mustapha jamiu sulayman's projet

CHAPTER ONE

1.1. GENERAL INTRODUCTION

Hypertension is usually defined by the presence of a chronic

elevation of systemic arterial pressure above a certain threshold

value. However, increasing evidence indicates that the

cardiovascular (CV) risk associated with elevation of blood

pressure (BP) above approximately 115 ⁄ 75 mm Hg increases in a

log-linear fashion. The progressive CV syndrome arising from

complex and interrelated etiologies. Early markers of the

syndrome are often present before BP elevation is sustained;

therefore, hypertension cannot be classified solely by discrete

BP thresholds. Progression is strongly associated with functional

and structural cardiac and vascular abnormalities that damage the

heart, kidneys, brain, vasculature, and other organs and lead to

premature morbidity and death. [1]

1.2. Calcium blockers in relation to fertility

Infertility is defined as the inability to achieve pregnancy

after one year of unprotected intercourse [2]. Conception is

normally achieved within 12 months in 80 to 85% of couples who

are not using contraceptive measures; which mean an estimated 15%

of couples attempting their first pregnancy, may experience

difficulty in conceiving. Some cases of male infertility are due

to anatomical abnormalities such as varicoceles, ductal

obstructions or ejaculatory disorders [3]. Infertility may also be

due to abnormal sperm morphology (tetratozoospermia) and

insufficient sperm motility [4]. A study recently carried out on

the sperm characteristics of infertile males at the University

College Hospital, Ibadan, Nigeria showed that abnormal semen

quality remains a significant contributor to overall infertility

with athenozoospermia being the most common seminal quality

abnormality [5]. A similar study that was done at the Nnamdi

Azikiwe University Teaching Hospital located in South-east

Nigeria showed that oligozoospermia (35.9%) and athenozoospermia

(32.3%) were the most common aetiological factors responsible for

male infertility [6]. There are evidences to show that sperm

counts have been declining over the last 50 years, with a

consequent increase in male infertility. [7]

Primary infertility may result from the use of various drugs.

This phenomenon may be the result of an effect on the

hypothalamic-pituitary-gonadal axis or a direct toxic effect on

the gonads. Some of the drugs are antineoplastic agents

(cyclophosphamide, chlorambucil, busulphan, and methotrexate),

glucocorticosteroids, hormonal steroids (diethylstilbestrol,

medroxyprogesterone acetate, estrogen, and the constituents of

oral contraceptives), antibiotics (sulfasalazine and

cotrimoxazole), thyroid supplements, spironolactone, cimetidine,

colchicine, marijuana, opiates, and neuroleptic agents [8].

Phenothiazines such as chlorpromazine and thioridazine, are

commonly used to treat schizophrenia and are known to cause

hyperprolactinaemia which induces hypospermatogenesis, impotence

and loss of libido in men [9].

1.3. AIMS OF STUDY

The present work is aimed at studying or evaluating the effect of

Nifedipine on the testis.

1.4. SCOPE OF STUDY

The research work is based on the analysis of the effect of the

Nifedipine, giving a group of adult male wistar rats a particular

dose of the Nifedipine to see on the testis. The research work

would be extrapolated towards finding the effect of the

Nifedipine on the following parameters:

1. Morphological changes of the testis (histological changes)

2. Sperm count

3. Sperm motility

4. Endocrine function of the testis (serum testosterone)

Comparative examination of the testes of the group exposed to the

minimum, maximum dose group as well as the control group would

show possible effects on the testes.

1.5. SIGNIFICANCE OF STUDY

The information gathered from the observations and results in

this study would help shed more light on the use of

antihypertensive (Nifedipine) and the effects of the drugs would

help one to make scientific suggestion as to whether the drugs

should be embraced or withdrew as one of the therapeutic drugs in

the management of hypertension.

1.6. STATEMENT OF PROBLEM

Nifedipine is widely used as an antihypertensive drug, effort

would be made, if it should be discourage or encourage among

hypertensive male of reproductive age, Nifedipine affect the male

reproductive system by blocking it functions

1.7. LIMITATION OF STUDY

Available laboratory equipment and facilities did not

favour the study.

Time factor was a constraint on the study.

Electricity was another huge factor on this study.

Lack of proper functioning of the apparatus and equipment

which altered the tissue processing procedure.

2. CHAPTER TWO

2.1. Hypertension

Hypertension is an important worldwide public-health challenge

because of its high frequency and concomitant risks of

cardiovascular and kidney disease. It has been identified as the

leading risk factor for mortality and is ranked third as a cause

of disability-adjusted life-years. [44]

2.2. Incidences

In the year 2001, high blood pressure accounted for “54% of

stroke, 47% of ischaemic heart disease, 75% of hypertensive

disease, and 25% of other cardiovascular disease worldwide” [10].

The negative impact of hypertension on health status is clear,

especially taking into account the disability, decreased quality

of life, and mortality associated with stroke and cardiovascular

disease. In 2001, 7.6 million deaths (13.5% of all deaths) and 92

million disability life-years (6% of total) were attributable to

systolic blood pressure greater than 115 mmHg . [10]

2.3. Drug of study

Name of the drug: Nifedipine

Nifedipine is a drug belonging to a class of pharmacological

agents, the dihydropyridine family (amlodipine, felodipine,

isradipine, nicardipine, nifedipine, and nisoldipine) the calcium

channel blockers.

Chemical Name: dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-

dihydropyridine-3,5-dicarboxylate. [11]

Molecular Formula: C17H18N2O6 Molecular Weight: 346.3

Figure 1 is the chemical structure of nifedipine. [11]

2.4. DESCRIPTION

Nifedipine (Nif) is a yellow, crystalline powder which is

practically insoluble in water and sparingly soluble in absolute

ethanol. It is sensitive to light, is the prototype compound of

the dihydropyridine class of calcium channel antagonists. Calcium

antagonists inhibit the influx of calcium ion through plasma

membrane channels and thus dilate vascular smooth muscle

contraction. NIF is a selective arterial dilator, and is used for

the treatment of hypertension, angina pectoris, and other

cardiovascular disorders [12].

The exposure of some drugs to light leads to photodecomposition.

These drugs undergo important chemical changes, accompained by

alternation in their activities and in some cases total loss of

their therapeutic activity [13].

NIF is very highly sensitive to photooxidation. NIF exposure to

ultraviolet-visible irradiation produces both aromatization in

the dihydropyridine moiety (turning it into a pyridine ring) and

a reduction of nitro group in to nitroso groups (NDNIF). In

addition, its exposure to ultraviolet light produces

dehydronifedipine (DNIF), NIF photodegradation products have no

pharmacological activity. Several studies related to its

photodecomposition have been reported. [14]

The Manufacturers of NIF products use light resistant coating

and/or packing to minimize their photodegradation. Long term

exposure to sunlight or artificial light may also occur if NIF

formulations are improperly stored by patients. Poor storage

conditions may potentially decrease clinical efficacy of NIF

products [15].

The tablets also contain the following excipients: purified talc,

povidone, lactose, carbomer 934P, hypromellose, silicon dioxide,

magnesium stearate, titanium dioxide, iron oxide red CI77491,

macrogol 4000 and Eudragit E100. The tablets are gluten free.

Furthermore, Nifedipine is a potent vasodilator, which relaxes

vascular smooth muscle probably by its inhibitory effect on the

transmembrane influx of calcium [16].

It has been demonstrated that this drug exerts a prompt and

marked hypotensive effect when administered to hypertensive

patients. [17]

Mechanism of Action

The precise means by which this inhibition relieves angina has

not been fully determined, but includes at least the following

two mechanisms:

1) Relaxation And Prevention Of Coronary Artery Spasm:Nifedipine

dilates the main coronary arteries and coronary arterioles, both

in normal and ischemic regions, and is a potent inhibitor of

coronary artery spasm. This property increases myocardial oxygen

delivery in patients with coronary artery spasm, and is

responsible for the effectiveness of nifedipine in vasospastic

(Prinzmetal’s or variant) angina. Whether this effect plays any

role in classical angina is not clear, but studies of exercise

tolerance have not shown an increase in the maximum exercise

rate-pressure product, a widely accepted measure of oxygen

utilization. This suggests that, in general, relief of spasm or

dilation of coronary arteries is not an important factor in

classical angina.

2) Reduction Of Oxygen Utilization:Nifedipine regularly reduces

arterial pressure at rest and at a given level of exercise by

dilating peripheral arterioles and reducing the total peripheral

resistance (afterload) against which the heart works. This

unloading of the heart reduces myocardial energy consumption and

oxygen requirements and probably accounts for the effectiveness

of nifedipine in chronic stable angina.

Pharmacokinetics

Nifedipine is almost completely absorbed after oral

administration. Plasma drug concentrations rise at a gradual,

controlled rate exhibiting zero order absorption kinetics after

nifedipine long acting tablet administration and reach a plateau

at approximately six hours after the first dose. For subsequent

doses, relatively constant plasma concentrations at this plateau

are maintained with minimal fluctuations over the 24 hour dosing

interval. At steady state, the bioavailability of nifedipine

extended release tablets is 86% relative to an immediate release

dosage form which has a systemic availability of 45 to 68%.

Administration of nifedipine long acting tablets in the presence

of food slightly alters the early rate of drug absorption, but

does not influence the extent of drug bioavailability. Markedly

reduced gastrointestinal retention times over prolonged periods

(i.e. short bowel syndrome) may, however, influence the

pharmacokinetic profile of the drug, which could result in lower

plasma concentrations. The pharmacokinetics of Nifedipine long

acting tablets are linear over the dose range of 30 to 180 mg, in

that plasma concentrations are proportional to dose administered.

There is no evidence of dose dumping in either the presence or

the absence of food. [18]

Distribution

Nifedipine is about 95% bound to plasma protein (albumin). [18]

Biotransformation

The active substance nifedipine is almost completely metabolised

in the liver, primarily by oxidative processes (the cytochrome

P450 enzyme CYP3A4). Some metabolic activity within the gut wall

may also contribute to the presystemic metabolism. These

metabolites show no pharmacodynamic activity. The main metabolite

is the hydroxycarbolic acid derivative (95%); the remaining 5% is

the corresponding lactone. [18]

Excretion/Elimination

Nifedipine is excreted in the form of its metabolites,

predominantly via the kidneys (60 to 80%), and about 5 to 15% is

excreted via the bile in the faeces. The unchanged substance is

recovered only in traces (below 0.1%) in the urine. [18]

Half-life

The terminal elimination half-life is 1.7 to 3.4 hours in an

immediate long acting formulation. In cases of impaired kidney

function, no substantial changes have been detected in comparison

with healthy volunteers. [18]

In cases of impaired liver function, the elimination half-life is

distinctly prolonged and the total clearance is reduced. A dose

reduction may be necessary in severe cases.

Patients on haemodialysis or chronic ambulatory peritoneal

dialysis have not reported significantly altered pharmacokinetics

of nifedipine.

Pharmacodynamics

Nifedipine inhibits the transmembrane influx of calcium ions into

cardiac and vascular smooth muscle. The contractile processes of

these tissues are dependent upon the movement of extracellular

calcium into the muscle cells through specific ion channels.

Nifedipine selectively inhibits the transmembrane influx of

calcium through the slow channel without affecting the

transmembrane influx of sodium through the fast channel to any

significant degree. This results in a reduction of free calcium

ions available within the muscle cells and an inhibition of the

contractile process. Nifedipine does not affect total serum

calcium. The specific mechanisms by which nifedipine relieves

angina and reduces blood pressure have not been fully determined

but are believed to be brought about largely by its vasodilatory

action. [18]

Common side effects

Common side effects of nifedipine include, Headache (7.3-7.9%).

dizziness (456.7%).

Lightheadedness (6.7%), giddiness (6.7%), nausea (6.7%). vomiting

(6.7%). Gastrointestinal distress (6.7%). flushing (5.8- 13.9%).

heat sensation (5.8- 13 -9%). peripheral edema (3.747%),

Hypotension. [19]

SYSTEM OF STUDY

On the male reproductive system

After life itself, fertility is probably the most highly prized

human possession, yet, while medical treatment of the individual

naturally demands priority, relatively little attention is paid

to the effects of treatment on reproductive function.

ANTIHYPERTENSIVE MEDICATIONS

The ultimate goal in treatment of the hypertensive patient is to

achieve the maximum reduction in the long-term total risk of

cardiovascular morbidity and mortality.

Classifications of antihypertensives

Agents useful in the chronic treatment of hypertension may be

classified into one of nine categories. [20]

They are in no particular order: 1) diuretics, 2) beta-blockers,

3) angiotensin converting enzyme inhibitors, 4) angiotensin

receptor antagonists, 5) calcium channel blockers, 6) alpha-

adrenergic blockers, 7) central alpha adrenergic agonists, 8)

direct vasodilators, and 9) peripheral adrenergic neuron

antagonists.

Calcium channel antagonists/blockers

Ca2+ channels allow passage of Ca2+ ions into the cytoplasm

through a selective pore which is opened in response to

depolarization of the cell membrane [21].

The Ca2+ flux creates a net inward, depolarizing current and the

resulting accumulation of Ca2+ in the cytoplasm can act as a

chemical trigger for secretion of hormones and neurotransmitters,

contraction of muscle and a variety of other Ca2+-sensitive

events. Thus, upon sensing membrane potential changes, Ca2+

channels simultaneously generate an electrical signal while

directly creating an intracellular chemical messenger. This dual

ability is unique among the family of ion channels and allows the

Ca2+ channel to play a variety of roles in excitation-secretion

and excitation-contraction coupling. It has now become clear that

versatility of function is reflected by diversity of the types of

Ca2+ channels on the membrane of individual cells. [22]

The term ‘‘calcium channel antagonists’’ refers to a chemically,

pharmacologically and therapeutically heterogeneous group of

drugs prominent both as cardiovascular therapeutic agents and as

molecular tools. The calcium antagonist consist of at least three

distinct classes of drugs as follows: 1,4-dihydropyridines

(prototype: Nifedipine ), phenylalkylamines :prototype

verapamil), and benzothiazepines (prototype:diltiazem). [23]

The cardiovascular activities of these drugs as

antihypertensive, anti-anginal and selective antiarrhythmic

agents are due to their interaction at one particular calcium

mobilization process—calcium entry through a voltage-gated

calcium channel of the L-type. Many studies have demonstrated

that in accord with their chemical heterogeneity these agents

interact at discrete receptor sites associated with a major

subunit of the channel. [24]

Iranloye et al, reported that Nifedipine appears to have a

deleterious effect on sperm functions in rats which is not

mediated by a change in testosterone secretion, Group 1 (control)

received distilled water; Group 2, received nifedipine 0.57

mg/kg; and Group 3, received 0.57 mg/kg and serve as a recovery

group and the treatment was done orally for 30 days. [25]

Amlodipine (as besylate, mesylate or maleate) is a long-acting

calcium channel blocker (dihydropyridine class) used as an anti-

hypertensive and in the treatment of angina [26].

Like other calcium channel blockers, amlodipine acts by relaxing

the smooth muscle in the arterial wall, decreasing peripheral

resistance and hence reducing blood pressure; in angina it

increases blood flow to the heart muscle.

The preliminary observations showed that administration of

Amlodipine lead to an abnormal morphology of the testis in that,

it had a deleterious effect on the seminiferous tubules in a dose

related fashion. By these preliminary observations, alteration in

the testis histology may have implication for infertility in man.

It could be observed from the result obtained in this study that

a high dose of 0.056mg/g/bodyweight of amlodipine gradually

produced an abnormal morphology of the testis while a higher dose

of 0.114mg/g/bodyweight led to a complete destruction of the

histological architecture of the testis when administered daily

for 8 weeks. The high doses were designed to represent human

exposure to high levels of amlodipine. Therefore, one can

conclude that high doses of amlodipine could lead to infertility

in man. [27]

Clinical nifedipine-associated infertility has been reported to

have negative effect on the acrosome. The acrosomal reaction is a

complex calcium-dependent process [28].

Premature spontaneous acrosome reaction prior to reaching the

oocyte, may lead to early sperm cell death. On the other hand,

the inability of sperm to undergo stimulated acrosome reaction in

response to oocyte investments and/or follicular fluid may lead

to the failure of sperm to fertilize the ovum. It has recently

been demonstrated that nifedipine, a calcium channel blocker, has

the capability of blocking acrosome reaction [29].

Benoff et al., reported a group of men taking nifedipine for

hypertension were found to have reversible disordered expression

of head-directed mannose-ligand receptors and low rates of

acrosome reaction during capacitating conditions. [30]

Following cessation of the drug, the acrosome reaction status

returned to normal and subsequently, pregnancy was achieved. [31]

Calcium ion has been shown to be a primary determinant of sperm

cell function, including progressive motility, hyper activated

motility, capacitation, and acrosome reaction; accordingly, a

number of Ca2+ - permeable channels and transporters have been

reported [32].

The therapeutic administrations of calcium antagonist for

hypertension control causes reversible male infertility

associated with an IVF Failure. A mechanism of inhibition of

sperm fertilizing potential through insertion of lipophilic

calcium ion antagonists into the bilayer of the sperm plasma

membrane. [33]

The possibility exists that the nifedipine used by the majority

of men in the control of hypertension has more of an adverse

effect on sperm function than verapamil, however, verapamil may

increase sperm motility and accelerate acrosome reaction [34].

However, a fundamental question is whether drugs blocking calcium

channel have any side effect that reduces male fertility as

available reports suggested that calcium ion mediates sperm

functions and fertilization process [35].

Furthermore, there are concerns about the induction of

infertility by CCB in males [36].

Recently, a particular study group reported that CCB appear to

have a reversible anti-fertility effect on male rats. Following

the exposure of male rats to CCB, a significant reduction in the

epididymal sperm count and motility was observed; which did not

occur through inhibition of the pituitary gonadal axis as

testosterone, follicle stimulating hormone and luteinizing

hormone levels remained unchanged [37].

Previously, many drugs have been shown to have adverse effect on

male fertility.

There is substantial evidence that CCB induces significant

oxidative stress in the testes, which appears to be responsible

for the adverse effects of decreased sperm count and motility

ultimately leading to reduced fertility in rats. [38]

Furthermore it was also reported by Olivari et al, that the

weight would be significantly less after chronic Nifedipine

therapy. [39]

ORGAN OF STUDY (TESTIS)

GROSS ANATOMY OF THE TESTIS

Introduction

The testes (testicles) are the male gonads—paired ovoid

reproductive glands that produce sperms (spermatozoa) and male

hormones, primarily testosterone. [40]

Shape: The testes (singular, testis) are paired, whitish, ovoid

organs.

Measurement: each measures about 4 cm (1.5 in.) long and 2.5 cm

(1 in.) in diameter, each testis weighs between 10 and 14 g. [41]

The testes are suspended in the scrotum by the spermatic cords,

with the left testis usually suspended (hanging) more inferiorly

than the right testis. [40]

Coverings: Two tissue layers, or tunics, cover the testes, the

outer tunica vaginalis is a thin serous sac derived from the

peritoneum during the descent of the testes. The tunica albuginea

(al''byoo-jin'e-a˘) is a tough fibrous membrane that directly

encapsulates each testis. [41]

The surface of each testis is covered by the visceral layer of

the tunica vaginalis, except where the testis attaches to the

epididymis and spermatic cord. The tunica vaginalis is a closed

peritoneal sac partially surrounding the testis, which represents

the closed-off distal part of the embryonic processus vaginalis.

The visceral layer of the tunica vaginalis is closely applied to

the testis, epididymis, and inferior part of the ductus deferens.

The slit-like recess of the tunica vaginalis, the sinus of the

epididymis, is between the body of the epididymis and the

posterolateral surface of the testis. [40]

The parietal layer of the tunica vaginalis, adjacent to the

internal spermatic fascia, is more extensive than the visceral

layer and extends superiorly for a short distance onto the distal

part of the spermatic cord. The small amount of fluid in the

cavity of the tunica vaginalis separates the visceral and

parietal layers, allowing the testis to move freely in the

scrotum. [40]

Fibrous inward extensions of the tunica albuginea partition the

testis into 250 to 300 wedge-shaped testicular lobules. Each

lobule of the testis contains tightly convoluted seminiferous

tubules that may exceed 70 cm (28 in.) in length if uncoiled. The

seminiferous tubules are the functional units of the testis

because it is here that spermatogenesis, the production of

spermatozoa, occurs. Spermatozoa are produced at the rate of

thousands per second—more than 100 million per day— throughout

the life of a healthy, sexually mature male. [41]

EPIDIDYMIS

The epididymis is an elongated structure on the posterior surface

of the testis.

Efferent ductules of the testis transport newly developed sperms

to the epididymis from the rete testis. The epididymis is formed

by minute convolutions of the duct of the epididymis, so tightly

compacted that they appear solid, the duct becomes progressively

smaller as it passes from the head of the epididymis on the

superior part of the testis to its tail. At the tail of the

epididymis, the ductus deferens begins as the continuation of the

epididymal duct. In the lengthy course of this duct, the sperms

are stored and continue to mature. The epididymis consists of:

Head of the epididymis: the superior expanded part that is

composed of lobules formed by the coiled ends of 12-14

efferent ductules.

Body of the epididymis: consists of the convoluted duct of

the epididymis.

Tail of the epididymis: continuous with the ductus deferens,

the duct that transports the sperms from the epididymis to

the ejaculatory duct for expulsion via the urethra during

ejaculation. [40]

[40]

BLOOD SUPPLY

The testicular artery arises from the aorta at the level of the

renal vessels. It anastomoses with the artery to the vas,

supplying the vas deferens and epididymis, which arises from the

inferior vesical branch of the internal iliac artery. This cross-

connection means that ligation of the testicular artery is not

necessarily followed by testicular atrophy. The pampiniform

plexus of veins becomes a single vessel, the testicular vein, in

the region of the internal ring. On the right this drains into

the inferior vena cava, on the left into the renal vein. [42]

LYMPH DRAINAGE

The lymphatic drainage of the testis obeys the usual rule; it

accompanies the venous drainage and thus passes to the para-

aortic lymph nodes at the level of the renal vessels. Free

communication occurs between the lymphatics on either side; there

is also a plentiful anastomosis with the para-aortic

intrathoracic nodes and, in turn, with the cervical nodes, so

that spread of malignant disease from the testis to the nodes at

the root of the neck is not rare. [42]

NERVE SUPPLY

The testis is supplied tenth thoracic (T10) sympathetic fibres

via the renal and aortic plexus. These convey afferent (pain)

fibres—hence referred pain from the testis to the loin. [42]

Histologically each testis is surrounded by a thick capsule of

dense connective tissue, the tunica albuginea. The tunica

albuginea is thickened on the posterior surface of the testis to

form the mediastinum testis, from which fibrous septa penetrate

the gland, dividing it into about 250 pyramidal compartments

called the testicular lobules. These septa are incomplete, and

there is frequent intercommunication between the lobules. Each

lobule is occupied by one to four seminiferous tubules enmeshed

in a web of loose connective tissue that is rich in blood and

lymphatic vessels, nerves, and interstitial cells, also known as

Leydig cells. Seminiferous tubules produce male reproductive

cells, the spermatozoa, whereas interstitial cells secrete

testicular androgens. During embryonic development the testes

develop retroperitoneally in the dorsal wall of the abdominal

cavity. They migrate during fetal development and become

positioned within the scrotum, at the ends of the spermatic

cords. Because of this migration, each testis carries with it a

serous sac, the tunica vaginalis, derived from the peritoneum.

The tunic consists of an outer parietal layer and an inner

visceral layer, covering the tunica albuginea on the anterior and

lateral sides of the testis.

SPERMATOGENESIS

Spermatogenesis is the process of formation of spermatozoids.

That is- the sequence of events in the seminiferous tubules of

the testes that leads to the production of spermatozoa.The

process begins with a primitive germ cell, the spermatogonium,

situated next to the basal lamina of the epithelium. At sexual

maturity, spermatogonia begin to divide by mitosis, producing

successive generations of cells. The newly formed cells can

follow one of two paths: they can continue dividing as stem

cells, also called type-A spermatogonia, if they differentiate

during progressive mitotic cycles to become type-B spermatogonia.

Type-B spermatogonia are progenitor cells that will differentiate

into primary spermatocytes. Soon after their formation, these

cells enter the prophase of the first meiotic division. Since the

prophase of this division takes about 22 days, the majority of

spermatocytes seen in sections will be in this phase. The primary

spermatocytes are the largest cells of the spermatogenic lineage

and are characterized by the presence of chromosomes in various

stages of the coiling process within their nuclei. From this

first meiotic division arise smaller cells called secondary

spermatocytes. The secondary spermatocytes are difficult to

observe in sections of the testis because they are short lived

cells that remain in interphase very briefly and quickly enter

into the second meiotic division. The division of each secondary

spermatocyte result into 2 cells that contain 23 chromosomes, the

chromatids. [43]

Spermiogenesis is the final stage of production of

spermatozoids. Spermiogenesis is the process by which spermatids

transform into spermatozoa, cells which are highly specialized to

deliver male DNA to the ovum. No cell division occurs during this

process.

Diagram showing the clonal nature of the germ cells

Structure of Spermatozoa

A mature sperm cell, or spermatozoon (sper-mat''o˘-zo'on) is a

microscopic, tadpole-shaped structure approximately 0.06 mm

long . It consists of an oval head and an elongated flagellum.

Although the head of a spermatozoon lacks mitochondria, it does

contain a nucleus with 23 chromosomes. The tip of the head,

called the acrosome (ak'ro˘-so ￣ m), contains enzymes that help the

spermatozoon penetrate the ovum. The body of the flagellum

contains numerous mitochondria spiraled around a filamentous

core. The mitochondria provide the energy necessary for

locomotion. The flagellum propels the spermatozoon with a lashing

movement. The maximum unassisted rate of spermatozoa movement is

about 3 mm per hour.

Semen

Semen, also called seminal fluid, is the substance discharged during

ejaculation (table 20.4). Generally, between 1.5 and 5.0 ml of

semen are ejected during ejaculation. The bulk of the fluid

(about 60%) is produced by the seminal vesicles, and the rest

(about 40%) is contributed by the prostate. Spermatozoa account

for less than 1% of the volume. There are usually between 60 and

150 million sperm cells per milliliter of ejaculate. In the

condition of oligospermia (ol''ı˘-go-sper'me-a˘), the male ejaculates fewer

than 10 million sperm cells per milliliter and is likely to have

fertility problems. [41]

A diagram of a spermatozoon. The histology of the epididymis

showing sperm in the lumina (50×). [41]

Function of testes

Production of spermatozoa.

Synthesis and release of testosterone.

The two testes form about 200 million spermatozoa per day.

Sertoli cells of the seminiferous epithelium also produce a

fructose-rich fluid that acts to nourish and transport the

newly formed spermatozoa from the lumen of the seminiferous

tubule to the extra testicular genital ducts.

Function of epididymis

Site where sperm mature

Stores sperm cells (20 days)

Expels sperm to the vas deferens during ejaculation

CHAPTER THREE

3.0 MATERIALS AND METHODS

The materials used in the accomplishment of this project

includes Nifedipine, Petri dish, Wistar rats (17),microtome, Water

bowl, Beaker, Calibrated syringe, Basket, Cannula, Animal cage,

Dissecting set, Condensation, Cotton wool, Oven, Water bath,

Plastic mould, Paraffin wax, Filter paper, Wooden block, Wash

hand basin, Measuring cylinder, weighing balance, Stationeries,

Light Microscope, Conical flask, Industrial salt (NaCl), Spatula,

Absolute alcohol, Dissecting kits, Xylene solution,

paraformaldehyde solution, formalin solution, specimen bottles,

Haematoxylin stain, Eosin stain, Slides.

3.1 BREEDING OF ANIMALS

Seventeen adult albino rats comprising of males only were

obtained with an average weight of 140 g. The rats after

procurement were subsequently housed in cages under light and

dark cycle at room temperature in the experimental animal house

of the department of anatomy, university of Ilorin. Proper

aeration was maintained and ensuring cross ventilation throughout

the animal house. They were fed with rat pelletized feed and

water was given. The feeds were obtained from Bendel feeds

Nigeria limited, Yoruba road, Ilorin. The rats were left to

acclimatize before treatment was carried out.

3.2 GROUPING OF ANIMALS

The rats were divided into three groups using a simple random

sampling as follows: two experimental groups and one control

group.

Control group (n=5), they were neither treated with 2ml of

distilled water.

Low dose group: (n=6), they were treated with 0.3mg/kg body

weight.

Higher dose group: (n=6), they were treated with 0.6mg/kg body

weight

3.3 PREPARATION OF NIFEDIPINE SOLUTION AND DOSAGE

Nifedipine tablets, each weighing 60mg, produced by Bayer

Schering Pharma AG 51368 Leverkusen, Germany, Nigeria Reg No: A4-

2373, manufacture date: 10-11-2010, Expiration: 10-11-2004.

5 tablets was slightly crushed and dissolved in distilled water

for about 4dyas for achieve total dissolution.

Calculation of dosage: since the therapeutic use of

Nifedipine is between 20mg to 90mg and since normal physiological

man weighs 70kg, therefore: 20mg70kg=0.3mgkg -1 therefore the

minimal dose given was 0.3mg/kg while the maximal dose group was

multiply by 2 therefore 2*0.3mg/kg ¿0.6mgkg -1

3.4 TREATMENT OF ANIMALS

Treatment was done orally using oral cannula and it lasted for 25

days.

Control group: 2ml of distilled water.

Low dose group: 0.3mg/kg body weight.

Higher dose group: 0.6mg/kg body weight.

Termination of treatment

After 25th day the rats were sacrificed to remove the male

reproductive system (testis and epididymis.

3.5 BODY AND ORGAN WEIGHT

The body weights were taken every 72 hours using weighing

balance.

3.6 SACRIFICE

At the end of the 25th day of treatment, the animals were

anaesthetized by giving katamine hydrochloride 25mg

interperitoneally (IP), then thoracolaparactomy was performed to

open the thorax and the abdomen, the epididymis was collected,

blood was taken from the heart through the right ventricle, total

body perfusion was done by using normal saline firstly to remove

the blood from vascular space followed by the introduction of 4%

paraformaldehyde for general body fixation which is then followed

by the removal of necessary organs.

3.7 TISSUE HOMOGENIZATION

The epididymis was homogenized in 1ml of normal saline mixed and

counted, and the collected blood was centrifuged at 2000 rpm for

20 min. The supernatant was subsequently collected and analysed

for hormonal bioassay (testosterone, LH, FSH).

HAEMATOLOGICAL ANALYSIS

The full blood count analysis include, packed cell volume (PCV),

white blood cell (WBC) count, red blood cell(RBC) count, and

blood film morphology, which may include differential count and

others.

The blood samples collected from the animals were stored in EDTA

bottles from which they were collected in time to run the

analysis.

PACKED CELL VOLUME PROCEDURE

The plain capillary tube was filled to 2/3 of its volume with the

blood and sealed at one end with plasticine. They were then

placed on the microhaematocrit centrifuge and centrifuged for 5

minutes at 10,000 rpm. After the centrifugation, the spun blood

was placed in the groove column of the microhaematocrit reader.

The packed cell volume was read as follows;

1. Allow the bottom level of the packed cells to stay at the

zero mark

2. Allow the top plasma level to stay at 100 mark.

3. Use the adjustable slanting mover to stay at the meniscus

level of the plasma/blood cells.

4. The reading of the pack can now be recorded.

WHITE BLOOD CELL COUNT PROCEDURE

0.02m/s of the blood sample of each rat was added to 0.38m/s of

diluting fluid (Turk’s solution) and allowed to mix well. The

counting chamber to be used is then cleaned and the cover slip

fixed. A few drops of the mixed blood sample and Turk’s solution

were charged into the counting chamber. The cells were allowed to

settle by placing on a firm bench for few minutes; four (4) big

squares of sixteen (16) smaller squares were counted.

The WBC count was calculated with the formula below

WBC = (number of cells counted ÷ depth of the counting

chamber) ×dilution ratio

Dilution ration for WBC count is 1:20

RED BLOOD CELL COUNT PROCEDURE

0.02ml of the blood sample of each rat was added to 4ml of

diluting fluid (formal citrate) and allowed to mix well for 2

minutes by tilting for proper reaction. After few drops had been

charged into the counting chamber as explained above, it is left

for 2 minutes before counting. The cells in 80 or 160 smaller

squares, including the cells that touch the bottom and left-hand

margins of the small square are counted.

The RBC count was calculated using the formula below:

RBC = (number of cells counted/volume counted) ×dilution

ratio×106

Dilution ratio is 1:200

LEISHMAN STAINING PROCEDURE

The slides were placed in a staining rack and the Leish-man stain

was applied for 2 minutes, few drops of 6.8pH was added and

allowed to act for 7 minutes. The slides where washed with 6.8pH

buffer solution and allow to air dry. The slides were then

examined and counting was done under the microscope at an (oil

immersion) objective magnification (×100).

SEMINAL ANALYSIS PROCEDURE

The semen analysis was done to determine the sperm count,

progressivity, motility and morphology.

In this study, epididymal sperm count, motility, morphology,

sperm motility was done immediately after semen collection,

whereby a rubber pipette was used to take small quantity of the

semen mixed with normal saline and drop it on a glass slide for

viewing under microscope, using an x40 objective magnification

with reduced light. Sperm counts were carried out using the

Neubeurs Haemocytometer counting chamber.

Progressivity was graded as follows:

A – Excellent forward directional movement (EFDM)

B – Good forward directional movement (GFDM)

C – Fair forward directional movement (FFDM)

D – Poor forward directional movement (PFDM)

(W.H.O, 1976).

HORMONAL ASSAY PROCEDURES

Similar procedure is required for all the hormones tested for,

which is analysed bellow

Immunoenzymometric assay: the essential reagents required for an

Immunoenzymometric assay include high affinity and specificity

antibodies (enzyme and immobilized), with different and distinct

epitope recognition, in excess, and in native antigen. In this

procedure, the immobilization takes place during the assay at the

surface of a microplate well through the interaction of

streptavidin coated on the well and exogenously added

biotinylated monocional anti-hormone assay for (LH, progesterone,

FSH) antibody.

upon mixing monoclonal biotinylated antibody, the enzyme label

antibody and a serum containing the native antigen, reaction

results between the native antigen and antibodies without

competition or steric hindrance to form a soluble sandwich

complex. The interaction is illustrated by the following

equation:

EnzAb(p) +AgLH +BtnAb(m) EnzAb(p).AgLH.BtnAb(m)

BtnAb(m)= Biotinylated Monoclonal Antibody (Excess Quality)

AgLH= Native Antigen (Variable Quantity)

EnzAb(p)= Enzyme labelled Antibody (Excess Quantity)

EnzAb(p)-AgLH-BtnAb(m) = Antigen-Antibodies Sandwich Complex

Ka = Rate Constant of Association

K-a = Rate Constant of Dissociation

Simultaneously, the complex is deposited to the well through the

high affinity reaction of streptavidinn and biotinylated

antibody.

This reaction is illustrated bellow:

EnzAb(p)-AgLH-BtnAb(m) + streptavidinnc.w. =immobilized complex

Streptavindinc.w. = streptavidin immobilized on well

Immobilized complex = Antibodies-Antigen sandwich bound.

After equilibrium is attained, the antibody-bound fraction is

separated from unbound antigen by decantation or aspiration. The

activity in the antibody-bound fraction is directly proportional

to the native antigen concentration. By utilizing several

different serum reference of known antigen values, a dose

response curve can be generated from which the antigen

concentration of an unknown can be ascertained.

REAGENT PREPARATION

1. Wash buffer

Dilute contents of wash concentrate to 100ml with distilled or

deionized water in a suitable storage container. Stored at 2-30C

for up to 60days.

2. Working Substrate Solution.

Pour the contents of the amber vial labelled Solution ‘A’ into

the clear vial labelled Solution ‘B’. Place the yellow cap on the

clear vial for easy identification. Mix and labelled accordingly.

TEST PROCEDURE

1. Format the microplate wells for each serum reference,

control and patient specimen to be assayed in duplicate.

Replace any unused microwell strips into the aluminium bag,

seal and stored at 2-8oC.

2. Pipette 0.050ml (50µl) of the appropriate serum reference,

control or specimen into the assigned well.

3. Add 0.100ml (100µl) of the assay hormone-Enzyme Reagent to

all wells.

4. Swirl the microplate gently for 20-30 seconds to mix and

cover.

5. Incubate 60 minutes at room temperature.

6. Discard the contents of the microplate by decantation or

aspiration. If decanting blot the plate dry with absorbent

paper.

7. Add 350ul of wash buffer decant or aspirate. Repeat two (2)

additional times for a total of three (3) washes. An

automatic or manual plate washer can be used.

8. Add 0.100ml (100µl) of working substrate solution to all

wells; always add reagents in the same order to minimize

reaction time differences between wells.

9. Incubate room temperature for fifteen (15) times.

10. Add 0.050ml (50µl) of stop solution to each well and

gently mix for 15-20 seconds.

11. Read the absorbance in each well at 450nm (using a

reference wavelength of 620-630nm to minimize well

imperfections) in a microplate reader. And the result should

be read within 30 minutes of adding the stop solution.

CALCULATION OF RESULT

A dose response curve is used to ascertain the concentration

of the hormone in unknown specimens.

1. Record the absorbance obtained from the printout of the

microplate reader.

2. Plot the absorbance for each duplicate serum reference

versus the corresponding hormone concentration hormone

concentration in mlU/ml on linear graph paper.

3. Draw the best-fit curve through the plotted points.

4. To determine the concentration of hormone for an unknown,

locate the average absorbance of the duplicates for each

unknown on the vertical axis of the graph.

0 1 2 3 4 5 6 7 8 9 10 11 120

0.51

1.52

2.5

Serum Testosterone

Conc. ng/ml

Absorbance

Graph 1: showing the standard curve used for the calculation of

the serum testosterone.

0 5 10 15 20 25 30 35 40 45 50 55 600

0.51

1.52

Serum Progesterone

Conc. ng/ml

Absorbance


the serum progesterone

0250

500

7501000

1250

1500

1750

2000

2250

2500

2750

3000

0

1

2

Serum Estradiol

Conc. pg/ml

Absorbance


the serum oestradiol

0 10 20 30 40 50 60 70 80 90 100

00.51

1.52

Serum FSH

Conc. mIU/ml

Absorbance


the serum Follicle stimulating hormone

0 25 50 75 100 125 150 175 2000

1

2

Serum LH

Conc. mIU/ml

Absorbance


the serum Luteinizing hormone

3.9 STATISTICAL ANALYSIS

Data were analysed by analysis of variance (ANOVA) and were

presented as Mean ± SEM. Using SPSS.

3.10 TISSUE PROCESSING FOR MICROSCOPY

The testis was harvested and fixed in both 4% paraformaldehyde

and formosaline solution for histopathological studies. The

summary of the steps used in fixation are:

Fixation: this is the first chemical treatment the tissue is

subject to. It is used for hardening of the tissue and decay

prevention. It prepares the tissue for sectioning and staining.

The testis tissue was fixed in 4% paraformaldehyde and some in

formosaline solution.

Dehydration: this is the removal of water from tissue during its

preparation for microscopical study, by placing it successively

in strong solutions of alcohol i.e. from 70%, 90%, and absolute

alcohol.

Clearing: this is the process of removing the cloudiness from

microscopical specimens after dehydration by means of two changes

in xylene. The xylene increases the transparency of the testis

tissue.

Infiltration/Impregnation: the tissues were transferred into two

changes of molten paraffin wax in an oven which was maintained at

about 55 °C for removal of the xylene. Each infiltration step was

left for about 1 hour each.

Embedding: this is the fixing of a specimen within a mass of firm

material in order to facilitate the cutting of thin sections for

microscopy. An embedding mould was used to keep the specimen

intact.

Sectioning: cutting of excess wax to expose the embedded tissues

preceded this part. The rotator microtome was adjusted to 4 µ and

the microtome knife was placed at an angle of about 45 °C to the

block. The ribbon obtained was placed on albumenized slide. Water

was added to spread the section, thereafter the slides were

placed on hot plate to dry off the water on the slide and melt

the wax leaving the section.

Staining and Mounting: the slides were stained with haematoxylin

and eosin (H & E) and mounted with DPX (Dextrin-tricresol

phthalate xylene), and cover slip is used.

CHAPTER FOUR

MORPHOLOGICAL OBSERVATIONS

Morphological differences were absent in the testis of the

experimental group when compared with the control group. The body

weights of both group A and B were seen to have gradual increase,

morphological changes were seen on the faeces by the watery

nature of the faeces.

Below is a table and bar chart showing the mean weights of the

rats during administration of the drugs and prior to sacrificing

(on the day of sacrifice).

Table showing the mean body weight of each group before, during

and prior to administration.

Days Control Low

dose(0.3)

High

dose(0.6)

Before Day 1

Day 3

Day8

213.76

219.38

217.66

191.73

203.80

203.22

193.77

202.2

221.18

During Day 13

Day 15

Day 17

Day 23

217.30

217.76

217.78

223.30

197.58

220.87

214.45

223.55

195.17

193.48

208.67

216.62

Day 27

Day 29

Day 31

228.42

231.44

233.20

229.23

231.55

198

222.62

226.43

208.67

Prior to

sacrifice

Day 36 234.05 234.05 227.20

Chart 1: Bar chart showing mean body weight of each group

before, during and prior to administration.

Day1 Day3 Day8 Day13 Day15 Day17 Day23 Day27 Day29 Day31 Day360

50

100

150

200

250

CONTROLLOW DOSEHIGH DOSE

SEMEN ANALYSIS

CONTROL LOW DOSE HIGH DOSE52

54

56

58

60

62

64

Chart showing the sperm count of the animals after

sacrifice; Sperm count (x106/ml):

GROUP MEAN±SEM

CONTROL 56.4000±10.19902

LOW DOSE 57.5333±11.99841

HIGH DOSE 63.1667±14.46554

Table showing the sperm count of the animals after sacrifice;

Sperm count (x106/ml):

WEIGHTS OF THE TESTES AFTER SACRIFICE

Table showing the Mean Value of testicular weight

Groups RIGHT (g) LEFT

CONTROL 1.1200±0.08602 0.8200±0.22226

LOW DOSE 1.0000±0.14376 0.8500±0.13601

HIGH DOSE 1.1500±0.18753 1.0500±0.16073

Value expressed as mean ± SEM; p<0.05.

CONTROL LOW DOSE HIGH DOSE0

0.2

0.4

0.6

0.8

1

1.2

1.4

RIGHTLEFT

Chart showing the testicular weight of the animals after

sacrifice

PROGRESSIVITY

CONTROL

DOSE GP

M3 M6 M9 M11 M17

BAD* A B A B

MINIMAL

DOSE GP

M1 M2 M4 M8 M10 M15

D B A D A B

MAXIMAL

DOSE GP

M5 M7 M12 M13 M14 M16

B D B A A A

Table showing the progressivity of each animal in the three

groups

(*): pathological (diseased)

PROGESTERONE OESTRADIOL0

20

40

60

80

100

120


Chart showing the hormonal analysis of the animal in the three

groups

FSH LH0

5

10

15

20

25

30



groups

TESTOSTERONE0

0.1

0.2

0.3

0.4

0.5

0.6

0.7



groups

HORMONES CONTROL LOW DOSE HIGH DOSE

PROGESTERONE 16.68±1.97646 23.1500±1.12153 21.2933±1.82049

OESTRADIOL 33.7600±7.69919 1.0625E2±5.6825

8

25.0000±3.87298

FSH 3.5600±0.50359 3.3000±0.29439 4.9200±0.05060

LH 19.6250±2.75397 10.4500±1.80555 25.7000±2.15870

TESTOSTERONE 0.3825±0.04779 0.3800±0.04761 0.6000±0.03651

Table showing the hormonal analysis of the animal in the three

groups

HISTOLOGICAL OBSERVATION

Two different magnifications where used for each group, so as to

obtain a detailed histological sequence of the seminiferous

tubules.

Photomicrograph of the transverse section of the testis of a rat

in the control group (control grp, H&E x256)

ST: Seminiferous Tubule, LC: Leydig cell


in the control group (control grp, H&E x640) ST: Seminiferous

Tubule, B.V: Blood vessel

LC

B.V


in the low dose group (H&E x256)

ST: Seminiferous Tubule


in the low dose group (H&E x640

Photomicrograph of the transverse section of the testis of a

Wistar rat in the high dose group (H&E x256)

SP: Seminiferous tubule, L: Leydig cell


in the high dose group (H&E x640)

S.T

ST

L

B.M

ST: Spermatids, SC: Spermatocytes, SG: Spermatogonium, M: Myoid

cells

B.M: Basement Membrane

HAEMATOLOGICAL OBSERVATIONS

The tables below show the mean results of haematological analysis

of the blood samples of in the three groups.

PCV

CONTROL 45.400±1.02956

LOW DOSE GROUP 49.3333±2.30458

HIGHER DOSE GROUP 54.0000±2.16025

Showing the average mean values of Pack cell volume found in the

blood analysis of control group, minimal dose group, and maximal

dose group

RBC

CONTROL 7.8000±0.19769



Showing the average mean values of Red Blood Cells found in the


dose group

Hb

CONTROL 13.9400±0.36000



Showing the average mean values of Haemoglobin found in the blood

analysis of control group, minimal dose group, and maximal dose

group

MCHC

CONTROL 29.6000±0.50990



Showing the average mean values of mchc found in the blood


group

MCV

CONTROL 65.8000±3.55528



Showing the average mean values of mcv found in the blood


group

WBC

CONTROL 6.5000±0.24900



Showing the average mean values of white blood cells found in the


dose group

NEUTROPHILS

CONTROL 10.4000±1.24900



Showing the average mean values of neutrophils found in the blood


group

LYMPHOCYTES

CONTROL 88.6000±0.97980



Showing the average mean values of lymphocytes found in the blood


group

EOSINOPHILS

CONTROL 1.2500±0.2500



Showing the average mean values of eosinophils found in the blood


group

PLATELETS

CONTROL 3.3060E2±47.64829

LOW DOSE GROUP 3.0983E2±20.16667

HIGHER DOSE GROUP 3.2433E2±29.85877

Showing the average mean values of platelets found in the blood


group, MEAN±SEM Statistically significant: P< 0.05

PVC RBC Hb MCHC MCV WBC NEUT LYMP EOS PLT0

50

100

150

200

250

300

350

CONTROLLOW DOSE GRPHIGH DOSE GRP

Chart showing the haematological analysis

CHAPTER FIVE

Male wistar rats were randomly selected to three groups, one of

which served as a control group and the other groups were treated

with Nifedipine at different dose (0.3 for low dose and 0.6 for

high dose mg/kg respectively.

There was no significant difference (p<0.05) in body weight

between the control group and the treated groups of rats where

weight change is concerned. However, this is not in correlation

with the report of Olivari et al, which reported that the weight

would be significantly less after chronic Nifedipine therapy. [39]

Even the comparison of right and left testicular weight between

the control and treated groups showed insignificant effect of

Nifedipine (p=0.528 and 0.375), respectively.

Weight changes may indicate physiological abnormalities in the

function of certain organs or systems, which could further

develop into serious health complications. Decrease in testicular

mass is an index of reproductive toxicity and could indicate

atrophic and degeneration of tissue. [45] It was thus important

that proper weight monitoring was done as one of the steps to

ensure that the rats were in good health. This served to show

also that patient undergoing Nifedipine therapy will very

unlikely experience drastic and unhealthy weight changes.

Sperm count was important to analyze the toxicity of the drug in

study on the spermatozoa production in the gonads of the rats.

Damage to cells involved in spermatogenesis or lowered sex

hormones could be reasons behind changes in sperm production. [46]

Rats in the control and treated groups (low dose and high dose)

had a mean sperm count of 56.4000±10.19902 x106/ml,

57.5333±11.99841 x106/ml and 63.1667±14.46554 x106/ml.

respectively, Despite a higher sperm count in the treated groups,

the difference between the three groups was insignificant

(p=0.726) to draw a conclusion that the drug affects sperm

numbers, by increasing the quantity of sperms based on the dose

given, thus, low dose increase slightly and at a higher dose,

increases highly.

Motility between rats of control and treated groups were similar

to each group. Clearly, sperm motility was not affected by the

treatment of nifedipine.

Sperm quality is dependent on variables of the sperm, the count,

motility, progressivity and morphology. While the increase in

sperm numbers will essentially increase the chances of a sperm

fertilizing an oocyte, this relation will not be of importance if

sperm motility was low. It is important that the sperm is motile

in a progressive manner to be able to move up the cervix and

along the fallopian tube to eventually encounter and fuse with an

oocyte. Low sperm motility is often attributed to chemical-

induced testicular toxicity. [45]

The three groups showed the same order in the number of abnormal

sperm according to types of abnormality – sperm with banana-like

form, folded on itself, without a hook, amorphous and lastly

double-tailed. No sperm of the last category was observed.

Sperm morphology influences the degree of its motility. The

energy required for the sperm to be motile is generated in the

axoneme, whereas the tail is required for the progressive

movement of the sperm. Hence, abnormalities in the anatomy of the

sperm will largely impair its movements. An individual’s sperm

morphology may also reveal the overall health of the testes since

its production is within the testis.[47]

The close-level of normality in the treated and control groups

showed that nifedipine does not affect the morphology of sperm,

rather it slightly increases the quantity of sperm, the overall

sperm analysis showed that Nifedipine did not adversely affect,

if at all, the quality of sperm.

The testosterone level was significantly increased (p=0.012) in

the high dose group when compare with the control. As

testosterone is required in the later stages of spermatogenesis,

this could possibly be one of the factors behind a slightly high

mean sperm count in the treated group. The comparative mean

concentration of testosterone between the control and treated

(higher dose group) also indicates that Leydig cells were not

damaged or adversely affected by Nifedipine.

The Follicle Stimulating Hormone level (FSH) was statistically

significantly (p=0.028) increased in the higher dose group when

compared with the control group, and would require a larger

sample size to test its accuracy. It has been proposed that FSH

levels increase as a result of seminiferous tissue damage due to

higher inhibin secretion.[48] FSH is inversely proportional to

spermatogonia population but the concentration of FSH is higher

in the treated group, which also had a higher sperm count than

the control group. Which value was statistically significant,

this could be due to a higher incidence of spermatogonia which do

not mature to become spermatozoa in the treated group. Elevated

FSH concentration could also point to germinal epithelial damage

and can be linked severe oligospermia or azoospermia of bad

prognosis. [48]

The Luteinizing Hormone (LH) level was significantly increased

(p=0.037) in the higher treated dose group compare with the

control group LH is an important hormone as it stimulates Leydig

cells to produce testosterone. The higher testosterone level in

the treated group (higher dose) might have indicated that LH

levels in the treated group were higher than the control too.

This is, however, not the case. First of all, LH concentration of

an individual cannot be determined with just a single test.

Instead, it has to be done several times over a period of time as

LH is secreted in bursts which vary from 30 minutes to 480

minutes. Secondly, it has a short half-life. Therefore, a single

determination of LH can only be 50% accurate. [48]

High levels of LH may indicate hyperthyroidism or androgen

resistance syndrome.[48] Despite some fluctuations with the

treated groups in sperm count, testis weight and hormone levels,

the difference could be used to draw a conclusion on such effects

as they were statistically significant. And above hormone results

were against the experiment carried out by Almaida et al, which

reported that calcium channel blockers had effect on the

testicular weight in that calcium channel blockers suppress

spermatogenesis [49]. Also discovered was that calcium antagonist

(amlodipine) used in the treatment of hypertension decreased the

plasma follicle-stimulating hormone (FSH) and testosterone but

not luteinizing hormone (LH) [50]

The results of my findings was also against Iranloye et al, that

reported, that Nifedipine appears to have a deleterious effect on

sperm functions in rats which is not mediated by a change in

testosterone secretion. [25]

Progesterone level is significantly increased (p=0.049) in the

treated group (higher dose) compared with the control group.

The oestradiol level was highly significant (p=0.049) in the

treated group (low dose) compared with the control.

Histologically, the results showed that the drug Nifedipine had a

destructive effect on the seminiferous tubule in a the low dose

related manner for the result showed a progressive destruction of

the seminiferous tubules which was against the high sperm count.

Haematologically the PCV was significantly increased (p=0.049) in

the treated group (higher dose) while the level all other

haematological parameters were statistically insignificant.

CONCLUSION

Many literatures had reported that antihypertensives especially

calcium blockers have deleterious effects on the reproductive

functions but with my extensive findings i realized that

Nifedipine which is the leading drug among the group of calcium

blockers do not have adverse effects on the testicular functions

based on the sperm count, testicular weight, and the hormonal

analysis.

But histologically speaking findings appears to be limited;

Nifedipine appears to have showed damage on the testicular

architecture in the lower dose group as this not certain because

the appeared damage might be artefacts as there was no

disorganization, degeneration or destruction of the testicular

histo-architecture.

However, the present study shows with substantial evidence that

CCB does not induces antifertility effects in the males.

Although, this study was unable to provide a substantial answer

to the microstructure of the testis.

After treating rats with 0.3 and 0.6mg/kg per dose of Nifedipine

for 28 days, statistical significant difference were seen between

the control and treated rat groups in major parameters it is

therefore concluded that Nifedipine did not adversely affect the

male reproductive health of treated rats. However, further

studies are warranted to draw a definite conclusion.

RECOMENDATION

It is recommended that further work should be done to elucidate

the toxicity effects of Nifedipine on the testis morphology of

male adult Wistar rats.

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