Interventions of Cetrorelix Acetate in Estrogen Beta Receptor

Expression and Histopathology in Rats Oviduct

Herlina Pratiwi

, Aulia Firmawati

, Diana Rahmayani Putri

, Albiruni Haryo

, Analis Wisnu

Wardhana

Embryology Laboratory, Faculty of Veterinary Medicine, Brawijaya University

Reproduction Laboratory, Faculty of Veterinary Medicine, Brawijaya University

Graduate Student of Veterinary Medicine, Faculty of Veterinary Medicine, Brawijaya University

Department of Pathology, Faculty of Veterinary Medicine, Brawijaya University

Department of Anatomy and Histology, Faculty of Veterinary Medicine, Brawijaya University

Keywords: Rat Ovarian Hypofunction, Cetrorelix Acetate, Estrogen Receptors, Oviduct Cilia.

Abstract: Ovarian hypofunction is pathologic conditions where is the ovary being abnormal. The abnormality of the

ovary can be induced by the abnormality of the endocrine that regulates the development of the ovary such

as, follicle-stimulating hormone (FSH) and luteinizing hormone LH. The production of FSH and LH in the

pituitary is determined with the Gonadotropin hormone (GnRH) stimulation. Development of rat ovarian

hypofunction models can be performed with the induction of cetrorelix acetate which has an antagonist

effect of GnRH. This research was conduct to know the effect of induction of cetrorelix acetate on rat

oviduct estrogen beta receptor expression and histopathology. The study used three groups of female rats

(Wistar strain) 8-10 weeks old and 150-180 gram weight, each group consisting of six rats. The first group

(control) without cetrorelix acetate, the second group treated with cetrorelix acetate 0.009 mg/kg BW and

the third group treated with cetrorelix acetate 0.0135 mg/kg BW. Observations of estrogen beta receptor

expressions (ERs β) are carried out with immunochemical methods, while observations of histopathological

changes of oviduct carried out by Hematocsilin-Eosin (HE stain). The results obtained indicate a significant

difference from the administration of the GnRH antagonists in the three treatment groups, among others, the

largest reduction of the expression of the estrogen receptor of the ES β by 59.2%, as well as the thinning of

the fallopian tubes and the reduced cilia. The conclusion of the study was Cetrorelix acetate as a GnRH

antagonist capable of lowering the beta estrogen receptor expression and reducing the number of cilia as

well as the viscosity of the wall lining of the fallopian tubes.

1 INTRODUCTION

The consumption need of protein from animals

source especially for meat per capita in Indonesia for

one day reaches 3.35 grams or 5.91%. This needs

every year increases. The largest increase in

consumption of livestock products experienced was

in 2016 about 32.17% (Ditjen PKH, 2017). The

increasing number of consumption of livestock

products, especially meat, can cause an increase in

import activities. One of the causes of the increase in

beef import activities in Indonesia is the low

reproduction performance of female cows in

Indonesia and reproductive disorders. One of the

most common examples of reproductive disorders in

female cows is ovarian hypofunction.

Sutiyono et al. (2017) explain that ovarian

hypofunction is a decrease in ovarian activity in

producing oosit or ovum. Ovarian hypofunction is a

pathologic condition caused by impaired secretion of

follicle-stimulating hormone (FSH) and luteinizing

hormone (LH) (Hermadi, 2015). Impaired secretion

of FSH and LH can be caused by decreased

secretion of Gonadotropin-Releasing Hormone

(GnRH) by the hypothalamus. According to

Wulandari (2013), GnRH functions to stimulate

FSH and LH secretion. FSH functions are to

stimulate follicular development and estrogen

secretion, while LH functions for the maturation of

de Graaf follicle and ovulation. The development

and function of the reproductive organs are highly

dependent on the secretion of FSH and LH in the

Pratiwi, H., Firmawati, A., Putri, D., Haryo, A. and Wardhana, A.

Interventions of Cetrorelix Acetate in Estrogen Beta Receptor Expression and Histopathology in Rats Oviduct.

DOI: 10.5220/0009587300620067

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 62-67

ISBN: 978-989-758-483-1

anterior pituitary controlled by GnRH in the

hypothalamus. The low secretion of GnRH in the

hypothalamus and low secretion of FSH-LH in the

anterior pituitary will cause anestrus animals

(Pemayun, 2010). One of the medicines that have an

effect on GnRH antagonists is Cetrorelix acetate.

This GnRH antagonist can cause a decrease in

ovarian function by suppressing binding between

GnRH and its receptors so it can inhibit the

synthesis of Follicle Stimulating Hormone (FSH)

and Luteinizing Hormone (LH) (Wen et al., 2010).

The inhibition of FSH and LH synthesis causes

inhibition of folliculogenesis (Sharif et al., 2016).

Delay in folliculogenesis can cause inhibition of

estrogen hormone synthesis which results in

decreased expression of estrogen receptors in tissues

(Caldon, 2014).

Estrogen is a steroid hormone that has functions

in many tissues in the body, including the oviduct.

The primary function of estrogen is for tissue

proliferation of the reproductive organs and other

tissues related to the reproductive system. The

morphology of oviduct epithelial cells is influenced

by ovarian hormones, one of which is estrogen

(Crow et al., 1994). GnRH antagonists that inhibit

estrogen synthesis cause a decrease in the bonds

between estrogens and its receptors, so it will make

the receptors inactive and not expressed. According

to Trisunuwati (2016), estrogens need estrogen

receptors to carry out their functions. In the oviduct,

estrogen hormone activity requires binding with

receptors to stimulate epithelial cell proliferation, so

the absence of estrogen receptors in the fallopian

tubes can cause inhibition of ciliary formation in the

fallopian tubes.

2 MATERIALS AND METHOD

The tools used include terumo® 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 include rabbit feed (pellets) SP®,

husks, and sufficient water, Phosphate Buffer Saline

(PBS), formaldehyde, alcohol, xylol, 0.9%

physiological NaCl, paraffin, Hematoxillin-Eosin

stain, entellan, 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).

The rats were acclimatized for 7 days for

adaptions, given rabbit feed and drinking water in an

adlibitum. The group was divided into 3 groups

which included a control group without

administration of Cetrorelix acetate, the first

treatment group (P1) with the administration of

0.009 mg/kg BW Cetrorelix acetate and the second

treatment group (P2) with 0.0135 mg/kg BW of

Cetrorelix acetate.

The vaginal swab preparations carried out by

dipping the cotton bud in physiological NaCl than

the rat placed in a dorsal lying position. The vaginal

swab done by inserting a cotton bud in the vagina by

rotating 360

, then cotton bud removed and swab on

the slide. The slide 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. The results of vaginal swabs are

observed under a microscope with a magnification

of 100x and 400x to see vaginal cells. The vaginal

swab was carried out before injected with the

cetrorelix acetate to equalize the estrous cycle of the

rat.

After the treatments, the rat was euatanated with

cervical dislocation method (University of

Melbourne Ethics and Animal Welfare Commission,

2016). The rat is vertically opened from the posterior

abdomen to the thorax cavity. The oviduct organs

are taken, then washed with physiological NaCl and

collected in pots containing 10% Formaldehyde.

The ovary is processed to block paraffin and then

continue with Haemotoxyline-eosin and

immunohistochemical stain. The changes in the

histopathological of the oviduct observed with the

number of cilia and the thickness of the fallopian

tube wall. The expression estrogen beta receptor was

carried out at 40 times magnification and 5 visual

fields (Le, 2005 and Okada et al., 2004). Estrogen

receptors in the oviduct observed in all layers of

cells. Expression of the estrogen beta receptor

observed by calculating the average number of

expressed cells using the ImmunoRatio application.

3 RESULT

The estrogen beta receptor expression in the oviduct

observed with immunohistochemical methods

(Table 1). This method will give a brown color to

the target cell. These receptor expressions vary

according to the stage of the ovarian cycle and peak

in the middle of the cycle (Amso et al., 1994; Pollow

Interventions of Cetrorelix Acetate in Estrogen Beta Receptor Expression and Histopathology in Rats Oviduct

et al., 1981). Estrogen hormones in the oviduct are

involved in the regulation of oviduct functions

themselves, such as oviduct fluid formation and

gamete transport (McDonell et al., 2002).

Functionally, estrogen beta receptor (ER-β)

functions in the growth of oviduct, regulation of

protein content and expression of growth factors.

Whereas ER-β functions in the process of gamete

transportation.

Table 1. Expression of ERs β in rat injected with cetrorelix

acetate

Group Average ERs β

expression (%)

Control group

54,01

P1 group

32,30

P2 group

22,03

Note: a, b, c notations indicate a significant difference

between one treatment and another.

Based on the estrogen beta receptor expression in

the table above, it can be seen that the average ERs β

expression in the treatment group (P1 and P2)

decreased compared to the control group. This data

also showed that the higher dose of cetrorelix acetate

has lowest expression of ERs β in rat. The

expression of β ERs can be seen in the Figure 1, 2

amd 3 below.

Figure 1. Expression of β ERs in rat oviduct of control

group (40 times magnification, cross section).

Figure 2. Expression of β ERs in rat oviduct of P1 group

(40 times magnification, cross section).

Figure 3. Expression of β ERs in rat oviduct of P2 group

(40 times magnification, cross-section).

The results of the vaginal swab that conducted

before euthanasia, showed that the rat was in the

metestrus phase. The oviduct in the metestrus phase

characterized by the thickening of the oviduct wall

and the formation of cilia (Restall, 1966). An

increasing number of ciliated cells and thick

fallopian mucosal layers occur in the estrous and

metestrus phases. Increased thickness of the

fallopian tube lining is caused by an increase in the

number of ciliated cells and secretory cells (Liputo,

2006). At the end of the metestrus phase, non-ciliary

epithelial cells and ciliated epithelial cells undergo

apoptosis, but most of the secretory granules remain

in the secretory cells which then revert to structural

changes and begin the process of ciliogenesis in the

next phase (Kress and Morson, 2007).

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

Figure 4. Histophatology of rat oviduct induced by

cetrorelix acetate, A. Control group, B. P1 group, and C.

P2 group. Haemotoxylin-eosin stain, 40 times

magnification.

Note: tunica adventitia (Serosa);

tunica media

(Muscularis); tunica

intima (Mucosa);

(L) : lumen

The control group showed thicken wall and

many cilia (Figure 4.A), wich group P1 and P2

reduce in-wall thick and the number of cilia in the

oviduct (Figure 4. B and C).

4 DISCUSSION

The expression of ER β decreased in the treatment

group P1 injected Cetrorelix acetate 0.009 mg/kg

BW and P2 injected Cetrorelix acetate 0.0135 mg/kg

BW. Decreased expression of β estrogen receptors

caused by injection of Cetrorelix acetate as

a Gonadotropin-Releasing Hormone (GnRH)

antagonist occurs because the cetrorelix acetate

competes with GnRH to bind to membrane receptors

on pituitary cells and control the release of Follicle

Stimulating Hormone (FSH) and Luteinizing

Hormone (LH), thereby delaying the LH surge and

ovulation (Rodney, 2013). Induction of Cetrorelix

acetate which is a GnRH antagonist causes

inhibition of estrogen synthesis that occurs during

the process of folliculogenesis it makes a decrease of

estrogen production. This decrease causes a

decreased binding of estrogen and its receptor so the

receptors are inactive and not expressed. These

results can be concluded that the injection of

Cetrorelix acetate can decrease the estrogen receptor

expression in the oviduct.

The reduced number of cilia and the thickness of

the oviduct wall caused by the injected by Cetrorelix

acetate, it caused by the suppressed secretion of

Follicle Stimulating Hormone (FSH), Luteinizing

Hormone (LH) and estrogen (Griesinger et al.,

2005). Decreased levels of FSH and LH can inhibit

folliculogenesis and ovulation (Cooke et al., 1998;

Hafizuddin et al., 2012). Inhibited folliculogenesis

by injection of cetrorelix acetate can cause impaired

estrogen hormone synthesis. Impaired synthesis of

estrogen hormones can affect the development of

reproductive organs including oviduct wall.

The function of estrogen is for epithelial cell

proliferation, secretion, and ciliogenesis (Verhage et

al., 1979; Donnez et al., 1985). Ciliogenic activity

and secretion are caused by estrogen which acts

through estrogen receptors on oviduct epithelial cells

(Lauschová, 1999; Listy and Chakravarti, 2011).

The mechanism of the hormone estrogen which can

affect the thickness of the oviduct can be explained

through estrogen activity in the cells making up the

oviduct. Estrogen activity in cells begins after

estrogen bonds in the cytosol. The estrogen and

receptor complex further diffuses into the cell

nucleus and attaches to DNA. The estrogen-receptor

complex binding with DNA induces the synthesis

and expression of mRNA in the form of protein

synthesis thereby increasing target cell activity,

which is indicated by cell proliferation (Johnson and

Everitt, 1984).

Increased thickness of the oviduct mucosal wall

caused by an increase in the number of cells making

up the fallopian tissue. Increasing the number of

cells both secretory epithelial cells and ciliary

epithelium can cause the oviduct mucosal layer to

Interventions of Cetrorelix Acetate in Estrogen Beta Receptor Expression and Histopathology in Rats Oviduct

get thicker. The thickness of the oviduct mucosal

wall affects individual fertility (Umami et. Al,

2014). This is consistent with the statement of Crow

et al. (1994) regarding the morphology of oviduct

epithelial cells affected by estrogen, which act

through their receptors, where the hormone estrogen

causes mucosal glandular tissue to proliferate and

increases the number of ciliated epithelial cells.

5 CONCLUSION

Injection of Cetrorelix acetate as GnRH antagonist

in the rat can reduce the expression of estrogen beta

receptor, decrease wall thickness and cilia in rat

oviduct with the best doses was 0.0135 mg/kg BW.

ACKNOWLEDGMENTS

We many thanks to LPPM Universitas Brawijaya

and Faculty of Veterinary Medicine, Universitas

Brawijaya for providing research assistance funds

and supporting the completion of this research.

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Interventions of Cetrorelix Acetate in Estrogen Beta Receptor Expression and Histopathology in Rats Oviduct