Study of Oviduct Expression Specificct Glycoprotein1 (OVGP1) on

Oocyte and Goat Follicles (Capra hircus)

Aulia Firmawati

, Herawati

, Herlina Pratiwi

, Latiffatul ‘Ainiyah

, and Regi Abdul Rozzaaq A. S.

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

Public Health Laboratory, Faculty of Veterinary Medicine, Brawijaya University, Indonesia

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

Undergraduate Bachelor Student, Faculty of Veterinary Medicine, Brawijaya University, Indonesia

Keywords: OVGP1, Liquid Semen, Goat, Membrane Integrity, Motility

Abstract: Oviduct Specific Glycoprotein (OVGP1) is a glycoprotein that has been identified as a protein secreted from

unciliated secretory epithelial cells in oviducts with a molecular weight of 65 kDa in goats. The expression of

this protein is very dependent on estrogen levels and the oestrus phase of the species. On the other hand,

glycoprotein plays an important role in the process of oocyte maturation, spermatozoa capitation, and early

embryonic development. The purpose of this research was to find the location of OVGP1 expression in

oocytes and follicles of kacang goat (Capra hircus), an endogenous goat from Indonesian . The methods used

in this study are: immunocytochemical techniques to see OVGP1 expression on oocytes and on goat follicles.

OVGP1 expression in goat oocytes (Capra hircus) observed using immunocytochemical techniques was

detected in the cumulus ooporus section, the zona pellucida, perivitelline space, and plasma oocyte membrane,

whereas in the follicle the OVGP1 expression observed using immunohistochemical methods showed that

OVGP1 was expressed in granulosa cells, external and internal theca cells.

1 INTRODUCTION

Polyspermia is the process of fusion of eggs by two

or more consecutive spermatozoa (polyspermy) is a

lethal condition in most organisms (Frank, 2000).

Polyspermi, the most common abnormality found in

fertilization, usually results in embryonic death.

Physiologically, the penetration of the cytoplasm of

an egg by more than one spermatozoa occurs in

various species including insects, reptiles, and birds,

whereas in mammals polyspermy is considered an

abnormal phenomenon that results in zygote failure

(Pepi et al., 2010). Mammals have several

mechanisms to reduce the incidence of polyspermy.

It is said that capacitation, spermatozoa transport

through several parts of the female channel (ie:

cervix, uterotubal junction), and reservoir of

spermatozoa in the oviduct regulates the number of

spermatozoa that reach the site of fertilization in

various species. In pigs, in particular, gamete

exposure to oviduct epithelial cells and oviduct

secretion can reduce the occurrence of polyspermy

(Gardner and Evans (2006). In addition to involving

the female reproductive tract, the egg itself blocks the

polyspermy to prevent fertilization by other

spermatozoa after the egg has first fertilized the egg.

Polyspermi inhibition in the egg occurs at two levels,

namely: 1) at the plasma level of the egg cell

membrane (oolemma) or vitellin and 2) on the outer

layer of the egg called the zona pellucida (ZP) in

mammals and vitelline envelope in non-mammals. At

the level of the zona pellucida, this defense is often

called a blockade zone (zone reaction) which involves

cortical exocytosis of granules after oocyte

penetration by spermatozoa, while at the oolemma

level it is called a vitellin block which involves Ca

signaling (Coy and Aviles, 2010). After the

penetration of spermatozoa, cortical granules (CG)

release their contents into the perivitelline space

(perivitelline space) in an event called a cortical

reaction. The removal of CG contents changes the

property of the zona pellucida known as the zone

reaction, thereby blocking polyspermy penetration

(Pepi et al., 2010).

Oviduct Specific Glycoprotein (OVGP1) is a

glycoprotein that has been identified as a protein

secreted from unciliated secretory epithelial cells in

oviducts with a molecular weight of 65 kDa in goats

Firmawati, A., Herawati, ., Pratiwi, H., ‘Ainiyah, L. and A. S., R.

Study of Oviduct Expression Speciﬁcct Glycoprotein1 (OVGP1) on Oocyte and Goat Follicles (Capra hircus).

DOI: 10.5220/0009586500490052

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 49-52

ISBN: 978-989-758-483-1

(Pratiwi, 2017). Expression of this protein is very

dependent on estrogen levels and the oestrus phase of

the species, therefore this protein is often referred to

as estrogen-dependent glycoproteins (OGPs).

McCauley et al., (2003) also mentioned that OVGP1

expression in rats and pigs was localized in the zona

pellucida, perivitelline space, and oocyte membrane

plasma taken from the oviduct (in vivo) in the pre

embryonic period.

In the study of Coy et al., (2008) states that

OVGP1 added to cattle and pig oocytes during oocyte

maturation in vitro is known to increase blockade of

the zona pellucida and can reduce the incidence of

polyspermy so that it can increase the success of

fertilization. Seeing the potential role of OVGP1

which is very supportive in fertilization and seeing

that research on OVGP1 in goats is still rarely done,

it is necessary to conduct research on OVGP1

expression in the ovaries and OVGP1 expression in

oocytes, in order to obtain supporting data on

increasing fertilization rates in the process of in vitro

fertilization.

2 MATERIALS AND METHODS

2.1 Tools and Materials

The research material in the form of ovaries was taken

from female goat reproductive organs waste obtained

from RPH in Malang, while oocytes were obtained

from aspirations from reproductive organs waste cut

in the RPH.

2.2 OVGP1 expression on Oocytes

The immunocytochemical technique used is the

avidin-biotin-peroxidase-complex (ABC) method.

Oocytes that are included in the quality of the

Fixation on top of polylysine glass objects that have

been given paraffin and vaseline at all four ends, then

covered with a glass cover while pressing slowly.

Oocytes are fixed in a solution of ethanol: acetic acid

with a composition of 3: 1, for 2-3 days. After

fixation, oocyte preparations are taken and then

placed on a tissue to dry. Oocytes are observed under

a microscope to see oocytes. After that, the aqua-

destilation is dropped using a 1cc disposable syringe

and left for 5 minutes. Then add PBS and wait for 5

minutes. After that, it drops with hydrogen peroxidase

block for 10 minutes. Dropped PBS twice for 5

minutes (the sauce is smoked slowly with tissue).

Trypsin drops for 15 minutes and puts an incubator.

Dropped with Ultra Violet Block for 5 minutes. Then

rinsed with PBS and immediately dropped with

OVGP primary antibody antibodies for 1 hour, after

that washed with PBS twice for 5 minutes. Drop the

Biotylated link (yellow) for 30 minutes. Then

dripping PBS twice for 5 minutes. Streptavidin (Red)

drops for 30 minutes and drops with PBS for 5

minutes. Dropped with chromagen DAB for 10

minutes. And PBS drops for 5 minutes. Then in drops

of aqua-destilation for 5 minutes after that, it was

stained with methylene green and flowed with water

for 5 minutes (Schmidt, 2001).

2.3 OVGP1 Expression on Follicles

Histological preparations of ovarian organs were with

xylol I, xylol II, absolute ethanol I, absolute ethanol

II, stratified ethanol (90%, 80%, 70%, 30%), and

distilled water for 1 x 5 minutes respectively, then

washed with PBS pH 7.4 3 times. Furthermore, the

preparations were mixed in 10 mM citrate buffer pH

6 and 1 mM EDTA pH 8 for 10-20 minutes at 90 oC

then the slides were washed using distilled water. The

next stage is the process of blocking tissue peroxidase

using 3% H2O2 in methanol for 10 minutes, then

washed with PBS 3 times. The next process is

blocking the slide with 1% skim milk in PBS-tween

for 30 minutes, then the slide is washed using PBS 3

times. The next slide is given with primary antibodies

in a ratio of 1: 100 in 1% skim milk and PBS-tween.

The slides are then stored at 4oC for approximately

24 hours. Then the slide was washed with PBS 3

times. The next process is the addition of secondary

antibodies with a ratio of 1: 300 in PBS that is left for

1 hour, then the slides are washed with PBS 3 times

(Hadi, 2015).

The histological preparations were then dropped

by SA-HRP (Strep Avidin Horse Radish Peroxidase)

in a ratio of 1: 500 in PBS and incubated for 45

minutes at room temperature. Then washed again

with PBS pH 7.4 3 times, then dropped with DAB

(Diamano Benzidine) and incubated for 30 minutes at

room temperature. The slides were then washed again

with PBS pH 7.4 3 times, then counterstaining the

slides with Mayer Hematoxyler for 10 minutes. The

preparations are then washed with running water for

10 minutes, then rinsed with distilled water and dried

for about 1 night. The final stage is the mounting

process by using the glass and then covered with a

glass cover. Subsequent results were observed using

a microscope with a magnification of 400x (Hadi,

2015).

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

3 RESULT AND DISCUSSION

3.1 OVGP1 Expression in Goat

Follicles

McCauley et al., (2003) mention Epihelliacell

OVGP1 Glycoprotein (EOGPs) in mice and pigs

localized in the zona pellucida, perivitelline space,

and plasma oocyte membranes taken from oviducts

(in vivo) and embryos. In this study, based on

immunohistochemical analysis shows that the

expression of OVGP1 (Oviduct Specific

Glycoprotein 1) in goat follicles is found in the

external theca cells, internal theca, liquor follicular,

granulosa, and zona pellucida. The results of OVGP1

expression in goat follicles can be seen in Figure 1

below.

Figure 1. OVGP1 expression in Goat Follicles

Information:

1. Theca external cells

2. Internal theca cells

3. Granulosa cells

4. Zona Pelusida

5. Liquor follicular

3.2 OVGP1 Expression on Goat

Oocytes

Based on the immunocytochemical analysis, OVGP1

expression on oocytes shows that there are cumulus

oophorus complexes, zona pellucida, and

perivitelline space. localized in part of the zona

pellucida, perivitelline space, and plasma oocyte

membrane is taken from the oviduct (in vivo) and the

embryo. The results of OVGP1 expression in goat

oocyte can be seen in Figure 2 below.

Figure 2. OVGP1 expression on Goat Oocytes

Information:

a) Cumulus oophorus Complex

b) Pelusida Zone

c) Perivitelin space

The OVGP1 expression of the results of this

study is in the internal and external parts of the theca.

OVGP1 which can be assumed also plays a role in the

process of follicular maturation along with GDF-9

which results from the maturation process of this

follicle that will produce estrogen synthesis. On the

part of granulosa cells that are expressed in follicles

and oocytes, this can play a role in the nourishment

process of eggs during follicle ripening and

maturation of oocytes from goats, whereas OVGP1

which is expressed in the zone of the pellucid and

perivitelline space shows that OVGP1 will bind to

TGF- β to inhibit polyspremia in the process of

fertilization. In addition, OVGP1 together with GDF-

9 late has been shown to play a role in the process of

folliculogenesis and oocyte maturation. Based on the

results of this study, it can be concluded that OVGP1

also plays a role in the process of follicular and oocyte

maturation along with GDF-9 and TGF-β as well as

zone and vitellin blockade. In addition to the role of

growth factors that directly interact with OVGP1 as a

receptor in the signaling process, there are also

important roles of the FSH, LH and estrogen

hormones that play an important role together with

OVGP1. Where the increase in OVGP1 levels in the

follicular phase is higher than in OVGP1 levels in the

luteal phase. It can be concluded that OVGP1 in

addition to playing a role in the process of

fertilization, polyspermia blockade, early embryonic

development, OVGP1 also plays an important role in

the process of preovulation, ovulation, and oocyte

maturation.

Study of Oviduct Expression Speciﬁcct Glycoprotein1 (OVGP1) on Oocyte and Goat Follicles (Capra hircus)

According to Buhi (2002), OVGP1 expression

depends on the stage of the estrous cycle and is

associated with circulating estrogen concentrations.

Estrogen will stimulate OVGP1 expression. The

synthesis of estrogen occurs from aromatization

enzymes that produce androgen hormones whic are

converted to estradiol

17β under the influence of

FSH (Liben, 2000). In the ovarian follicle, the

hormone estrogen will be formed by granulosa cells

through enzymatic reactions. The activity of these

granulosa cells is related to the activity of the

hormones FSH and estrogen. OVGP1 can indirectly

increase some of the effects of the FSH related to the

activity of GDF-9 aromatization in granulosa cells

and the promotion of DNA synthesis in follicular

cells (Laheri et al., 2017). OVGP1 expression is

influenced by hormonal levels while still in the ovary,

so that OVGP1 expression is related to hormonal

influences (Buhi, 2002). OVGP1 expression in the

zona pellucida indicates that OVGP1 will later bind

to TGF-β which is TGF- this is a receptor of OVGP1

which will then cause complex bonding so as to

increase defense of the zone and vitellin blockade so

as to prevent polyspermia.

4 CONCLUSIONS

OVGP1 expression in goat follicles is expressed in

the external theca cells, internal, granulosa, zona

pellucida and liquor follicle while OVGP1 expression

in oocytes is expressed in the cumulus oophorus

complex, the zona pellucida, and perivitelline space.

ACKNOWLEDGMENTS

We would like to thank the Faculty of Veterinary

Medicine, Universitas Brawijaya for providing

research assistance and support funds until the

completion of the research.

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