2008 Kyung Hee University Press 67

Oriental Pharmacy and Experimental Medicine 2008 8(1), 67-72

www.opem.org

OPEM

Morphine induced inhibition of the activities of accessory reproductive ducts

in male rats

Ramesh L Londonkar1, Sharangouda

2 and Saraswati B Patil

2,*

1Department of Biotechnology, Gulbarga University, Gulbarga-585106, Karnataka, India;

2Department of

Zoology, Gulbarga University, Gulbarga-585106, Karnataka, India

SUMMARY

Adult male albino rats were treated with 0.5 mg and 0.75 mg morphine/100 g body weightintraperitoneally for 30 days. All the animals were autopsied on 31st day. Epididymis and vasdeferens were dissected out, weighed and processed for histological and biochemical studies.Morphine has caused a reduction in the weight of epididymis and vas deferens in both the dosesof drug treated groups. The total cholesterol content is increased while protein, DNA and RNAcontents and epididymal sperm counts are decreased. The acid phosphatase content is decreased10.12 ± 0.11 in caput, 9.26 ± 0.30 in cauda of epididymis and in vas deferens 8.14 ± 0.15 in 0.5 mgtreated groups and in 0.75 mg treated rats shows 9.52 ± 0.27 in caput, 9.14 ± 0.18 in cauda ofepididymis and in vas deferens 7.84 ± 0.11is decreased, whereas alkaline phosphatase isincreased. The surface epithelial cell height of these ducts is reduced and secretory activity isinhibited with the disruption of epithelial cell projections. The gravimetric and histometricchanges of epididymis and vas deferens may be due to non-availability of androgens in morphinetreated rats.

Key words: Morphine; Epididymis; Vas deferens; Rat

INTRODUCTION

Morphine, a narcotic analgesic opiate has been

discovered by Sertuner in 1806. It binds to opioid

receptors in Central Nervous System (CNS) and

exerts its chief pharmacological actions on the

central nervous system (Russell et al., 1989). Morphine

administered to female rats during parturition

inhibits the uterine contractibility, disturbs the

normal maternal behavior in lactating rats (Russell

and Spears, 1984). Like other CNS influencing

drugs morphine is also known to inhibit the release

of follicular stimulating hormone (FSH) and luteinizing

hormone (LH) from the pituitary, acting through

hypothalamus blocking the neural stimulus to the

gonadotrophin releasing hormone (Blake, 1974;

Blake, 1978; Anderson et al., 1982). According to Jaffe

and Martin (1985), opioids act on the hypothalamus

and inhibit the release of gonadotrophin releasing

hormone (GnRH) and corticotrophin releasing factor

(CRF), thus decreasing the circulating concentrations

of LH, FSH, adrenocorticotrophic hormone (ACTH)

and β-endorphin.

Epididymis is an important site at which the

spermatozoa undergo progressive morphological

and physiological changes. It provides a favourable

milieu for acquiring the motility, fertilizing ability,

storage and survival of spermatozoa (Kasturi et al.,

1995). The vas deferens in addition to its role in

*Correspondence: Saraswati B Patil, Department ofZoology, Gulbarga University, Gulbarga-585106,Karnataka, India. E-mail: sharanjpatil@gmail.com

DOI 10.3742/OPEM.2008.8.1.067

2008 Oriental Pharmacy and Experimental Medicine 8(1), 67-72

68 Ramesh L Londonkar et al.

transport of spermatozoa is also involved in

maturation and survival of spermatozoa (Orgebin-

Crist, 1969; Orgebin-Crist and Danzo, 1975).

Therefore, the epididymis and vas deferens

instead of remaining mere passage ducts have

an important role in male reproduction and

fertility. Any drug influencing the CNS activities

can also modify the function of gonads and in

turn accessory organs. As the development and

function of epididymis and vas deferens

depends directly on gonadal activities, it is of

interest to study the effect of morphine on the

accessory reproductive organs.

MATERIALS AND METHODS

Healthy male albino rats of Wistar strain from

inbred colony, weighing 180 - 200 g, of 80 - 90 days

old were maintained at room temperature of 20 ±

28oC with lighting schedule of 12 h light and 12 h

darkness. They were maintained in individual

cages and divided in groups each containing six

animals and fed with balanced diet as described by

CFTRI (Central Food and Technological Research

Institute) Mysore, Karnataka, India and water ad

libitum.

The animals were divided into following groups

Group-I: Received 0.2 ml saline/100 g body weight

i.p. for 30 days and served as control.

Group-II: Received 0.5 mg morphine/100 g body

weight i.p. for 30 days in 0.2 ml saline.

Group-III: Received 0.75 mg morphine/100g body

weight i.p. for 30 days in 0.2 ml saline.

All the animals were sacrificed by cervical dislocation

after 24 h of the last injection. The epididymis and

vas deferens were dissected out immediately and

separated from adherent tissue, weighed up to the

nearest mg on electronic balance. Organs from one

side of each animal were fixed in Bouin’s fluid for

histological studies. They were embedded in paraffin,

sectioned at 5 µ, stained with Ehrlich hematoxylin

and Eosin. The micrometric measurements like

diameter of epididymis, its epithelial cell height

and lumen diameter of vas deferens were made

from randomly chosen 20 sections appearing

round at cross sections from each group using

ocular and stage micrometers. The sperm count

from cauda epididymis was done by using

haemocytometer (Kempinas and Lamano- Carvalho,

1987). Organs from other side were used for

biochemical estimations.

The total cholesterol (Peters and Vanslyke, 1946),

protein (Lowry et al., 1951), nucleic acids (Glick,

1985) and phosphatases (Toussaky and Shorr, 1953)

of epididymis and vas deferens were estimated

and “Student’s t-test” was employed for statistical

analysis.

RESULTS

Changes in the epididymis

Gravimetric changes (Table 1)

The weight of caput and cauda epididymis is

decreased significantly (P < 0.05) with 0.5 mg of

morphine treatment and highly significantly (P <

0.001) with 0.75 mg of morphine treatment when

Table 1. Effect of morphine on gravimetric and histometric changes of epididymis

GroupsWeight of epididymis

(mg/100 g body weight)Diameter (µm) Epithelial cell height (µm) Caudal

sperm count(million/ml)Caput Cauda Caput Cauda Caput Cauda

Control 395.84 ± 2.05 289.68 ± 2.12 2,645.24 ± 25.50 2324.45 ± 22.26 31.31 ± 1.40 32.45 ± 0.79 58.25 ± 4.34

0.5 mg 380.27 ± 3.01 277.52 ± 2.36 2,486.35 ± 22.54 2,238.81 ± 25.320 26.20 ± 21*0 29.83 ± 0.56 49.43 ± 5.00

0.75 mg 352.40 ± 6.10 263.45 ± 3.20 2,291.73 ± 29.80 2,214.27 ± 18.090 23.15 ± 1.38 27.75 ± 0.50* 46.72 ± 3.25**

Dose: 0.5 mg and 0.75 mg/100 g body weight. Duration: 30 days. Minimum 6 animals were maintained in eachgroup. Each datum represents the mean ± S.E. *P < 0.05, **P < 0.01.

Morphine induced inhibition of the activities of accessory reproductive ducts in male rats 69

2008 Oriental Pharmacy and Experimental Medicine 8(1), 67-72

compared to saline treated controls.

Biochemical changes (Table 3)

Protein

There is significant (P < 0.005) decrease in the protein

content of caput and cauda epididymis due to

0.5 mg morphine treatment, but it is highly

significant (P < 0.001) due to 0.75 mg morphine

treatment.

Cholesterol

The total Cholesterol content of caput and cauda

epididymis is increased significantly (P < 0.05) with

0.5 mg morphine treatment and highly significantly

(P < 0.01) with 0.75 mg morphine treatment when

compared to control group.

Nucleic acids

The DNA and RNA content of caput and cauda

epididymis is reduced almost significantly (P <

0.005 with 0.05) with 0.5 mg morphine treatment

and highly significantly (P < 0.001) with 0.75 mg

morphine administration.

Phosphatases

The acid phosphatase is decreased significantly

(P < 0.05) in caput and non-significantly in cauda

epididymis due to 0.5 mg morphine administration.

But highly significant reduction (P < 0.01) in

caput and significant (P < 0.005) reduction in

cauda epididymis is observed with 0.75 mg

morphine treatment. There is a significant increase

(P < 0.05) in alkaline phosphatase activity of caput

and non-significant increase in cauda epididymis

with 0.5 mg morphine treatment. Highly significant

(P < 0.01) increase in both caput and cauda

epididymis is observed after the administration of

0.75 mg morphine.

Histometric changes (Table 1)

The epithelial cells of saline treated control group

are tall, healthy, columnar and filled with secretary

material. But those of morphine treated cells are

smaller, disrupted and are with less secretary

material. The diameter of caput epididymis is

reduced almost significantly (P < 0.05) with 0.75 mg

morphine treatment.

Sperm morphology and number

The cauda epididymal sperms of normal rat shows

sickle shaped head and straight tailpiece. But in

morphine treated rats the sperms are abnormal as

their head region reduced and the tail is wrinkled

or coiled. A significant reduction (P < 0.05) in the

sperm count of cauda epididymis with 0.5 mg

morphine and highly significant (P < 0.01) reduction

with 0.75 mg morphine is observed.

Changes in vas deferens

Gravimetric changes (Table 2)

A non-significant decrease with 0.5 mg morphine

and significant decrease (P < 0.01) with 0.75 mg

morphine is observed in the weight of vas deferens.

Biochemical changes (Table 3)

Protein

There is non-significant decrease in the protein

content of vas deferens due to 0.5 mg morphine

treatment is observed but it is almost significant

(P < 0.05) due to 0.75 mg of morphine treatment.

Table 2. Effect of morphine on gravimetric and histometric changes of vas deferens

GroupsWeight of Vas Deferens(mg/100 g body weight)

Diameter (µm)Epithelial cell height

(µm)Diameter of lumen

(µm)

Control 83.19 ± 1.8 278.00 ± 22.13 28.00 ± 1.28 225.32 ± 2.61

0.5 mg 076.85 ± 1.99 251.62 ± 26.00 27.16 ± 1.24 236.20 ± 4.25

0.75 mg 0052.31 ± 1.60**0200.28 ± 15.10**

021.38 ± 1.05** 243.21 ± 3.55**

Dose: 0.5 mg and 0.75 mg/100 g body weight. Duration: 30 days. Minimum 6 animals were maintained in eachgroup. Each datum represents the mean ± S.E. *P < 0.05, **P < 0.01.

2008 Oriental Pharmacy and Experimental Medicine 8(1), 67-72

70 Ramesh L Londonkar et al.

Cholesterol

Though the total cholesterol content of vas

deferens is increased with both the doses, it is

significant (P < 0.01) only with 0.75 mg morphine

treatment.

Nucleic acids

The DNA and RNA content of vas deferens is

reduced in both the treated groups, but it is

significant (P < 0.05) only with 0.75 mg morphine

treatment.

Phosphatases

Acid phosphatase activity of vas deferens is

significantly (P < 0.01) reduced but the alkaline

phosphatase activity is significantly increased (P <

0.01) with 0.75 mg morphine treatment. Similar

changes are observed with 0.5 mg morphine

administration.

Histometric changes (Table 2)

Morphine administration has caused the reduction

in the diameter of vas deferens and its epithelial

cell eight, thereby increasing the lumen diameter.

Table 3. Biochemical changes in epididymis and vas deferens

Contents GroupsEpididymis

Vas deferensCaput Cauda

Protein (mg/100 mg)

Control 016.46 ± 0.22 015.80 ± 0.17 14.86 ± 0.12

0.5 mg 0 15.78 ± 0.16*013.82 ± 0.21* 13.60 ± 0.15*

0.75 mg 0 15.23 ± 0.09**0 12.56 ± 0.21** 12.82 ± 0.60*

Cholesterol (µg/100 mg)

Control 146.45 ± 2.21 100.05 ± 2.12 78.64 ± 2.21

0.5 mg 158.89 ± 3.13 0 124.56 ± 2.64** 82.51 ± 3.30

0.75 mg 178.58 ± 5.11*0 149.11 ± 20.20** 89.26 ± 1.00**

DNA (µg/100 mg)

Control 186.10 ± 2.91 162.30 ± 3.21 153.61 ± 3.60

0.5 mg 173.61 ± 4.00* 150.00 ± 3.11* 148.36 ± 8.42

0.75 mg 165.00 ± 2.21** 146.22 ± 3.20** 140.00 ± 3.82*

RNA (µg/100 mg)

Control 868.30 ± 7.52 791.31 ± 5.42 772.43 ± 4.01

0.5 mg 0835.01 ± 9.50* 758.60 ± 4.23* 759.62 ± 4.27

0.75 mg 0 824.10 ± 4.72** 735.02 ± 2.31** 753.32 ± 3.50*

Acid Phosphatase (µg/100 g)

Control 011.48 ± 0.43 10.42 ± 0.39 0 8.60 ± 0.13

0.5 mg 010.12 ± 0.11 9.26 ± 0.30 8.14 ± 0.15

0.75 mg 0 9.52 ± 0.27 9.14 ± 0.18* 7.84 ± 0.11**

Alkaline Phosphatase (µg/100 g)

Control 0 5.75 ± 0.10 5.33 ± 0.09 4.23 ± 0.19

0.5 mg 0 6.26 ± 0.12* 5.67 ± 0.14 4.51 ± 0.28

0.75 mg 0 6.64 ± 0.09** 6.21 ± 0.16** 4.96 ± 0.74*

Dose: 0.5 mg and 0.75 mg/100 g body weight. Duration: 30 days. Minimum 6 animals were maintained in eachgroup. Each datum represents the mean ± S.E. *P < 0.05, **P < 0.01.

Fig. 1. Cross section of the cauda epididymis ofnormal adult rat showing tall healthy and secretaryepithelial cells. The surface villi are projectingtowards the lumen. The nuclei of the cells givepseudostratified appearance and spermatozoa arespermatozoa are clearly seen in the lumen. H & EStaining (400×). AC, Apical Cell; BC, Basal Cell; HC,Halo Cell; L, Lumen; N, Nucleus; PC, Principal Cell;SP, Spermatozoa; V, Villi.

Morphine induced inhibition of the activities of accessory reproductive ducts in male rats 71

2008 Oriental Pharmacy and Experimental Medicine 8(1), 67-72

These changes are significant only with 0.75 mg

morphine administration.

DISCUSSION

The male accessory reproductive organs play an

important role in the sperm maturation, motility

and formation of semen (Orgebin-Crist, 1969;

Hamilton, 1975; Martin and Sloan 1977). Spermatozoas

formed in the seminiferous tubules are transported

from the testis into epididymis and remain in the

duct system for varying periods of time before

being ejaculated (Gaddum and Glover, 1965).

During this period they acquire motility and

fertilizing capacity in the epididymis (Gaddum

and Glover, 1965).

Opioids by acting through the hypothalamus

inhibit the release of GnRH and CRF, thus decreasing

the circulating concentrations of LH, FSH and

ACTH (Reisine and Pasternak, 1996). The epididymis

and the accessory glands of reproduction depend

on testicular androgens (Price and William Ashman,

1961). Testosterone being an important factor for

the maintenance of accessory sex organs (Ojeda

and Urbanski, 1994). In turn the synthesis and

release of androgens depends on the availability of

pituitary gonadotrophins like FSH and LH/ICHS

(Connel and Eik-Nes, 1868; Johnson and Ewing,

1971; Hanson et al., 1973).

Morphine being a µ-opioid receptor against

inhibits the release of pituitary gonadotrophin

(Barraclough and Sawyer, 1955; Bruni et al., 1977),

which is essential for the androgen synthesis.

Therefore, reduction in the weight of epididymis

and vas deferens is observed in the present study.

As the androgen regulate the synthesis of DNA,

RNA and protein (Brooks, 1981; Duggan and

North, 1983; Martin, 1984). The decrease in the

DNA, RNA and protein contents after morphine

treatment supports the reduced androgen productivity

by the testis. Significant increase in the cholesterol

content of these ducts indicates the hampered

conversion of androgen from cholesterol, which is

dependent on the availability of pituitary

gonadotrophins.

Decrease in the acid phosphatase activity and

increase in the alkaline phosphatase activity after

morphine treatment may be due to decline in the

endogenous androgen production (Kasturi et al.,

1995). It is also one of the factors for the suppression of

spermatogenesis, which is evidenced by the

lowered sperm count in the cauda epididymis after

morphine treatment.

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