norepinephrine (NE) induced changes in SOD levels. *p value < 0.05 as compared with corticosterone with LH ** p value < 0.05 as compared with nor epinephrine with LH *** p value < 0.05 as compared with corticosterone and nor epinephrine with LH

* ** ***

136

0

0.05

0.1

0.15

0.2

LH +Corticosterone 100

nM

LH + NE 10 µM LH+ Corticosteron100 nM+NE 10 µ

SODU/well

Control AA 100 µM+AT 10ug/ml

Figure 3.33: Effect of ascorbic acid (AA) and alpha tocopherol (AT) combination on stress hormones corticosterone and norepinephrine (NE) induced changes in SOD levels *p value < 0.01 as compared with corticosterone with LH ** p value < 0.05 as compared with nor epinephrine with LH *** p value < 0.05 as compared with corticosterone and nor epinephrine with LH

* ** ***

137

DISCUSSION

Stress is an extensively prevalent problem, the contributing factors of which

include changed life styles along with industrialization and pollution (Eskiocak et

al., 2005). Stress participates in the pathophysiology of various diseases (McEwen,

2000). Stress of various origins has inhibitory effect on male reproductive

functions (Hardy et al., 2005). We examined the effects of stress by the in vivo

study using mesh wire restraint immobilization stress protocol on adult male

Sprague Dawley rats. This immobilization stress model, serves as a mixture of

psychological and physical stressors by isolating the experimental animal from its

group and by restricting its movements (Pacak and Palkovits, 2001). Restraint

stress as comprehended by shansky et al., not only stimulates a number of

neurological changes but may also induce various molecular, biochemical and

hormonal changes (Shansky et al., 2006).

We also designed an in vitro model of isolated and cultured Leydig cells to

explore the effects of two important stress hormones in rat, i.e., corticosterone and

nor epinephrine on testosterone synthesis by Leydig cells. The stress induced

changes in lipid peroxidation and superoxide dismutase activity were probed. The

individual as well as combined effects of antioxidants, ascorbic acid and alpha

tocopherol to prevent the stress induced alterations in above parameters were

investigated in both in vivo and in vitro models.

Our results of acute stress have shown that LH levels remained unchanged. It is

supported by various studies which claim that acute stress mediated effects are not

via changes in hypothalamo pituitary gonadal axis, therefore there is no change in

Chapter 4

138

LH as a result of acute stress. Acute stress affects the steroidogenesis at the level of

Leydig cells only. However, stress exposure of more than ten days may result in

decline of LH levels (Maric et al., 1996). Hence the suppression of whole

hypothalamo pituitary gonadal axis by acute stress is ruled out by the data of

present study.

Stress of various origins suppresses male reproductive functions (Ozawa et

al, 2002). Exposure of our experimental animals to restraint stress for six hours

resulted in rise in corticosterone and a fall in testosterone. These findings are in

accordance with study by Dong et al., done on mice in which they exposed the

mice to immobilization stress for three hours with resultant fall in testosterone and

rise of corticosterone, the predominant steroid of rodents (Dong et al., 2004). We

made it sure that timings for sampling remain the same between 11 am and 12

noon, for sampling serum corticosterone in control and experimental groups

(Debigare et al., 2003).

The group of rats exposed to restraint stress, showed a significant rise in the

corticosterone. The literature supports the concept that even two hours of

immobilization stress to rats may decrease the testicular steroidogenesis and also

there is an increase in the level of serum corticosterone. It is thought that this rise

of corticosterone is responsible for decline in testosterone (Hales and Payne, 1989).

A study done by Kosti et al., revealed that immobilization stress decreases the

testosterone levels but hypothalamo pituitary gonadal axis is not affected (Kosti et

al., 1997). Another study by Taylor et al., points towards the fact that

immobilization stress causes a decline in testicular androgens (Taylor et al., 1994).

139

An important point of consideration here is that, not only immobilization

stress, but stress of multiple origin like various diseases, exercise and even

psychosocial interactions may also result in suppression of male reproductive

functions (Chrousos and Gold, 1992). For example, testosterone levels are lower in

patients of Cushing’s syndrome which were further depressed by dexamethasone

given for diagnostic reasons (Contreras et al., 1996; Vierhapper et al., 2000).

Similarly, in humans the severe psychological stress due to death of a relative or

spouse lowers sperm count which is most likely caused by stress induced decline in

testosterone (Fenster et al., 1997).

As testosterone is produced by Leydig cells, we designed an in vitro model

of isolated and cultured Leydig cells with three hours incubation time, to

investigate the effects of stress hormone corticosterone on Leydig cells. When we

incubated the Leydig cells with corticosterone 100 nM, there was a significant

decline in synthesis of testosterone. Therefore, it is evident that corticosterone

decreases the steroidogenesis at Leydig cell level.

Synthesis of testosterone is dependent on optimal functioning of Leydig

cells, located in testicular interstitium and stress induced escalation in

corticosterone can induce apoptosis in Leydig cells. However, the apoptotic effect

is evident only after 12 hours of stress exposure; therefore the fall in testosterone as

a consequence of apoptosis induced damage to Leydig cells shows no correlation

with our study (Gao et al., 2002). Acute immobilization stress induced fall in

testosterone is also suggestive that these effects may be produced by corticosterone

are non-genomic, because it involves relatively rapid response which may not

involve the protein synthesis. These responses may include change in permeability

140

of ion channels, involvement of cAMP and calcium channels (Wehling, 1997;

Dong et al., 2004). Steroidogenic acute regulatory (StAR) protein is also thought

to be suppressed by glucocorticoids (Wang et al., 2000).

In order to investigate the mechanism of action of corticosterone, we used

phosphodiesterase inhibitor, 3 iso butryl -1-methyl xanthine (IBMX). It blocks the

activity of phospodiesterases which may cause hydrolysis of cAMP. Therefore, we

can say that IBMX increases the availability of cAMP at cell level by decreasing its

breakdown. The cAMP is involved in biosynthetic pathways of testicular

steroidogenesis (Soderling et al., 1998). Initially we observed that, IBMX produced

significant rise in basal testosterone synthesis at concentration of 1 mM, which is in

accordance with literature (Shiow et al., 1997). Also IBMX led to increased

testosterone production in the presence of LH.

When corticosterone was added to the well containing Leydig cells with

IBMX, there was a fall in concentration of testosterone as compared to IBMX

alone stimulated cells. Similarly, the effects of stimulation of LH and IBMX both

were also diminished by corticosterone. Based on these findings, it is suggested

that corticosterone would have decreased the cAMP production in Leydig cells,

(both basal and LH stimulated) due to which the effects of IBMX were not evident.

These findings support the study done by Dong et al, in which they measured

cAMP and found that direct action of corticosterone is to decrease cAMP. We

used IBMX to check the cAMP instead of measuring cAMP levels (Dong et al,

2004).

141

Addition of pregnenolone increased the production of testosterone. It is due

to the fact, that pregnenolone is an important precursor in the pathway of testicular

steroidogenesis, because after the conversion of cholesterol to pregnenolone, it is

converted to progesterone with the help of 3 beta hydroxyl steroid dehydrogenase

(3βHSD), which by action of 17 α hydroxylase is converted to 17 α hydroxyl

progesterone. Action of C-17-20 lyase will result in formation of androstenedione,

which is then converted to testosterone (Zirkin, and Chen, 2000).

In our study, the pregnenolone was unable to replenish the fall of

testosterone synthesis induced by corticosterone, because addition of corticosterone

resulted in a decline in testosterone synthesis both in basal and LH stimulated cells,

in which pregnenolone was added.

It is well know that steroidogenesis involve conversion of cholesterol to

pregnenolone as a rate limiting step catalyzed by the mitochondrial cholesterol side

chain cleavage enzyme (Payne and Hales, 2004) For rapid regulation of steroid

synthesis, transfer of cholesterol from outer to inner mitochondrial membrane

involves steroid acute regulatory (StAR) protein (Stocco, 2001). Inability of

pregnenolone to replenish decrement in testosterone induced by corticosterone is

suggestive that synthesis of pregnenolone is not affected or we can say that activity

of StAR protein may not be affected by corticosterone at Leydig cell level during

three hours of incubation. Therefore we suggest that corticosterone could have

inhibited the enzymes like 3βHSD or 17 α hydroxylase or C-17-20 lyase down in

steroidogenesis pathway. But 3βHSD inhibition is excluded by study done by Orr

et al, in which they estimated the levels of progesterone and 17 α hydroxyl

progesterone. Their study revealed that after 03 hours of immobilization stress,

142

nine fold rise of corticosterone was accompanied by the increase in the levels of

progesterone by 33 percent while there was 47 percent decline in levels of 17 α

hydroxyl progesterone. This suggests that conversion of progesterone to 17 α

hydroxyl progesterone is seriously impaired by corticosterone. It means that the

enzyme involved in above step is blocked by corticosterone, which they confirmed

by estimation to be 17 α hydroxylase. Also it was suggested that the activity of C-

17-20 lyase is also suppressed. (Orr et al., 1994) Our study validates the findings

of Orr et al., that enzymatic pathways are also blocked by stress hormones.

However, we obtained this inference on cultured Leydig cells by adding

pregnenolone and corticosterone together, in the well containing cultured Leydig

cells contrast to Orr et al., because they measured levels of these enzymes.

Along with increase in the glucocorticoids, another neuroendocrine

component of stress response include increased secretion of epinephrine and nor

epinephrine from sympathetic nervous system and adrenal medulla (Carrasco and

VanderKar, 2003). Restraint stress exposure to our experimental animals

significantly increased the nor epinephrine levels. Increase in catecholamines like

nor epinephrine is well known entity of stress, which results in stimulation of

adrenal cortex and locus coeruleus, with resultant increase in nor epinephrine

(Montoro et al., 2009). Increase in nor epinephrine is also reported after cold

induced stress (Dronjak et al., 2004). Similarly, immobilization stress may result in

rise in catecholamines like nor epinephrine (Collu et al., 1984). Immobilization

stress is thought to decrease the catecholamines stores in central and peripheral

tissues (Dronjak and Gavrilovic, 2006). Therefore, the rise of nor epinephrine

143

confirms that our stress protocol was efficient enough to produce the effects of

stress.

Involvement of catecholamines in testicular steroidogenesis is an important

fact to be considered because a direct autonomic pathway of innervation from

spinal cord to testicular interstitium may become active during stressful conditions

(Lee et al., 2002). Fewer studies point towards role of catecholamines during stress

at Leydig cell level (Hardy et al., 2005). Catecholamines are generally assumed to

increase steroidogenesis in vitro (Mayerhofer et al., 1993). Beta blockers which

decrease sympathetic activity decrease testicular steroidogenesis (Khan et al.,

2004). Now, stress involves rise of nor epinephrine and fall in testosterone values.

Therefore, we investigated the role of nor epinephrine at Leydig cell level.

Nor epinephrine significantly increased the testosterone production in LH

stimulated Leydig cells in our study. These findings are in accordance with studies

done by Anakwe and Moger who have shown that catecholamines stimulate

testicular steroidogenesis in vitro on isolated cultured Leydig cells. They have

shown that the beta agonist isoproterenol, increased steroid production to 174

percent after three hours of incubation of Leydig cells. Similar proportionate

increase was observed with LH (Anakwe and Moger, 1986). Likewise, the in vitro

study done by Meyerhofer et al, on isolated Leydig cells of Siberian Hamster have

shown that nor epinephrine is one of the most potent stimulus to stimulate

testosterone production in vitro (Mayerhofer et al., 1993).

As mentioned before, that phosphodiesterase inhibitor IBMX increases the

availability of cAMP, therefore we exposed the purified Leydig cells with nor

144

epinephrine in the presence of IBMX. Addition of nor epinephrine with IBMX,

significantly stimulated the testosterone production in Leydig cells. However nor

epinephrine failed to produce an upsurge in IBMX stimulated testosterone

production in the presence of LH in our study. It shows that presence of LH

decreased responsiveness of Leydig cells to nor epinephrine. One reason may be

this, that the Leydig cells may respond after delay to nor epinephrine in the

presence of IBMX and LH both and three hours culture was insufficient to produce

such effect. There could also be a possibility that enough stimulation and release of

cAMP was achieved by IBMX alone and adding of LH has given no additional

surge (Cooke et al., 1982). From this, it is suggested that nor epinephrine have

stimulated the cAMP pathway and presence of IBMX resulted in more potent

steroidogenesis at Leydig cell level.

On the other hand, there were insignificant effects produced by addition of

nor epinephrine to pregnenolone added wells, both in LH stimulated and non-

stimulated Leydig cells. Thus, we can suggest that nor epinephrine may be

affecting the enzymes like 17 α hydroxylase or C-17-20 lyase down in

steroidogenesis pathway. We are also uncertain about the effect of nor epinephrine

on 3 ß HSD activity. The measurement of levels of these enzymes after exposure of

Leydig cells to nor epinephrine can give further clue to the mechanism of action of

nor epinephrine.

However, investigators have worked on other possible mechanisms of nor

epinephrine which may involve the presence of alpha and beta receptors on Leydig

cells (Mayerhofer et al., 1992). That’s why; studies have shown that beta blockers

145

like atenolol and propranolol may decrease the testicular steroidogenesis (Khan et

al., 2004; Mayerhofer et al., 1992).

Another important point of consideration regarding actions of

corticosterone and nor epinephrine is that, in vivo both are increased after stress and

as a result the testosterone decreased. However, in vitro study revealed that

corticosterone decrease testicular steroidogenesis while nor epinephrine increase it.

The query is how do they interact? To solve this, we added both corticosterone and

nor epinephrine to our wells containing LH stimulated Leydig cells. The

combination of both, resulted in a significant fall of testosterone, similar to that

occurs in in vivo after acute stress.

The question arises that if nor epinephrine is increasing testicular

steroidogenesis in vitro, then why stress decreases the levels of testosterone in

males. One of our in vitro experiments data helps to resolve this problem, that

when we added both corticosterone and nor epinephrine, the effects of

corticosterone were predominant with a resultant fall in testosterone levels.

Another possible explanation is that in vivo, the rise of nor epinephrine can

induce vasoconstriction which may restrict the access of LH to testicular

interstitium to act on Leydig cell, with consequent fall in levels of testosterone in

blood (Ogilive and Rivier,1998; Turnbull and Rivier, 1997).

As LH is also not changed in acute stress, which means hypothalamo

pituitary gonadal axis is not involved in acute stress, therefore there is another

possibility that stress may cause release of chemical substances in circulation or

cause their local production in testis that directly decreases the testicular

146

steroidogenesis (Selvage et al., 2004). Also the investigators have worked out an

efferent neural pathway from hypothalamus to testis (Lee et al., 2002; Selvage and

Rivier, 2003). It is proposed that this pathway work independently from pituitary

axis (Turnbull and Rivier 1997). This pathway was mapped in a retrograde fashion

from testis via spinal segments T10 - L1, L5 - S1 to brain stem. Hypothalamic areas

may include para ventricular nucleus and lateral hypothalamus (Gerendai et al.,

2000).

Some researchers are also of the opinion that activation of this pathway

leads to inhibition of Leydig cell’s responsiveness to gonadotropins. Interestingly,

this pathway is activated by intra cerebroventricular injection of beta agonists like

nor epinephrine (Turnbull and Rivier, 1997; Ogilvie and Rivier, 1998). Therefore,

it is possible that stress and disturbed emotions via this efferent pathway could also

adversely affect the testicular steroidogenesis and the isolated increase of testicular

steroidogenesis by nor epinephrine (shown by in vitro studies) is largely nullified

by these mechanisms and net result of stress is decrease in testicular

steroidogenesis.

Another important point of consideration is that immobilization stress

protocol as followed by us, can lead to oxidative stress (Nadeem et al., 2006).

Oxidative stress may occur due to imbalance between prooxidants and

antioxidants. This imbalance may result from either a decrease in level of

antioxidants or an increased production of prooxidants (Halliwell and Whiteman,

2004). Increase in the production of reactive oxygen species and generation of free

radicals constitute the oxidative stress. When reactive oxygen species exceed body

natural antioxidant defense, and protective mechanisms become unable to prevent

147

stress induced damages, then damage to macro molecules such as DNA, proteins

and lipids occur (Bartsch and Nair, 2000).

The reactive oxygen species most commonly target polyunsaturated fatty

acids of cell membrane with resultant oxidation of lipids called lipid peroxidation

(Makker et al., 2009). The degradation of lipid peroxides of the biological

membranes may result in formation of a number of aldehydes such as

malondialdehyde (MDA), which may cause damage to cells by reacting with

proteins, lipids and nucleic acids (Sim et al., 2003).

Endogenous antioxidant systems that may act as free radical scavengers

may have important role to combat against stress induced lipid peroxidation. One

of important endogenous antioxidant defense system is an enzyme, superoxide

dismutase (SOD). The SOD helps body to combat with the deleterious effects of

reactive oxygen species (Jones et al., 1981). The levels of SOD may thus indicate

the activity of natural intrinsic anti-oxidant defense system of body, and

considering its importance, we estimated the levels of SOD in this study.

Exposure of rats to the restraint sessions is a time tested classic model to

induce stress (Shansky et al., 2006). In present study, the restraint stress to Sprague

Dawley rats for six hours in mesh wire restrainer led to the increase in levels of

MDA, which suggests that the stress protocol has resulted in per oxidative damage

with increased lipid peroxidation. In addition there was fall in antioxidant enzyme

activity as manifested by fall in SOD levels in group II thus, suggesting the balance

in favor of oxidative stress. As the testicular tissue is highly susceptible to stress

because testicular membranes are rich in polyunsaturated fatty acids (Chainy et al.,

148

1997). Similar findings were obtained by in vitro results of our study, where

addition of corticosterone and corticosterone plus nor epinephrine together has

resulted in rise of lipid peroxidation in terms of elevated MDA levels and fall in

antioxidant enzyme activity as is evidenced by reduced SOD levels.

Therefore, it can be said that the oxidative stress induced damage to testis at

cellular level could also be responsible for the decline in levels of testosterone in

our study.

As antioxidants obtained from dietary sources constitute an essential

component of human antioxidant defense system (Agarwal and Prabakaran, 2005).

Many epidemiologic and clinical trials have reported that supplementation with

antioxidant vitamins is associated with a reduction in the incidence of chronic

disease morbidity and mortality (Weber et al, 1996; Enstrom, 1997; Gey, 1998).

Therefore we investigated the effects of supplementation of two important, FDA

approved dietary antioxidants i.e., ascorbic acid and alpha tocopherol, both in vivo

and in vitro.

In our in vivo component of study, the supplementation of rats with ascorbic

acid 500 mg/L drinking water for one month was unable to prevent restraint stress

induced rise in corticosterone and decline in testosterone. Also there was

insignificant change in nor epinephrine levels. Release of LH was also unchanged

in ascorbic acid supplemented group. However, despite insignificant change in

MDA, SOD activity increased in ascorbic acid supplemented group. This shows

that antioxidant capability via increase in SOD is enhanced but stress induced lipid

peroxidation was not prevented by ascorbic acid administration.

149

High levels of ascorbic acid are thought as a defense line against oxidative

stress. Also increased testosterone levels are reported after ascorbic acid

supplementation at a dose of 500 mg/kg/day (Sonmez et al., 2005). Ascorbic acid

supplementation in high doses is shown to reduce oxidative stress and consequent

damage in arsenic induced stress in mice (Chang et al., 2007). Smokers are at

increased risk of oxidative stress, and various studies have shown that

supplementation with ascorbic acid, decreases stress with consequent reduced lipid

peroxidation as is evidenced by decline in MDA levels (Naidoo and Lux 1998;

Wen et al, 1997).

Our previous study has shown that ascorbic acid do not affect the basal

testosterone levels in rats (Lodhi et al., 2009). Although numerous studies point

towards antioxidant role of ascorbic acid, but some studies also point that ascorbic

acid may not ameliorate or prevent all stressors, like alloxan induced stress was

increased by ascorbic acid supplementation (Dillard et al., 1982).

In our study, there was insignificant difference in levels of MDA between

the stressed group and ascorbic acid alone supplementation group, exposed to same

stressor for same duration. This is in accordance with a study on male humans in

which they have suggested that ascorbic acid has no effect on MDA concentration

(Bayer-Eder et al., 2004). But the levels of SOD were increased in ascorbic acid

supplemented group, which suggested the increased antioxidant capability of

ascorbic acid. Therefore we cannot deny the antioxidant role of ascorbic acid,

contrary to the study done by Anderson et al. in which they had shown that

ascorbic acid increases the per oxidative damage (Anderson et al, 1994).

150

In our in vitro experimental model, addition of different doses of ascorbic acid to

the wells containing the cultured Leydig cells have shown no significant effect,

rather high doses adversely affected testosterone synthesis. However, the

corticosterone induced decline in testosterone synthesis was restored to normal by

addition of ascorbic acid. The addition of ascorbic acid also restored decline in

testosterone induced by combination of corticosterone and nor epinephrine.

Ascorbic acid decreased MDA insignificantly, while SOD activity was

increased significantly in the presence of LH. Therefore, based on these findings it

is suggested that in basal conditions, without exposure to stress hormones, ascorbic

acid increases antioxidant enzyme activity.

Ascorbic acid alone in our study had no favorable effect on MDA level in

our study wells containing LH and Leydig cells, but when combined with alpha

tocopherol, the antioxidant effect of alpha tocopherol got enhanced, which suggests

some synergism in their actions. This can be explained on the basis of available

literature which supports that ascorbic acid increases the availability of alpha

tocopherol to the body. For example, when ascorbic acid is added to cultured

hepatocytes, it prevented the loss of alpha tocopherol and thus increased its

availability (Halpner et al., 1998).

Among stress hormones, corticosterone alone and in combination with nor

epinephrine caused a significant rise in MDA and fall in SOD. But, nor epinephrine

has caused no significant changes in MDA and SOD, based on which we can infer

that norepinephrine is not causing lipid peroxidation. The derangements in levels of

151

MDA and SOD were not seen in those corticosterone containing wells in which we

added ascorbic acid.

As reviewed by Padayatty et al., many studies suggest that ascorbic acid

has failed to prove prevention against oxidative stress in humans, but it has shown

antioxidant capability in the in vitro experiments (Padayatty et al., 2003). Increase

in plasma concentration of ascorbic acid increases its concentration in adrenal

gland and this local increase in ascorbic acid has been assigned an interesting role

of quenching the oxidants which are produced during steroidogenesis (Rapoport et

al., 1995). Therefore, based on our in vitro experiments, it is suggested that

ascorbic acid can act as an efficient antioxidant at Leydig cell level also.

However, the results of in vivo component of our study are not favoring the

international studies in which ascorbic acid is proved as an antioxidant. Ascorbic

acid is thought to prevent the oxidative stress induced derangements, like study by

Ergul et al., which narrates that ascorbic acid may reduce the oxidative damage to

liver caused by anti tuberculous drug isoniazid (Ergul et al 2010). This difference

could be due to dose difference, because Ergul and colleagues used a dose of 100

mg/kg/day via oral route but we used ascorbic acid in a dose of 500mg/l drinking

water.

Another study done by Sengupta et al., reveals that ascorbic acid restored

the cadmium induced decline in testosterone levels. However, they used ascorbic

acid via subcutaneous route in a dose of 400 mg/100 g BW for 48 h period (200 mg

at 24 h before and 200 mg at 24 h after cadmium injection). This difference of

152

route and dose of administration of ascorbic acid in our case could be responsible

for inefficiency of ascorbic acid to reduce stress effects (Sengupta et al., 2004).

This is supported further by another study of Peters et al., which have given

two different doses of ascorbic acid, 500 mg/day and 1500 mg/day to marathon

runners before race and estimated the levels of stress hormones, cortisol and

adrenaline immediately after the race. It was observed that ascorbic acid at 1500

mg/day dose significantly reduced the both cortisol and adrenaline, while 500

mg/kg was ineffective. Based on this we can say that although ascorbic acid can act

as free radical scavenger, but very high doses are required to have antioxidant

effect. (Peters et al., 2001)

High doses are recommended because high doses are not toxic and it is well

known fact, discovered long ago that the excessive ascorbic acid is rapidly

eliminated in urine within 24 hours (Sneer et al., 1968). Also a comprehensive

review has strongly suggested that ascorbic acid RDA should be increased for more

beneficial effects (Carr and Frei, 1999). Therefore, based on our findings, it is

suggested that ascorbic acid at high doses can act as antioxidant to prevent

oxidative stress induced infertility.

Supplementation of the rats of group IV with alpha tocopherol for one

month before stress exposure, significantly prevented restraint stress induced rise

of corticosterone and fall in testosterone. However LH and nor epinephrine

remained unchanged. There was also positive effect on oxidative stress with

reduced lipid peroxidation and consequent fall in MDA and rise of antioxidant

capability in terms of rise of SOD activity.

153

Alpha tocopherol is an important component of anti-oxidant defense system

of our body and protects the tissues against oxidative damage induced by reactive

oxygen species (Naziroglu et al., 2003). Our findings are indicative that oxidative

stress induced by restraint has resulted in decline in testosterone, which was

significantly protected by alpha tocopherol and oxidative stress effects were also

reduced as is evidenced by values of MDA and SOD. However, alpha tocopherol

decreased only corticosterone, while it has no effect on nor epinephrine levels.

Our results to assess antioxidant preventive role of alpha tocopherol are

supported by various international studies which have confirmed the anti-oxidant

role of alpha tocopherol. For example, administration of alpha tocopherol protects

the rats against various stressors like homocystine induced oxidative stress

(Sonmez et al., 2005), Cadmium induced toxicity (Ognjanovic, et al., 2003),

nicotine induced damaging effects (Hijazi et al., 2002) and ethane dimethane

sulfonate induced testicular toxicity (Sahinturk, et al., 2007).

Similarly some recent studies also point towards this antioxidant role of

alpha tocopherol. A study by Hong et al., has suggested that supplementation of the

diets of goat with alpha tocopherol significantly increased SOD activity while

MDA levels were decreased in testicular tissue. Therefore it is suggested that alpha

tocopherol protect their testis from peroxidative damage (Hong et al., 2010). In

another study, intra peritoneal administration of alpha tocopherol in a dose of 50

mg/kg/day to streptozotacin induced diabetic rats significantly lowered the MDA

and increased SOD activity in the nervous tissue of rats (Kabay et al., 2009).

154

Alpha tocopherol on cultured Leydig cells in the presence of LH stimulated

the testosterone synthesis in a dose of 10 µg/ml. However, high doses lead to

suppression at Leydig cell level. This is supported by research work of Mather et

al., which claims that alpha tocopherol has stimulant effect on testosterone

synthesis by Leydig cells (Mather et al., 1983) Some authors believe that alpha

tocopherol may decrease the plasma testosterone levels (Hartman et al., 2001) but

in our view, in this study, Hartman et al have given alpha tocopherol to older men

in which testosterone is already lower than normal.

In the cultured Leydig cells, alpha tocopherol decreased MDA

insignificantly, while SOD activity was increased significantly in the presence of

LH. Therefore, based on these findings, it is suggested that in basal conditions,

without exposure to stress hormones, alpha tocopherol increases antioxidant

enzyme activity. However, alpha tocopherol prevented decline in testosterone

synthesis in the presence of corticosterone and there was fall in MDA and rise in

SOD activity, when we added alpha tocopherol to corticosterone and NE added

wells.

Alpha tocopherol is mandatory to combat against stress. It was verified by

experiments of Burczynski et al, in which they given the rats a diet deficient in

alpha tocopherol and rat adrenal mitochondria showed increased lipid peroxidation

after exposure to stress (Burczynski et al., 1999). Our in vitro results are also

pointing the preventive roles of alpha tocopherol against corticosterone induced

derangements. It is also well supported by literature that alpha tocopherol can

reduce lipid peroxidation in cultured Leydig cells and may increase testosterone

synthesis (Chen et al., 2005).

155

The effectiveness of alpha tocopherol as an anti-oxidant to prevent the

testicular tissues against various diseases is well supported by literature (Sahinturk

et al., 2007). If we see effects of ascorbic acid and alpha tocopherol, we can see

that alpha tocopherol exerted more potent effect in reducing lipid peroxidation and

our results are in agreement with research outcomes of Massaeli et al., who also

narrated that alpha tocopherol is more potent in this regard (Massaeli et al., 1999).

Therefore, based on our in vivo and in vitro findings, we hereby suggest that alpha

tocopherol is very effective anti-oxidant against restraint stress induced

derangements in testicular steroidogenesis.

Supplementation of our experimental animals with a combination of

ascorbic acid and alpha tocopherol for one month before stress exposure,

significantly prevented restraint stress induced rise of corticosterone and fall in

testosterone as shown in table 3.4. However LH and nor epinephrine remained

unchanged in this group too. Oxidative stress was also reduced as narrated by

reduction in lipid peroxidation and consequent drop in MDA and escalation of

antioxidant capability as expressed by rise of SOD activity.

These findings are supported by various international studies.

Krishnamoorthy et al., have reported that Polychlorinated biphenyls - PCB

(Aroclor 1254) which is an endocrine disruptor present in paints, oils etc., has

damaging effects on reproductive parameters of rats. The sperm count and motility

decreased with evident emblems of oxidative stress in terms of raised MDA and

fall in SOD activity. However, supplementation with ascorbic acid and alpha

tocopherol combination effectively restored the fertility parameters as well as stress

markers to their normal physiological range. Therefore, they narrated these

156

antioxidants as excellent tools to normalize both the enzymic and non-enzymic

antioxidant activity (Krishnamoorthy et al., 2007). The preventive role of ascorbic

acid and alpha tocopherol supplementation against various types of stressors is well

documented in literature (Akturk, et al., 2006; Judge et al., 2007).

As the steroid levels were decreased in our study and testosterone was

significantly increased in this combination supplemented group. Similar research

outcomes are narrated in the study of Schroder et al., in which they have seen that

in athletes, combination of antioxidants results in statistically significant increase in

testosterone and a fall in glucocorticoid activity seen (Schroder et al., 2001). In

ascorbic acid and alpha tocopherol supplemented group, serum MDA levels were

decreased. These results are similar to the study of Guney et al., in which rats were

exposed to fluoride toxicity and supplementation with ascorbic acid and alpha

tocopherol reduced the levels of MDA (Guney et al., 2007b). Therefore ascorbic

acid and alpha tocopherol combination is effective in ameliorating the oxidative

stress, manifested by the decline in MDA levels and increase in SOD activity.

An important point of consideration evident from our study data is that, rise

of testosterone and fall of corticosterone is in almost same significance range in

alpha tocopherol supplemented group, but considering the values of MDA and

SOD, we can see that there is more significant fall in MDA and rise in SOD in the

group of rats supplemented with a combination of ascorbic acid and alpha

tocopherol as compared to alpha tocopherol alone. Therefore, it is suggested that,

combination of antioxidants ascorbic acid and alpha tocopherol has synergistic

effect in reducing oxidative stress.

157

Although some authors are of the opinion that antioxidants combination

have no added advantage like that of study by Huang et al., in which they have

given antioxidants in combination to humans and their results revealed no added

advantage of combination than either alone (Huang et al., 2002). Contrary to this,

many studies are favoring our findings. When ascorbic acid was added to cultured

hepatocytes, it prevented the loss of alpha tocopherol and thus increased its

availability (Halpner, et al., 1998). Another study shows that ascorbic acid

supplementation lead to enhanced levels of alpha tocopherol (Eichenberger et al.,

2004). Also, the documented evidence is there, that alpha tocopherol increases

tissue ascorbic acid concentration (Sztanke et al., 2004). Therefore this could be

the reason of our results that both antioxidants in combination affected more

potently and led to an increase in testosterone with more marked reduction in lipid

peroxidation as suggested by decreased MDA levels.

Our in vitro findings are confirming our in vivo results, that combination of

ascorbic acid and alpha tocopherol in the presence of stress hormones not only

prevented fall in testosterone, but also has positive effect on oxidative balance.

Therefore, the combination of antioxidants is effective in the presence of stress

hormones in decreasing lipid peroxidation in terms of fall in MDA and an

increased anti-oxidant i.e., SOD activity.

Our in vitro results are endorsed by various research works. A study on

cultured erythrocytes by Durak et al., has revealed that an organophosphate

pesticide Malathion induce oxidative stress in erythrocytes and combination of

ascorbic acid and alpha tocopherol prevented the malathion induced oxidative

stress (Durak et al., 2009).

158

Similarly a study by Murugesan et al., in which they have noted the harmful

effects of an important environmental contaminant i.e., polychlorinated biphenyl

called Aroclor 1254, on Leydig cell steroidogenesis. They performed experiments

in vivo on rats and in vitro on cultured Leydig cells of rat. Aroclor caused oxidative

stress and contrary to our studies, the levels of LH were also reduced by Aroclor.

The reason is that, oxidative stress exposure was for thirty days, leading to chronic

stress and chronic stress may affect the hypothalamo pituitary gonadal axis as

discussed before. The results of this study have substantiated that co-

administration of ascorbic acid and alpha tocopherol prevented the decrement in

testosterone levels induced by Aroclor 1254 (Murugesan et al., 2007). Therefore,

based on the results of this group and in vitro component where we added both

antioxidants, it is proposed that the combination of antioxidants (ascorbic acid &

alpha tocopherol) is useful in preventing the stress induced decline in testicular

steroidogenesis as well as the derangements in MDA/SOD balance and thus confer

protection against the oxidative stress.

As mentioned before, exposure of our experimental animals to restraint

stress for six hours caused increased lipid peroxidation as is evidenced by rise in

MDA and there was also fall in SOD activity. If there is increase in MDA, it is an

evidence of enhanced lipid peroxidation (Lata et al., 2004). Increase in lipid

peroxidation may result in rise of glucocorticoid activity as a result of stimulation

of stress axis (Gupta et al., 2004). Similarly a fall in antioxidant enzyme activity

showing decline in SOD also points towards oxidative stress (Kleczkowski et al.,

2003). Therefore, we can propose that our experimental protocol induced the

oxidative stress, as a result of which there was decline in testicular steroidogenesis.

159

The oxidative stress has multi-dimensional damaging effects on body.

Oxidative stress involves generation of free radicals, which are atoms and

molecules with an unpaired electron. These free radicals have the capability to

damage molecules that are important for functions of the cell, resulting in impaired

functions. Superoxide radicals are formed during reduction of oxygen and site of

formation is inner mitochondrial membrane. The can initiate the chain reaction in

fatty acids of phospho lipids which may lead to lipid peroxidation in membrane

(Evans, 2000).

The peroxidation of membrane lipids can be very detrimental because it is

self propagating chain reaction and oxidation of few molecules of lipids may result

in significant damages. This lipid peroxidation may modify the biological

properties of membranes, like the degree of fluidity of membrane. It can also lead

to inactivation of various receptors and enzymes which may lead to increased tissue

permeability and normal cellular functions are blighted (Dalle-Donne et al., 2006;

Zaidi et al., 2003). Such damage by oxidative stress is due to production of reactive

oxygen species which may damage the macromolecules including DNA (Guney et

al., 2007a).

Free radicals which are generated in response to stress may also lead to an

increase in protein oxidation (Dohm and Louis, 1978). Various reactive oxygen

species may include superoxide anions, hydroxyl radicals, and hydrogen peroxide

etc. These reactive oxygen species released during oxidative stress are recognized

as damaging agents for various components of steriodogenic pathways including

steriodogenic enzymes (Murugesan et al., 2005).

160

At cell level, various mechanisms are operating to decrease the damage or

neutralize the effects of oxidative stress. Although these intrinsic mechanisms are

normally operating in body to respond to stressor in order to neutralize the adverse

effects of stress, but restraint stress is capable of generating severe oxidative stress

which may disrupt the defense mechanism and reactive oxygen species are

liberated (Zaidi et al., 2003). Also, there is documented evidence that stress of

various origins may cause depletion of antioxidant sources of body (Sconberg et

al., 1993). Our results have also shown similar trend with a significant fall in SOD

activity.

One of the antioxidant which we investigated was ascorbic acid which has

the ability to react with various reactive oxygen species like superoxide,

hydroperoxyl radicals, aqueous peroxyl radicals, nitrogen peroxide, nitroxide

radicals and hypochlorous acid (Buettner, 1993). Ascorbic acid is hydrophilic and

is a very important free-radical scavenger in extracellular fluids. It traps the

radicals in the aqueous phase and thereby confers protection of bio membranes

from per oxidative damage (Harapanhalli et al., 1996). Ascorbic acid is thought to

interact with plasma membrane too by donating electrons to alpha tocopheroxyl

radical and a trans plasma membrane oxidoreductase activity (May, 1999) as

reviewed by Padayatty et al., ascorbic acid by donating electron is a reducing agent

and its antioxidant actions are also dependent on this property, because by donating

electrons it prevents oxidation of other compounds. Reactive and harmful free

radicals react with ascorbic acid and form less reactive ascorbyl radical which is

then reduced to dehydroascorbic acid which may be hydrolyzed or reduced back to

161

ascorbic acid. Reduction of reactive free radicals with less reactive radicals is

called free radical scavenging property of ascorbic acid (Padayatty et al., 2003).

The other antioxidant of our study alpha tocopherol is considered as a major

chain breaking anti-oxidant and as first line of defense against oxidative stress. It is

also recognized as a potent scavenger of free radicals like superoxide and per

oxynitrite. One of its core functions is to scavenge lipid peroxyl radicals by virtue

of which, it protects the polyunsaturated fatty acids (PUFAs) from oxidation. If

sufficient quantity of alpha tocopherol is accessible at site of oxidative stress where

there is free radical generation, it would neutralize the deleterious effects of ROS

(John et al., 2001). Alpha tocopherol supplementation provides more than normal

levels of alpha tocopherol in blood, and thus reacts with membrane phospholipids

bilayers, thereby breaking the chain reaction initiated by ROS. By these actions,

alpha tocopherol could have protected Leydig cell membrane structures against

oxidative injury (Therond et al., 1996). The improvement of antioxidant enzyme

activities could be the result of the free radical scavenging effects of alpha

tocopherol (Gurel et al., 2005).

Both antioxidants are important to combat against stressors of various

origins. For example, when alpha tocopherol deficient diet was given to guinea

pigs that were ascorbic acid deficient, death occurred in few days (Hill et al.,

2003). Documentary evidence is there that simultaneous administration of ascorbic

acid and alpha tocopherol maintains the expression of steriodogenic enzymes in

Leydig cells (Murugesan et al., 2007). Along with its antioxidant effects, ascorbic

acid is also involved in the regeneration of tocopherol from tocopheroxyl radicals

in the membrane. Therefore, ascorbic acid and alpha tocopherol can have

162

interactive but protective effects against generation of reactive oxygen species and

lipid peroxidation (Stoyanovsky et al., 1995). The antioxidants supplementation

may also protect the protein structures (Schroder et al., 2001) receptors and

enzymes which are then not allowed to be reactivated by ROS, and there is

stabilization the cell membranes.

Due to these abilities of antioxidants, Leydig cells were protected from

detrimental effects of stress with consequent protection against derangements in

testicular steroidogenesis induced by oxidative stress. Therefore, it is concluded

that the antioxidants ascorbic acid and alpha tocopherol confer protection at Leydig

cell level against restraint induced oxidative stress.

163

Limitations of our study

1 Due to non-availability of male Sprague Dawley rats at National Institute of

health, we had to perform in vivo component of project with 16 rats in each

group instead of 30 rats per group.

2 We have investigated effects of stress hormones in short term culture only.

It is due to the reason that the cell culture technique was first time established

by us in our institution and we had to rely on 3 hours incubation, because

facilities for long term incubation of cultured Leydig cells are not yet

established.

3 We had also a plan to see the effects of androstenedione while investigating

the mechanism of action of corticosterone and nor epinephrine on cultured

Leydig cells, but unfortunately we could not import it and it was not available

locally.

164

Conclusion

The data of our study led us to conclude that:

1. Acute stress suppresses steroidogenesis at Leydig cell level.

2. Corticosterone in acute stress decreases the Leydig cell steroidogenesis by

inhibiting cAMP as well as enzymatic pathways whereas nor epinephrine

increases the testosterone synthesis at Leydig cell level by stimulating

cAMP or the enzymes involved in testosterone synthesis.

3. Ascorbic acid at low dose is not effective as antioxidant, but alpha

tocopherol is an effective antioxidant against restraint stress induced

derangements in testicular steroidogenesis.

4. Combinations of antioxidants, ascorbic acid and alpha tocopherol prevents

the stress induced fall in testicular steroidogenesis and have synergistic

effect in reducing oxidative stress by decreasing lipid peroxidation and

increasing the antioxidant enzyme activity.

Chapter 5

165

Future directions

1. Mechanism of action of nor epinephrine needs further clarification. The

measurement of levels of cAMP in the presence of nor epinephrine in

cultured Leydig cells or working on other intra cellular pathways with the

help of blockers may further reveal the insights into the mechanism of

action of nor epinephrine.

2. Stress response in rats fed on antioxidants deficient diet needs to be

elucidated which will further explain the definite role of these antioxidants

to combat the stress induced derangements.

3. The possible role of genetic basis (especially DNA) in the stress response

needs further elucidation.

166

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ANNEXURE-A

Composition of Pelleted Diet for Rats

INGREDIENTS Weight

1. Wheat Flour 2.85 Kg

2. Wheat Brawn 2.85 Kg

3. Dried skimmed milk Powder 2.00 Kg

4. Soyebean Oil 0.50 Liter

5. Mollasen 0.15 Kg

6. Fish meat 1.50 Kg

7. Salt (common). 0.05 Kg

8. Vitamins/ Minerals/amino acids (Premix)*. 0.10 Kg

________________________________________________________

Total weight. 10.00 Kg

This diet is prepared at National Institute of Health (N.I.H), Islamabad according to

the standard approved by the Universities Federation for Animals Welfare.

* = Composition of premix is given in the next appendix-2

200

ANNEXURE-B

Composition of Vitamins/ Minerals/amino acids (Premix)

Mixed in the Diet, for Rats.

INGREDIENTS Weight

1. Vitamin A 10,000 IU/Kg*

2. Vitamin D 5,000 IU/Kg

3. Vitamin E 50 mg/Kg

4. Choline 800 mg/Kg

5. Methionine 500 mg/Kg

6. Sodium chloride 5 g/Kg

7. Dibasic Calcium Phosphate 9.5 g/Kg

8. Zinc Sulphate 24 mg/Kg

9. Potassium Iodide 3 mg/Kg

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Total weight = amount of the premix added in 10 kg of the Pelleted diet prepared.

* = amount added/ kg of the diet prepared.