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EFFECT OF CLOMIPHENE CITRATE ON THE OVARY OF

ADULT RAT

Abdullah.G.Alkushi, Mustafa. M. Sinna , Badr, A. Azab* , , Gamal,

E. AbdEl. Salam*, Essam, M. Eid* and Naser A. ElSawy**

Department of Anatomy, Faculty of Medicine, Umm Al-Qura University, Makkah, K.S.A *Anatomy Department, Benha Faculty of Medicine, Benha University

**Department of Anatomy and Embryology Faculty of Medicine, Zagazig University, Egypt

ABSTRACT This study evaluated the effect of clomiphene citrate on the ovary of adult rat. Eighty

selected female rats with regular estrous cycle checked with vaginal smear once daily.

Materials and methods: These rats were divided into two groups. Control group (40

rats) were injected by physiological saline 2m1 subcutaneous for 10 consecutive days.

The treated group was divided into two subgroups. Subgroup (A) was injected

subcutaneously by clomiphene citrate in low dose (0.3mg/kg body weight/day) for 10

consecutive days. Subgroup (B) was injected subcutaneously by clomiphene citrate in

high dose (3.0 mg/kg body weight/day) for 10 consecutive days. The rats of control

and treated groups were killed 24 hours after the last injection. The right ovary of

each rat was prepared for light and electron microscopes. The diameters of non atretic

and atretic follicles were measured by ocular micrometer in the serial sections of the

ovary at magnification of 100 and the numbers of non atretic, atretic follicles and

corpora lutea were counted. Statistical analyses were made using the t-test among the

studied groups.

Results: The total follicle numbers were 18.2 ± 2.6 for control group, 25.1 ± 4.23 for

group 2 and 28.3 ± 2.9 for group 3. The numbers of graffian, antral and multilaminar

follicles increased significantly in both experimental groups when compared with the

control groups (p<0.05), however there were no significant differences in follicle

numbers and number of unilaminar primary follicles among the experimental groups.

The diameters of non atretic and atretic follicles were measured by ocular micrometer

in the serial sections of the ovary at magnification of 100 and the numbers of non

atretic, atretic follicles and corpora lutea were counted. Statistical analyses were made

using the t-test among the studied groups. clomiphene citrate low dose does not affect

the number of non atretic follicles ranging from. 51-400 μm in diameter, but it

decreased the number of non atretic follicles larger than 400 μm in diameter. It

increased the number of atretic follicles ranging from 51 to 400 μm in diameter. The

high dose of clomiphene citrate increased the number of non atretic follicles ranging

from 51to 400 μm in diameter and decreased the non atretic follicles larger than 400

μm in diameter. The combination also increased the number of atretic follicles

ranging from 51to 600 μm in diameter. Both doses inhibited the ovulation rate

through decreasing the number of mature follicles larger than 400 μm in diameter,

increasing the atresia of follicles and decreasing the number of first type of corpus

luteum.

Conclusion: In spite of Clomiphine induced folliculogenesis in rat ovary and

increased the number of growing ovarian follicles but cause apoptosis and decrease

oocyte quality.

Keywords Clomiphene Citrate, Ovulation Induction, Oocyte Quality

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INTRODUCTION

Clomiphene citrate is the traditional first-line treatment for chronic anovulation that

characterizes polycystic ovary syndrome (PCOS) with half-life two weeks increasing

ovulation rate to a 60-85% and pregnancy rate to 10-20% per cycle [1]. Polycystic

ovaries (PCO) are endowed with an abnormally rich pool of growing follicles from

classes 1–5 (until 5 mm), probably due to intraovarian hyperandrogenism that

promotes excessive early follicular growth. Second, the selection of one follicle from

the increased pool of selectable follicles and its further maturation to a dominant

follicle does not occur, an abnormality that is called ―follicular arrest‖ (FA). The

reasons for this last phenomenon are unknown, although inhibition of the local effects

of FSH seems pivotal [2]. Both follicle abnormalities may be linked since we recently

reported a negative correlation between the small (2–5 mm) and larger (6–9 mm)

antral follicle number (FN) at ultrasound [3].

However, 20%–25% of PCOS women fail to ovulate with incremental doses of

clomiphene citrate (CC). In addition, clinical data revealed a discrepancy between

ovulation rates(75%–80%) and conception rates (30%–40%) during CC treatment [4].

CC is a synthetic compound composed of two isomers, enclomiphine and

zuclomiphine [5] Chemically, CC is a nonsteroidal triphenylethylene derivative that

exhibits both estrogen agonist and antagonist properties [6]. Estrogen agonist

properties are seen only if the endogenous estrogen level is extremely low. Otherwise,

CC acts as a competitive estrogen antagonist. Approximately 85% of an administered

dose is eliminated after approximately 6 days, although traces may remain in the

circulation for much longer [7]. Available evidence indicates that enclomiphene is the

more potent isomer and the one primarily responsible for the ovulation-inducing

actions of CC [8]. Enclomiphene levels rise rapidly after administration and fall to

undetectable concentrations soon thereafter. Zuclomiphene is a less active isomer; it is

cleared far more slowly [9].

CC is a very cheap and easily available drug and hence it is poor man‘s medicine for

the treatment of anovulatory dysfunction worldwide. More than 80% of women

ovulate when they are treated with CC [10]. There is a higher than expected incidence

of miscarriage in the conception cycle after CC treatment in humans [11]. Such a

discrepancy is believed to be due to anti-estrogenic effect of CC, particularly at the

level of ovary, cervical mucus and endometrium in human [12]-[14]. The anti-

estrogenic effects of CC may affect final stages of folliculogenesis by inducing

apoptosis in encircling granulosa cells and oocyte in ovary in vitro. This was further

strengthened by animal studies that CC treatment induced granulose cell as well as

oocyte apoptosis in rats in vivo [15].

The anti-estrogenic effects of CC and the mechanism by which CC exerts its direct

action at the level of ovary remains poorly understood. One study suggest that CC

induces apoptosis in human granulosa cells cultured in vitro [16] and reduces estradiol

17β as well as progesterone synthesis in rat [17] and in humans [18]. These finding

were further supported by animal studies that CC induces granulosa cell apoptosis and

reduced level of estradiol 17β in ovary as well as circulation in rat [19] as well as in

monkey [20].

The developed hypoestrogenic condition after CC treatment may induce generation of

ROS and thereby deterioration of oocyte quality by inducing apoptosis. This

possibility was further strengthened by observations that CC treatment increased

hydrogen peroxide (H2O2) level and reduced catalase activity in ovary. The increased

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level of H2O2 induced bax protein expression and DNA fragmentation both in

cumulus-granulosa cells and oocytes in rat as well as in human Animal studies

suggest that CC induces accumulation of ROS in ovary possibly by inhibiting catalase

activity leading to oocyte apoptosis in rat [21] - [23]. Further, studies in monkey as

well as in cattle suggest that gonadotropins directlystimulate PGE2 synthesis in

granulosa cells required for follicular rupture that results in ovulation [24]-[26].

There are several reports demonstrating various effects of clomiphene citrate as It is

the most commonly and first line of medicine used for the induction of ovulation in

anovulatory women worldwide,[27] and in spite of high ovulation induction with the

use of CC, the pregnancy rate is much lower. Such a discrepancy could be due to the

peripheral anti-estrogenic effect of CC, particularly at the level of ovary,

endometrium and cervical mucus.[28] Also old animals studies suggest that

clomiphene citrate inhibits ovulation in the normally cycling rats [29]. and monkeys

[30]. During final stages of folliculogenesis and ovulation ovary generates reactive

oxygen species (ROS) [ 31 ] Overproduction of ROS in the follicular fluid leads to

oxidative stress that may affect oocyte quality and thereby ART outcome [32]. These

increased levels of ROS are scavenged by enzymatic as well as non-enzymatic

antioxidants[ 33 ]. Vitamin D3 reduces H2O2 level by increasing catalase activity and

protects against CC-induced oocyte apoptosis [34].

So, the aim of this study is to evaluate the effects of clomiphene citrate on the ovary

of adult albino rats by light and electron microscopes

MATERIAL AXD METHODS

This study included 100 adult female albino rats weighing from (200-250gm). They

were kept individually in cages (30.5xl2xl0cm) with food (rice, chick, pea & wheat),

water ad libitum. After 15 days of acclimatization in the laboratory (25°C, light on 6

AM- 6 PM), the rats were checked with vaginal smears once daily between 10 -

11AM to determine the estrous cyclicity. Eighty rats with regular estrous cycle were

selected in this study. Strict care and cleaning measures were important factors for

keeping the animals in a normal healthy state.

The rats were divided into two groups: Group I (control group) included 40 rats. They were injected by physiological saline

2m1/day subcutaneously for 10 consecutive days.

Group II (treated group) included 40 rats. They were subdivided into two subgroups.

Each subgroup included 20 rats.

Subgroup (A) was injected subcutaneously by clomiphene citrate in low dose of 0.3

mg/kg body weight! day for 10 consecutive days.

Subgroup (B) was injected subcutaneously by clomiphene citrate in high dose of 3.0

mg/kg body weight! day for 10 consecutive days.

*Drug: Clomiphene citrate powder was obtained from pharmaceutical Arabic

company. It was suspended in physiological saline. For low dose, 10mg clomiphene

citrate was dissolved in 30ml saline. Each l ml = 0.3mg. For high dose, 10mg

clomiphene citrate was dissolved in l0ml saline. Each l ml = 1mg.

The rats of control and treated groups were killed by cervical dislocation 24 hours

after the last injection. The right ovary of each rat was removed and prepared for light

and electron microscopes. The right ovary of each rat was fixed in Bouin‘s fluid for

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about three days to achieve proper degree of hardening of soft tissue. Then the

specimens were dehydrated in ascending grades of alcohol (70%, 90%, 96% and

100%). After complete dehydration, the specimens were cleared in xylene,

impregnated in 3 changes of soft paraffin wax, each for one hour and then

impregnated in three changes of hard paraffin. Finally, the specimens were embedded

in hard paraffin. Serial sections 5-7 μm thick were cut. Some sections were treated

through haematoxylin and eosin stains. While other sections were subjected to

Masson‘s Tricbrome which is the best stain for demonstrating collagenous tissue and

smooth muscle fibers. Collagen fibers appear green-blue in colour, while the smooth

muscle fibers appear red in colour [35]. The diameter of all non atretic follicles was

measured by calibrated ocular micrometer in the serial sections of the ovary at a

magnification of 100. With the basement membrane of the stratum granulosum as the

boundary of the follicles, the mean of two perpendicular diameters measured was

taken as the mean diameter. The follicles were classified according to their diameters

into five classes: 30-50, 51-200, 201-400, 401-600 and > 600μm . The number of each

class was counted. The diameter of the atretic follicles were measured using the mean

of the maximum diameter and a diameter at right angle to it. The number of each

class of atretic follicles was counted.

Statistical analysis was made using the t-test. All comparisons were made between the

clomiphene treated and control groups. [36].

The atretic follicles were characterized by shrinkage, luteinization and loss of

nucleus. The corpora lutea were divided into three types. The first type was formed

during the current cycle, with a fluid-filled cavity. The second type was one cycle

older, without central cavity. The third type was two or more cycles older, with

degenerated pycnotic and vacuolated lutein cells, accompanied by large numbers of

fibrocytes and inflammatory cells. The number of each type of corpora lutea was

counted. The ovulation rate during the period of treatment was obtained from the

number of first and second types of corpora lutea [37].

For electron microscope, small pieces of the same extracted right ovary were fixed in

2% glutaraldehyde in phosphate buffer at pH 7.4. After 20 hours of fixation, the

specimens were washed in cooled 0.15 M Na cocodylate / Hcl buffer and postfixed in

1% osmium tetroxide. The specimens were then dehydrated and embedded in

Durcopan. Semithin sections were stained with 1% toluidine blue and used for

orientations in ElM. Ultrathin sections were mounted on the copper grids, stained with

4% uranyl acetate and 2% lead citrate and examined by a transmission electron

microscope [38].

RESULTS

The non atretic follicles The structure of non atretic follicles in the control and treated groups was found to be

similar. The growing follicle in the studied group is formed of primary oocyte which

consists of well defined cell membrane, pale cytoplasm and rounded or oval nucleus

with prominent nucleolus (Fig. 1,2 and 4). The cell membrane of oocyte is surrounded

by a pale zone called zona pellucida through which the microvilli of the cell

membrane protrude inside it (Fig. 2 and 5). Outside the zona pellucida, there is a layer

of cuboidal cells called corona radiata (Fig. 1,2 and 5). The granulosa cells surround

the oocyte. Each granulosa cell has a large irregular deep nucleus with pale cytoplasm

(Fig. 1,2 and 5). In between these cells, there is eccentric cavity called follicular

cavity (Fig. 2 and 4). In mature Graafian follicle, there is a large cavity which

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surrounds completely the oocyte and cumulus oophorus (Fig. 3), the granulosa cells

are surrounded by theca folliculi which contains fusiform stroma cells and connective

tissue (Fig. 1 and 2).

The non atretic follicles in control and treated groups are classified according to their

diameters into five classes. The first class ranges from 30 to 50pm in diameter. The

second class ranges from 51 μm to 200 μm in diameter. The third class ranges from

201 to 400 μm in diameter. The fourth class ranges from 401 to 600 μm in diameter.

The fifth class is more than 600 μm in diameter. The number of non atretic follicles

are counted. Statistical analysis were made using the t-test between the control and

treated groups. From table (1) it was found that the clomiphene injection at a dose of

0.3mg/kg body weight/day for 10 days (low dose decreases the number of non atretic

follicles ranging from 30 to 50 μm in diameter, 401-600 μm in diameter and larger

than 600μm in diameter. This decrease is highly significant (P<0.001). This low dose

not significantly affect the number of non atretic follicles ranging from 51 -400 μm in

diameter. From table (2) it was found that the clomiphene injection at a dose of

3mg/kg body weight/day for 10 days (high dose) increases the number of non atretic

follicles ranging from 30 to 50 μm in diameter (P < 0.05), 51 to 200 μm in diameter

(P < 0.05) and 201 to 400 μm in diameter (P < 0.001). These increases are statistically

significant. While combination decreases the number of non atretic follicles ranging

from 401 to 600 μm in diameter (P<0.001) and larger than 600 μm in diameter (P <

0.001). These decreases are statistically highly significant.

The atretic follicles : The morphological evidences of follicular atresia are noted in

the control and 2 treated groups. These changes are found in the oocytes and the

surrounding granulosa cells. The oocyte becomes shrinked with irregular cell

membrane and vacuolated cytoplasm (Fig. 6 and 7). In another ‗oocyte, the cell

membrane is irregular. The cytoplasm contains large vacuoles with loss of its nucleus

and the zona pellucida disappeared (Fig. 8). In another atretic follicle, the oocyte is

degenerated to form a small atretic body. The space of oocyte is infiltrated by

inflammatory cells (Fig. 9). The granulosa cells are infiltrated by inflammatory cells

and contained areas of degeneration (Fig. 7 and 9). In electron micrograph, the

mitochondria in the cytoplasm of oocyte are vacuolated (Fig 10). The cytoplasm of

granulosa cells contains many vacuoles of different sizes, but its nucleus is still intact

(Fig. 11). The atretic follicles in the control and treated groups are classified

according to their diameters into five classes as the non atretic follicles. Their

numbers are counted and statistical analyses are made using the t-test between control

and treated groups. From table (3) it was found that the clomiphene injection at a dose

of 0.3 mg/kg body weight/ day for 10 days (low dose) increases the number of atretic

follicles ranging from30 to 50 μm in diameter (P < 0.001), 51-200 μm in diameter (P

<0.001), 201- 400 μm in diameter (P < 0.05). while this low dose decreases the

number of atretic follicles ranging from 401 to 600 μm in diameter (P<0.001). This

decrease is statistically highly significant. The atretic follicles ranging from 30-50μm

and larger than 600 μm in diameter are absent in the control group. From table (4) it

was found that the clomiphene injection at a dose of 3mg/kg body weight/day for 10

days( high dose) increases the number of atretic follicles ranging from 51- 200 μm in

diameter (P <0.001), 201-400 μm in diameter (P<0.001) and 401 -600 μm in diameter

(P<0.001). These increases are statistically highly significant. The atretic follicles

ranging from 30 to 50 μm in diameter are absent in this treated group.

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The corpora lutea: Their different types are found in the control and 2 treated groups

and their structures are similar in these studied groups. Structurally, the corpora lutea

are divided into three types. The first type is formed of granulosa lutein cells without

clear cell boundaries. It contains a fluid filled cavity without connective tissue inside

it (Fig. 12). This type is formed during the current cycle. The second type of corpus

luteum is formed of granulosa lutein cells without central cavity (Fig. 13). This type is

formed during one cycle older. The third type is formed of degenerated granulosa

cells and fibrous connective tissue (Fig. 14). The granulosa lutein cells contain many

vacuoles in their cytoplasms. In between these lutein cells, there are fibroblasts with

spindle shaped nuclei (Fig. 15). Some lutein cells contain pycnotic nucleus and many

lamellae in its cytoplasm (Fig. 16). This type of corpus luteum is formed two or more

cycles older. From table (5), it was found that the clomiphene citrate in low dose

decreases the number of first type of corpora lutea (p <0.001), but does not markedly

affect the number of second and third types of corpora lutea. From table (6), it was

found that the clomiphene injection at a dose of 3mg/kg body weight/day for 10 days

(high dose) decreases the number of first type of corpora lutea (p< 0.001), but slightly

increases the number of 2‘ and 3rd types, which is statistically not significant.

Fig.(1): A photomicrograph of a section of adult control rat ovary (with regular

estrous cycle) showing : Non atretic growing follicle deep to the germinal epithelium

(e). It contains primary oocyte with cell membrane (m), pale cytoplasm (y), central

nucleus (n) and prominent nucleolus (u).The oocyteis surrounded by granulose

cells(r) and the theca folliculi (t). Note the interstitial cells (i) and the stroma cells

(s).(Masson ‗s Trichrome X 200).

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Fig.(2): A photomicrograph of a section of adult rat ovary treated by 0.3 mg

clomiphene citrate (low dose) showing : Non atretic growing follicle contains primary

ooeyte with cell membrane (m), pale cytoplasm (y), nucleus (n). it is surrounded by

zona pellucida (z), corona radiata (d), granulosa cells (r), theca folliculi (t). In

between the granulosa cells, there is eccentric follicular cavity (v). Note the stroma

cells (s)and interstitial cells (i). (Masson ‗s Trichrome X200).

Fig.(3): A photornicrograph of a section of adult rat ovary treated by 3 mg

cloiniphene citrate (high dose) showing : Non atretic mature Graffian follicle (g)

which protrudes near the surface epitheliuin (e). It contains secondary oocyte (o)

which is surrounded by zona pellucida, corona radiata, granulosa cells (r) and large

follicular cavity (v). Note multiple corpora lutea (c). (Masson ‗s Trichrome X 100).

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Fig.(4): Electron micrograph of a section of control adult rat ovary (with regular

estrous cycle) showing primary oocyte (arrow) with oval nucleus (n) and pale

cytoplasm (y). It is surrounded by multiple granulose cells (r) with irregular nuclei

(n1). Note a cavity (v) between the granulosa cells.(Magnification X 4000).

Fig.(5): Electron micrograph of a section of adult rat ovary treated by 0.3 mg

clomiphene citrate showing part of nucleus (n) of primary oocyte. Its cell membrane

forms microvilli (*) which extend inside the zona pellucida (z). Note the multiple

granulose cells (r) with irregular nuclei. .(Magnification X 4000).

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Fig.(6): photomicrograph of a section of adult control rat ovary (with regular estrous

cycle) showing The atretic follicle contains shrinked primary oocyte with irregular

cell membrane (m). its cytoplasm contains a cavity (v). Note the presence of zona

pellucida (z), corona radiata (d), granulosa cells (r), interstitial cells (i), stromal cells

(s), and evident theca folliculi (t) (Masson ‗S Trichrorne X400).

Fig. (7): A photornicrograph of a section of adult rat ovary treated by 3 mg

clomiphene citrate (high dose) showing: atretic oocyte is shrinked with irregular cell

membrane (m). The zona pellucida (z) and the corona radiata (d) are still present. In

between the granulosa cells (r), there are some inflammatory cells (arrows). Note the

part of follicular cavity (v). (H & E X4OO):

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Fig. (8): A photomicrograph of a section of adult rat ovary treated by 0.3mg

clomiphene citrate showing :the atretic oocyte is shrinked with irregular cell

membrane (m). Its cytoplasm contains large cavity (v), with degeneration of its

nucleus. The zona pellucida is disappeared. In between the granulosa cells (r), there

are multiple small cavities (v). (Masson ‗S Trichrorne X 400).

Fig. (9) : A photomicrograph of a section of adult rat ovary treated by 0.3mg

clorniphene citrate showing : Degeneration of the oocyte that is in the form of small

atretic body (b). The spaces of oocyte is infiltrated by inflammatory cells (arrows).

The zona pellucida and corona radiata are disappeared. Note the patches of cavitations

(v) in the granulosa cells (r). (Masson ‗S Trichrome x 400).

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Fig.(10): Electron micrograph of a section of adult control rat ovary (with regular

estrous cycle) showing: Atretic oocyte containing vacuolated mitochondria (m) in its

cytoplasm. The nucleus (n) contains nucleolus (u). (Magnification x 2000).

Fig.(11): Electron micrograph of a section of adult rat ovary treated by 3 mg

clomiphene citrate (high dose) showing : Multiple granulosa cells (r) (of atretic

follicle) with active irregular nuclei (n). Their cytoplasm contain multiple vacuoles (v)

of different sizes. (Magnification x 2000).

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Fig.(12): A photomicrograph of a section of adult control rat ovary (with regular

estrous cycle) showing : The corpus luteum (c1) with a cavity (v) filled with fluid.

The cell boundaries of the granulosa lutein cells (r) are ill defined. The corpus luteum

is surrounded by connective tissue (t). (Masson ‗s Trichrome x 100).

Fig.(13): A photomicrograph of a section of adult rat ovary treated by 0.3mg

clomiphene citrate showing : Large size of corpus luteum (C2) without cavity. Note

others small corpora lutea (c). (Masson ‗S Trichrone X 100).

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Fig.(14): A photomicrograph of a section of adult rat ovary treated by 3 mg

clomiphene citrate (high dose) showing : The corpus luteum contains multiple patches

of degeneration (d)of granulosa lutein cells (r). Its central part contains fibrous tissue

(f and small cavity (v). (Masson ‗S Trichrome X200).

Fig.(15): Electron micrograph of a section of adult rat ovary treated by 3 mg

clomiphene citrate (high dose) showing Multiple granulosa lutein cells (r) in corpus

luteum. One of them contains many vacuoles (v) which surround the nucleus (n).

Note the fibroblast (0 with spindle shaped nucleus. (Magnification x 1500).

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Fig.(16): Electron micrograph of a section of adult control rat ovary (with regular

estrons cycle showing: Evidences of degeneration of granulosa lutein cell (g) in the

corpus luteurn. The nucleus (n) is pycnotic with shrinkage of its size. The cytoplasm

contains many lamellae (L). Note other intact granulosa lutein cells (r).(Magnification

x 4000).

Table (1): Comparison between the number of non atretic follicles per ovary in the

control and treated groups by 0.3mg/kg body weight / day clomiphene citrate (low

dose) for 10 consecutive days.

Studied group Control group

( n=40)

Treated group 0.3 mg /

kg (n= 20)

T P

Non atretic follicle X +SD X +SD

30-50μm 23.4 +5.71 15.25 +3.01 7.24 <0.001

51-200, μm 95.1 +23,67 101.05 +19.99 1.02 >0.05

201-400, μm 13.95 +3.46 16 +4.38 1.86 >0.05

401-600 μm 12.3 +3.19 3.1 +0.79 17.23 <0.001

> 600μm 9.35 +2.38 1.12 +0.33 21.45 <0.001

P=0.05 - P 0.05 non significant.-- P < 0.05 - significant.-- P< 0.01-Highly significant.

Table (2) : Comparison between the number of non atretic follicles per ovary in the

control and treated groups by 3 mg clomiphene citrate for 10 consecutive days.

Studied group Control group

( n=40)

Treated group 3 mg /

kg (n= 20)

T P

Non atretic follicle X +SD X +SD

30-50μm 23.4 +5.71 29 +6.73 2.85 < 0.05

51-200, μm 95.1 +23,67 110.95 +24.73 2.37 < 0.05

201-400, μm 13.95 +3.46 20.35 +4.52 11.45 <0.001

401-600 μm 12.3 +3.19 6.7 +1.63 9.01 <0.001

> 600μm 9.35 +2.38 1.75 +0.79 18.29 <0.001

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Table (3): Comparison between the number of atretic follicles per ovary in the control

and treated groups by 0.3mg/kg body weight/day clomiphene citrate (low dose) for 10

consecutive days.

Studied group Control group

( n=40)

Treated group 0.3 mg /

kg (n= 20)

T P

Atretic follicle X +SD X +SD

30-50μm 0 +0 25.2 +5.32 21.2 <0.001

51-200, μm 3.1 +0,9 12.4 +2.66 15.19 <0.001

201-400, μm 12.5 +2.87 15.5 +3.27 3.49 < 0.05

401-600 μm 5.15 +1.25 2.8 +0.7 9.33 <0.001

> 600μm 0 +0 1 +0 0 0

Table (4):Comparison between the number of atretic follicles per ovary in the control

and treated groups treated groups by 3 mg clomiphene citrate (high dose) for 10

consecutive days

Studied group Control group

( n=40)

Treated group 3 mg /

kg (n= 20)

T P

Atretic follicle X +SD X +SD

30-50μm 0 +0 0 +0 0 0

51-200, μm 3.1 +0,9 14.95 +4.01 13.07 <0.001

201-400, μm 12.5 +2.87 20.8 +5.55 6.28 <0.001

401-600 μm 5.15 +1.25 8 +2.2 5.37 <0.001

> 600μm 0 +0 1 +0 0 0

Table (5) : Comparison between the number of corpora lutea per ovary in the control

and treated groups by 0.3mglkgm body weight/day clomiphene citrate (low dose) for

10 consecutive days.

Studied group Control group

( n=40)

Treated group 0.3 mg /

kg (n= 20)

T P

No. of corpus luteum X +SD X +SD

First type 4.15 +1.03 1.8 +0.62 11.02 <0.001

Second type 3.95 +1.65 3.25 +1.81 1.45 > 0.05

Third type 8.15 +2.36 9.55 +4.46 1.32 > 0.05

Table (6): Comparison between the number of corpora lutea per ovary in the control

and treated groups treated by 3 mg clomiphene citrate (high dose) for 10 consecutive

days

Studied group Control group

( n=40)

Treated group 3 mg /

kg (n= 20)

T P

No. of corpus luteum X +SD X +SD

First type 4.15 +1.03 1 +0 19.34 <0.001

Second type 3.95 +1.65 4 +0.92 0.15 > 0.05

Third type 8.15 +2.36 10.02 +4.3 1.81 > 0.05

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DISCUSSION In the present study, the clomiphene citrate was injected in normal cyclic rats at a low

dose (0.3mg/kg/day) in one group and at a high dose (3.0mg/kg/day) in another group.

Both doses did not affect the structure of non atretic follicles. This finding was similar

to the results of Birkenfeld, et a!. (1985) who studied the effect of clomiphene citrate

on the rabbit ovary and found that the folliculogenesis appeared normal and the

morphology of follicles were within normal ranges.

In the present study, the low dose of clomiphene citrate did not affect the number of

non atretic follicles ranging from 51 to 400pm in diameter. In contrast, the high dose

of clomiphene citrate increased the number of non atretic follicles ranging from 30 to

400 μm in diameter. In both doses, the clomiphene citrate decreased the number of

non atretic follicles larger than 400 μm in diameter. These results were in agreement

with that of Nakano, et al. (1982). Sahu (1987) reported that the low dose of

clomiphene citrate decreased the number of non atretic follicles larger than 400 μm in

diameter. The high dose of clomiphene citrate increased the number of follicles

between 201 to 400 μm in diameter and decreased the number of non atretic follicles

larger than 400 μm in diameter. Nakano, et al (1982) reported an inhibition of follicle-

stimulating hormone (F.S.H.) induced ovarian follicular growth by cis-clomiphene in

hypophysectomized immature female rats.

In the present-study, both doses of clomiphene citrate did not affect the structure of

atretic follicles. The signs of atresia were noticed in control and treated groups. These

signs were in the form of shrinked oocyte, loss of its nucleus, vacuolated cytoplasm

and vacuolated mitochondria. The granulosa cells were infiltrated by inflammatory

cells and contained areas of degeneration. These results were similar to that of Sahu

(1987) and Devine, et al. (2000) who reported that the atresia of follicles revealed

nuclear segregation, cytoplasmic and nuclear condensation. The granulosa cells

contained vacuolated cytoplasm with loss of cristae in mitochondria.

In this work, the low dose of clomiphene citrate increased the number of atretic

follicles ranging from 30 to 400 μm in diameter, while it decreased the number of

atretic follicles ranging from 401 to 600 urn in diameter. In contrast, the high dose of

clomiphene citrate increased the number of atretic follicles ranging from 51 to 600 μm

in diameter, these results coincide with Sahu (1987) and Moon, et a!. (1989) who

reported that the clomiphene citrate increased the atresia of follicles and cancel the

cessation of estrous cycle. Sahu (1987) reported that the low dose of clomiphene

citrate increased the atresia of small sized follicles less than 200 μm in diameter.

While the high dose of clomiphene citrate increased the atresia in the medium sized

follicles ranging from 201 to 400 μm in diameter. From the results of this study and

previous studies, the clomiphene citrate inhibited the follicular growth and stimulated

the atresia of follicles. These results can be explained by Biljan, et al (1999) and

Bostrom, et a!. (2000). They proved that the clomiphene citrate has both estrogenic

and anti-estrogenic effects. Estrogen reduces the number of ovulated oocytes in rats

and induces atresia of the preovulatory follicles. This action is produced by

clomiphene citrate, so it has estrogenic effect. Estrogen also is responsible for

repeated cell division of follicular cells and so stimulate the growth of follicles along

with FSH and LH. This action is against the action of clomiphene citrate which

inhibits the follicular growth, so it has antiestrogenic effect.

In the present study, the low and high doses of clomiphene citrate did not affect the

structures of corpora lutea when compared with control group. These findings were in

line with the results of Patton and Stouffer (1991) who described the structures of

Vol 65, No. 12;Dec 2015

42 Jokull Journal

three types of corpora lutea. Also, the low and high doses of clomiphene citrate did

not affect the number of second and third types of corpora lutea. So the ovulation rate

during the initial period of treatment did not affected. In contrast, both doses of

clomiphene citrate decreased the number of first type of corpus luteum. So the

ovulation rate decreased during the final period of treatment. These results were

similar to that of Sahu (1987) who suggested that the mechanism by which this drug

acts on the corpus luteum is not clear, but maybe due to its estrogenic effect.

In conclusion: In adult rats with regular estrous cycle, both doses of clomiphene

citrate decreased the ovulation rate through: decreasing the number of mature follicles

larger than 400 μm in diameter, increasing the atresia of follicles and decreasing the

number of first type of corpus luteum. In the wild rat 0.3 mg clomiphene /kg was

mostly effective on small, nonantral follicles (< 200 μm ), while 3 mg clomiphene /

kg mainly acted on pre-antral to just -antral follicles (201 — 400 μm ), and 10 days of

treatment with clomiphene with the doses employed was not sufficient to inhibit or

decrease ovulation in rats

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