Expression of Estrogen Beta (Ers Β) Receptor and Ovarian

Histopathology Changes in Rats (Rattus Norvegicus) Ovarian

Hypofunction Model

Aulia Firmawati

, Mitra Artha Kurnia Hutabarat

and Herlina Pratiwi

Reproduction Laboratory, Faculty of Veterinary Medicine, Brawijaya University, Indonesia

Undergraduate Students of the Faculty of Veterinary Medicine, Brawijaya University, Indonesia

Embryology Laboratory Faculty of Veterinary Medicine, Brawijaya University, Indonesia

Keywords: ERs β, Folliculogenesis, GnRH antagonists, Ovarian hypofunction, Rats

Abstract: One of the reproductive disorders that are often found in breeders in Indonesia is ovarian hypofunction.

Ovarian hypofunction is a pathological condition in the ovary that is characterized by a decrease in ovarian

function that causes inhibition of folliculogenesis and failure of ovulation. The purpose of this study was to

develop ovarian hypofunction animal models through cetrorelix acetate induction and observe their effects on

the expression of estrogen beta receptors (Ersβ) and histopathological changes in the ovaries. This study used

three groups of female Wistar strains (Rattus norvegicus), with ages 8-10 weeks, and body weight 150-180

grams. The treatments in this study included a control group (KN) without cetrorelix acetate, the first group

(P1) was treatment group with an injection of cetrorelix acetate 0.009 mg/kg BW, and the second group (P2)

was treatment group with an injection of cetrorelix acetate exposure 0.0135 mg/kg BW. Expression of beta

estrogen receptors (Ersβ) in the ovaries was analyzed by immunohistochemical methods, and the data were

analyzed using the BNJ test (p <0.05). The ovarian histopathological changes were analyzed by the

hematoxylin-eosin (HE) staining method, then analyzed qualitatively. The results of this study indicate that

the treatment groups P1 and P2 with GnRH antagonists differ significantly compared to the negative control

group. The P2 treatment group had the highest reduction in estrogen receptor expression with Ers β by 92.2%.

The result of histopathological in P1 and P2 treatment groups were able to inhibit the development of antral

follicles. The conclusion of this study is that cetrorelix acetate as GnRH antagonists can reduce estrogen

receptor expression and inhibit folliculogenesis in ovarian histopathology.

1 INTRODUCTION

The highest incidence of reproductive disorders in

community farms in Indonesia is ovarian

hypofunction. The incidence rate of ovarian

hypofunction in East Java in 2010-2017 was 9.28%

(Hermadi, 2015), while ovarian hypofunction in

Rembang Regency, Central Java, was 6.25%

(Sutiyono et al., (2017) and in the Regency Enrekang,

South Sulawesi Province at 71% (Yahya, 2017) and

in Jambi province it was reported that the incidence

of ovarian hypofunction had the second- highest

percentage of events at 19.32% (Rosadi et al., 2018).

Ovarian hypofunction is a pathological condition

characterized by a decrease in ovarian activity is

showing signs of lust and producing ovum. Ovum

produced under conditions of ovarian hypofunction

generally has low fertility, making it difficult and

even cannot be fertilized despite good quality

spermatozoa. Ovarian hypofunction is the most

frequent reproductive disorder due to

mismanagement of feed and reproductive hormone

mechanisms (Pradhan and Nakagoshi, 2008).

According to Gitonga, (2010) Cows that experience

ovarian hypofunction are often found with the

occurrence of silent heat, sub estrus (lust without

ovulation), irregular lust cycle and the onset of

postpartum lust. Ovarian hypofunction that is not

treated immediately can cause ovarian atrophy that is

irreversible.

In conditions of ovarian hypofunction Ovarian

histopathological features can show ongoing

folliculogenesis in the ovary. Ovarian follicles based

on their development are divided into several levels

including primordial follicles, primary follicles,

Firmawati, A., Hutabarat, M. and Pratiwi, H.

Expression of Estrogen Beta (ERs ) Receptor and Ovarian Histopathology Changes in Rats (Rattus norvegicus) Ovarian Hypofunction Model.

DOI: 10.5220/0009586400410048

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 41-48

ISBN: 978-989-758-483-1

growing follicles, and de Graaf follicles. Disrupted

folliculogenesis can eliminate follicles in various

stages and then develop into follicular atresia.

Ovarian hypofunction will show a delay in the

development of primary follicles, secondary follicles,

and the presence of atresia follicles in quite high

numbers. Folliculogenesis that occurs is difficult to

reach the ovulation stage so that no tertiary follicles,

de Graaf follicles or corpus luteum are found

(Hestianah et al., 2014).

The production of the hormone estrogen

continues to increase when de Graaf follicles will

ovulate. The effect of the hormone estrogen in tissue

is closely related to the activation of the estrogen

receptor that the tissue has. Obstructed ovulation will

certainly affect the production of the hormone

estrogen followed by activation of its receptors. The

production of the hormone estrogen can decrease

because follicles do not develop thereby reducing

estrogen receptor activation in some target tissues.

Estrogen can diffuse into ovarian, uterine, and

mammary glands systemically (Caldon, 2014).

Ovarian hypofunction can be caused by the

disruption of hormonal regulatory mechanisms by

suppressing reproductive hormone synthesis. One of

the causes of disruption of the reproductive hormone

mechanism is the use of GnRH antagonists namely

Cetrorelix acetate in humans which aims to suppress

ovum production so that it can cause a decrease in

ovarian function or ovarian hypofunction. GnRH

antagonists are indicated to suppress the luteinizing

hormone surge (LH) that is too early in women. LH

suppression occurs due to a decrease in the number of

activated GnRH receptors on gonadotropin cells

(Beckers and Reila, 1997).

2 MATERIALS AND METHODS

2.1 Tools and Materials

The tools used in this study include therumo® 1 cc

syringes, terumo® 3 cc syringes, blades, surgical

scissors, anatomical tweezers, serological tweezers,

surgical boards, petri dishes, and pins, microtomes,

incubators, and optilab microscopes.

Materials used in this study include feed in the

form of rabbit pellets (SP®), husks, and sufficient

water, Phosphate Buffer Saline (PBS), formalin,

alcohol, xylol, 0.9% physiological NaCl, paraffin,

Hematoxillin-Eosin dye, and entellan, PBS, primary

antibody ERS β brand abcam® (ab288), secondary

antibody labeled peroxidase, normal Horse serum

2.5% brand abcam® (ab7484), hydrogen peroxide,

methanol and chromogen diaminobenzidine

tetrahydrochloride (DAB) brand abcam® (ab64238).

2.2 Preparation of Animal Ovarian

Hypofunction Models

Rats were acclimatized for 7 days to be able to adapt

to the new environment. Rats are given feed in the

form of rabbit pellets (SP®) and the provision of

drinking water by adlibitum. Rats were divided into 3

treatment groups including: control group (KN), the

untreated group was only given a placebo NaCl for 17

days. The treatment group was a cetrorelix acetate

dose of 0.009 mg/kg BW given for 17 days (P1). The

third group was the treatment group with a dose of

cetrorelix acetate of 0.0135 mg/kg BW.

2.3 Vaginal Swab Preparations

The method of making vaginal swab preparations

carried out in this study by immersing cotton bud in

physiological NaCl then the mice to be swab vagina

will be placed in a dorsal lying position, after which

a vaginal swab is carried out by inserting a cotton bud

soaked physiologically NaCl in the vagina by rotating

360

. Then, the cotton bud is removed on the slide

and allowed to dry and then fixed using alcohol. The

preparations that have been fixed with alcohol and

which have dried then are stained with Eosin

Negrosin for 15 minutes, then rinsed with running

water with a small flow of water and rinsed slowly.

After that, the results of vaginal swabs are observed

under a microscope with a magnification of 100x and

400x to see vaginal cells. In this study, a vaginal swab

was carried out before interfering with the GnRH

antagonist to equalize the estrous cycle of rats before

starting treatment in this study.

2.4 Collect of Ovary Organs

The rats were euthanized by cervical dislocation.

Dislocation is a condition where the joints completely

change position without contact with each other

(Manuabada and Putu, 2017). Neck dislocation is a

physical euthanasia technique by separating the joints

between the skull and brain from the spinal cord in

the vertebrae. This technique is done by placing the

mouse in a ventral fall position, the nape is held with

tweezers, then pulled at the base of the tail and the

whole body until the mouse dies (Isbagio, 1992).

The rats that have died are dissected vertically

from the posterior abdomen to the anterior region and

then opening the abdominal and chest cavity. The

ovaries were taken together with the uterus, then

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

separate the ovaries from the mesovarium. The

ovaries are prepared from the surrounding fat and

washed with physiological NaCl.

2.5 Preparation of Ovarian

Hhistopathology

The histological preparations consist of fixation,

dehydration, clarification, paraffin infiltration,

embedding, sectioning, sticking to glass objects and

coloring. Histopathological changes that will be

observed in the description of ovarian histopathology

are folliculogenesis, histopathological changes

include necrosis, inflammation or other cell damage.

2.6 Observation of Ovarian Estrogen

Receptor Beta (ERsβ) Expression

Observation of the expression of estrogen beta

receptors (ERs β) was observed using the

immunohistochemical method. Ovarian

histopathological preparations without coloring are

incubated for > 24 hours to facilitate

deparaffinization. The preparations are soaked in

xylol 3 times for deparaffinization, removing paraffin

from the tissue as much as possible. The preparations

are then soaked in stratified alcohol in sequence

(95%, 90%,80%, 70%) for rehydration, ie re-entering

the liquid into the tissue making it easier to stain, then

stored in the refrigerator 30

C for > 24 hours. The

preparations are washed in distilled water 3 times

each for 3 minutes. The preparations are immersed in

3% hydrogen peroxide mixed with methanol in a

room temperature humidity chamber for 40 minutes,

then washed in PBS for over 3 x 3 minutes. The

preparations were incubated with 50% normal serum

horse as much as 50 µL for one night in a humidity

chamber at 3oC and rinsed again with PBS for 3 x 3

minutes.

Preparations added 40 µL (primary antibody)

anti-ERs β brand abcam® (ab288), for one night at

3oC, then rinsed again with PBS for 3 x 5 minutes.

The preparations are added with abcam® brand anti-

receptor secondary antibodies (ab7484), 40 µL for 60

minutes at room temperature, then washed with PBS

for 3 x 3 minutes. The preparation is dripped with one

drop peroxidase, left for 40 minutes, then washed

with PBS for 3 x 3 minutes.

The preparation is rinsed again with distilled

water. Preparations were added chromogen DAB

(3,3-diaminobenzidine tetrahydrochloride) abcam®

(ab64238) as much as 40 µL for 20 minutes at room

temperature then washed in distilled water 3 x 5

minutes and countered with hematoxylin for 1 minute

at room temperature. The preparation was washed

again in the water at pH 8, 3 x 1 minute and let it dry,

mounting using an entelan, then the preparation was

observed with an optical microscope.

ERs β in the ovary are observed by calculating the

average number of cells expressing β ERs.

Observation of β ERs in the ovary can be observed in

theca cells and granulosa cells. ERs β calculation is

done at 400x magnification with 5 visual fields per

slide and then analyzed with the help of Immunoratio

software (Ridwan et al., 2015).

3 RESULTS AND DISCUSSION

Effect of GnRH Antagonists on Estrogen Beta

Receptor Expression (ERsβ) in the Ovary Expression

of beta estrogen receptors (ERsβ) in the ovary can be

observed by immunohistochemical (CPI) methods.

Ovarian cells that express β ERs will be colored easily

brown to dark brown with a certain area. Expression

of beta estrogen receptors (ERs β) in the ovary will be

expressed in the cells of granulosa cells and germinal

epithelium. Physiologically, ERs β show more

expression than α ERs in the ovary (Wang et al.,

2000).

In this study, the expression of estrogen receptors

is expressed in the theca cells of ovarian follicles. The

estrogen receptor's expression is colored light brown

to dark brown, this indicates that the theca cells

express the presence of estrogen that binds to the

estrogen receptor in the theca cell. According to Cui

et al., (2013) theca cells are cells that synthesize the

formation of estrogen in cooperation with granulosa

cells during the process of folliculogenesis and

ovarian follicle maturation. Estrogen produced will

stimulate proliferation and differentiation of theca

cells and granulosa cells in ovarian follicles in the

process of ovarian follicle maturation. Theca cell

differentiation is influenced by several factors, one of

which is the Bone Morphogenic Proteins (BMP) and

Growth Differentiation Factor-9 (GDF-9) expressed

by estrogen-estrogen receptor binding in the

transcription process (Young et al., 2010).

The average calculation result of ERs β expression

obtained in the control group was 62.58 ± 1.4, the P1

group was 33.80 ± 1.6, and the P2 group was 4.87 ± 1.6.

ERs expression of β in the P1 and P2 groups decreased

compared to the control group.

Data obtained from the expression of ERs β showed a

greater control group (62.58 ± 1.4) when compared to

the P1 group (33.80 ± 1.6) and P2 group (4.87 ± 1.6).

The ERs β expression values can be seen in table 1.

Expression of Estrogen Beta (ERs ) Receptor and Ovarian Histopathology Changes in Rats (Rattus norvegicus) Ovarian Hypofunction

Model

Table 1. Effects of GnRH antagonists on the expression of

ERsβ in rats ovarian hypofunction

Group

Average Expression of ERs β

standard of deviation

KN (placebo)

62,58 ± 1.4

P1 (cetrorelix acetat

33,80 ± 1.6

0.009 mg/Kg BW)

P2 (cetrorelix acetat

4,87 ± 1.6

0.0135 mg/Kg BW)

Information :

The notation a, b, c shows that there is a significant difference

between one treatment and another

Based on the table above shows that the higher the

dose of GnRH antagonists, the lower the expression

of ERs β in rat ovaries, the decrease in estrogen

receptor beta receptor is due to the response of GnRH

antagonists, which gives inhibition of anterior

pituitary secretion to secrete primer reproductive

hormones. Cetrorelix acetate as one of the GnRH

antagonists competes with GnRH in the body to bind

to membrane receptors on pituitary cells and inhibits

the release of Follicle Stimulating Hormone (FSH)

and Luteinizing Hormone (LH), thereby delaying LH

surge, and it will result in inhibition of ovulatory

processes (Rodney, FSH) and Luteinizing Hormone

(LH).

The existence of obstacles in these pituitary cells

causes obstacles in the process of folliculogenesis.

Hampered the process of eating folliculogenesis will

result in a decrease in estrogen production which will

then cause a decrease in the binding of estrogen

hormones to estrogen receptors, one of which is ERs

Beta. With this decreased hormone-receptor binding,

the expression of beta ERs becomes inactive and

unexpressed, this is in accordance with the results in

this study that decreased expression of beta receptors

in rat ovaries treated with GnRH antagonists. In this

case, the administration of cetrorelix acetate affects

the decrease in expression of beta estrogen receptors

in the ovary. The β ERs expression in ovaries can be

seen in (Figure 1.)

In this study, it was shown that the decrease in the

ERs expression of the control group was greater than

the P1 and P2 groups. Decreased expression of ERs β

in the treatment groups P1 and P2 are caused by

GnRH antagonistic intervention. Exogenous GnRH

antagonists that are competitive with endogenous

GnRH in binding to estrogen receptors will inhibit

endogenous GnRH to inhibit FSH and LH secretion

in the anterior pituitary. This inhibition of FSH and

LH causes a decrease in the level of FSH and LH in

the blood. The effect of FSH secretion inhibition

inhibits the process of folliculogenesis and inhibits

ovulation due to low levels of LH in the blood. So that

this causes estrogen which is synthesized during the

formation, development, maturation of follicles until

ovulation occurs. It will experience a disruption in

secreting the hormone estrogen in the blood

circulation so that it experiences inhibition of

folliculogenesis and inhibition of ovulation.

Figure 1. Expression of β ERs by the Imunnohistochemical

method in the ovary (400x) Information:

1: Control (placebo)

2: Group P1 (Cetrorelix acetate 0.009 mg / kg BW)

3: Group P2 (Cetrorelix acetate 0.0135 mg / Kg BW)

: Estrogen Receptor β

In addition, a decrease in estrogen production will

cause the lack of estrogen hormone binding with

estrogen receptors both ERs β and ERs that is formed

so that gene expression also decreases and can cause

no emergence of estrous performance. ERs β work is

known to affect gamete transport, development, cell

growth in the reproductions channel with the presence

of GnRH inhibition, which can cause gamete

transport disorders, one of which is the disruption of

the process of folliculogenesis, LH surge disruption

which causes obstruction of ovulation, cell growth

and the growth of the female reproductive tract.

3.1 Effects of GnRH Antagonists on

Changes in Ovarian Histopathology

In this study, we did a vaginal swab first to determine

the last reproduction phase before the euthanized.

And based on the results of a vaginal swab conducted

before euthanasia showed experimental animals in

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

the control group (KN), group P1, and group P2 was

in the metestrus phase. Physiologically in the

metestrus phase is characterized by the development

of de Graaf follicles that have ovulated to the corpus

luteum. The process of maturation of the corpus

luteum produces progesterone whose concentration

will peak on the 6th day after ovulation. Progesterone

plays a role in influencing the reproductive tract to

maintain pregnancy in the event of fertilization

(Akbar, 2010).

The control group showed a follicle that had just

ovulated which was called the corpus haemoragicum

which would develop into the corpus luteum (Figure

2A). Follicles that successfully reach ovulation are

dominant follicles that have gone through 2 to 3

follicular waves during the estrous cycle period.

Dominant follicles produce estrogen which can

suppress the growth of other small follicles

(Jainudeen and Hafez, 2000). Besides copus

haemoragicum, corpus luteum and primary,

secondary and atresia follicles are also found. This

shows that the control group that was only given a

placebo still showed normal ovarian activity.

A follicular wave is defined as a process of

follicular growth that is synchronous with several

small follicles. One of these small follicles is selected

to grow into a dominant follicle, while the other small

follicles will stall their growth into follicular atresia.

Atresia follicles that are formed will cause a new

follicular wave that is the second follicular wave. The

dominant follicle in the second follicular wave will

become anovulatory, while the dominant follicle in

the third wave will ovulate (Siregar, 2010). Other

follicles such as primary follicles and secondary

follicles encountered in the negative control group

indicate a new wave of follicles in the ovaries.

P1 group by giving Cetrorelix acetate dose 0.009

mg/kg BW showed that there was a small number of

tertiary follicles observed and the formation of atresia

follicles was like the control group. This can be

caused when the animal is given induction of GnRH

antagonist ovarian status in the mouse model of

ovarian hypofunction cell death occurs in the

development of follicles de graf into corpus luteum.

Physiologically, de graf follicles that have failed in

ovulation then the de graf follicles will be eliminated

and turn into follicular atresia. Whereas in the P2

group that was given a Cetrorelix acetate dose of

0.0135 mg/kg BW the primary and secondary

follicles were observed in large amounts compared to

follicular development. The growth of primary and

secondary follicles classified as quite a lot in the P2

group compared to the P1 and K groups can be

assumed that in the group given the GnRH antagonist

intervention experienced inhibition of

folliculogenesis so that the primary and secondary

follicles do not experience follicular development

into the antral follicle. In addition, the P3 group also

found a lot of follicles that experience necrosis.

Necrosis is the first step of ovarian cell damage

that can result in apoptosis (cell death) so that it will

decrease the secretion of the hormone estrogen and

decrease the expression of ERs. An image of the

changes in ovarian histopathology in mice with

ovarian hypofunction models can be seen in Figure 2.

GnRH antagonists induced in the treatment group

gave a difference between the treatment group and the

negative control group. The increasing number of

primary and secondary follicles in the negative

control group, the P1 treatment group, and the P2

treatment group occurs because the growth of primary

and secondary follicles does not depend on GnRH so

that the induction of GnRH antagonists has no effect

on the preantral follicle (Puspitasari, 2011). The

development of primordial follicles into primary

follicles and primary follicles into secondary follicles

depends on the nutrition provided (Ramadhani et al.,

2017). The decrease in the number of tertiary follicles

in groups P1 and P2 shows that GnRH antagonists are

able to inhibit the development of antral follicles that

depend on GnRH. The increase in the number of

atresia follicles in the antral follicle along with the

increasing dose of GnRH antagonist shows that the

induction of GnRH antagonist is able to increase the

number of atresia follicles.

Expression of Estrogen Beta (ERs ) Receptor and Ovarian Histopathology Changes in Rats (Rattus norvegicus) Ovarian Hypofunction

Model

Figure 2. Histological features of folliculogenesis in the

ovaries of Rats (HE, 40x); FA (Atretic follicular); FP

(Primary follicles); FS (Secondary follicles); FT (Tertiary

Follicles); FD (De Graaf Follicular); CH (Corpus

Haemoragicum); N (Necrosis)

Description :

(A) K : Control (placebo)

(B) P1: Cetrorelix acetate (0.009 mg / Kg BW)

Follicles that undergo a degenerative process

before reaching the ovulation stage are called

follicular atresia. This degenerative process occurs

normally to select follicles, so only follicles that

contain healthy oocytes are ovulated. According to

Hsueh et al., (1994) there are 3 theories that explain

the causes of atresia in a follicle. The first theory

explains that follicles experience atresia due to

deficiencies in the genetic component, oocyte

cytoplasm, somatic cells, and follicular environment.

The second theory explains that the follicle is exposed

to teratogenic factors that carry the follicle into the

degenerative pathway. The third theory explains that

atresia is the fate of all follicles when going through a

critical stage in the follicle unless the critical stage

coincides with FSH stimulation, the follicle will

continue to develop and become a dominant follicle.

An increase in the number of follicular atresia with

increasing dose of GnRH antagonist shows decreased

FSH stimulation due to induction of GnRH antagonist

and does not coincide with the critical stage of the

follicle. Durlinger et al., (2000) research on atresia

follicles in pre-ovulatory follicles due to GnRH

antagonist states that evaluation of atresia follicles is

done by observing the thickness of the granulosa cell

layer which is getting thinner. Cumulus oophorus in

de Graaf follicles also experiences thinning. Atresia

follicles in the growing follicles and de Graaf follicles

will cause the following changes: (1) The oocyte

becomes the first degenerated structure then

disappears; (2) The zona pellucida swells and

disappears most recently; (3) The granulosa cells

degenerate so that the granulosa membrane

boundaries become irregular because the cells are

scattered and disappear as well; (4) Internal theca cells

in the follicle will become theca lutein cells,

eventually the atresia follicles will become the corpus

albikan (Hestianah et al., 2014).

Further research on the use of GnRH antagonists

in ovarian stimulation for IVF programs is ongoing.

According to Macklon and Fauser, (2000) the initial

step of the IVF program, namely the administration of

GnRH antagonists, aims to utilize dominant follicles

that develop from primordial follicles to secondary

(preantral) follicles. Dominant follicles are follicles

with granulosa cells which are more sensitive to FSH

stimulation. This causes the less FSH to be stimulated,

the greater the potential for FSH to only affect the

dominant follicle. Non-dominant follicles will lack

FSH then become follicular atresia. A decreased FSH

concentration will leave a dominant follicle ready for

maturation. GnRH antagonists were given at a dose of

0.0135 mg/kg BW in the P2 group causing damage in

the form of necrosis of the antral follicle in the ovary.

This is in accordance with research Safitri et al.,

(2012) regarding the condition of ovarian

hypofunction caused by malnutrition showing

histopathological conditions in the form of necrosis,

congestion, and edema in ovarian follicles.

Degeneration and necrosis of ovarian follicles indicate

that there is no development in ovarian follicles. This

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

is in accordance with the case study of Kesler and

Gaverick, (1982) which states the condition of ovarian

hypofunction is caused by hormonal deficiency and

imbalance resulting in anestrus and estrus which is not

accompanied by ovulation. This hormonal imbalance

affects the function of the anterior pituitary so that the

production of FSH and LH is low which causes the

ovaries to not develop.

According to Griesinger et al., (2005) GnRH

antagonists were able to reduce FSH levels by 75%,

LH levels by 84%, and estrogen until undetectable

within 36 hours. The activity of the hormone estrogen

requires binding to receptors to stimulate stromal and

epithelium cell proliferation. and β receptors work

homologously with each other and have a high affinity

for estrogen. Decreased estrogen levels due to the

administration of GnRH antagonists in the treatment

groups P1 and P2 will reduce estrogen binding

estrogen receptors thereby reducing the expression of

estrogen receptors in both ERs β and α. This causes

the transcription process that occurs because estrogen

bonds and estrogen receptors decrease and cause gene

expression to also decrease so that it interferes with

cell proliferation in the target cell. The proliferation of

disturbing cells will cause inhibition of

folliculogenesis in the ovary (Cooke et al., 1998). This

inhibition of folliculogenesis will result in decreased

estrogen levels. A decrease in estrogen levels and

estrogen bind with ERs causes no positive feedback to

the hypothalamus so that GnRH does not stimulate the

anterior pituitary to produce LH, and LH surge does

not occur so ovulation will never occur (Hafizuddin et

al., 2012).

4 CONCLUSIONS

GnRH antagonists were able to reduce ERsβ

expression in white rats in the P2 treatment group at

a dose of 0.0135 mg / Kg BW and GnRH antagonists

were able to inhibit the development of ovarian

follicles namely tertiary follicles and de Graaf

follicles in the ovarian histopathology picture in the

P1 and P2 treatment groups and cause the occurrence

of necrosis in the ovarian histopathology picture of

the P2 treatment group approaching the condition of

ovarian hypofunction.

ACKNOWLEDGMENTS

We would like to thank to LPPM Universitas

Brawijaya and the Faculty of Veterinary Medicine,

Universitas Brawijaya for providing research

assistance and support funds until the completion of

the Ovarian Hypofunction Rats Model with the

antagonist GnRH Induction.

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ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019