Effect of Human chorionic Gonadotrpin (hCG) on in vitro oocyte maturation in freshwater cyprinid,

Barilius vagra

Pakistan Congress of

Zoology

April, 2011

Amina zuberi & Samina Jalali Department of Animal sciences

Faculty of biological sciences

Quaid-i-Azam University

Pakistan Congress of

ZoologyApril, 2011

Like other vertebrates, final oocyte maturation in teleosts

occurs prior to ovulation

It consists of the migration and breakdown of the germinal

vesicle (GVBD), chromosome condensation, and formation

of the first polar body.

Terminates in ovulation

Pakistan Congress of

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April, 2011

Introduction

Cont.

Morphological and cytoplasmic changes in the follicular component

during oocyte maturation include

the increase number of organelles including mitochondria, Golgi

bodies and endoplasmic reticulum and free ribosomes

Follicular somatic cells cytodifferentiation that cooperate in the

production of steroidal mediators (MIS) (Kayaba et al., 2001;

Srijunngam et al., 2005; Unal et al 2005).

Retraction of microvillar process (contact between microvilli of

granulosa cells and pore canals of zona radiata (Kayaba et al., 2001)

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Several studies have determined the effectiveness of various gonadotropin and pituitary preparations to induce oocyte maturation

in vivo (Haniffa et al.,2000 ; Miwa et al., 2001; Rehman et al.,2001; Chuda et al 2002; Mishra and Joy, 2006) and

in vitro (Skoblina, 2009; Sorbera et al., 1999;, Kagawa et al., 1994; Patino & Thomas, 1990 ; Miwa et al., 2001;Rehman et

al.,2001) in many species.

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Cont.

Gonadotropin induction of maturation

It is a two step process Making the oocyte sensitive to maturation inducing steroids

(MIS)

Induce MIS synthesis in follicle cells surrounding the oocyte

In majority of teleost 17, 20ßP appeared as the most potent MIH, whereas a number of other ovarian steroids including estradiol, corticosteroids and deoxycorticosteroids have also been found to have equal and in some cases even greater potency as MIS in vitro in several species.

Pakistan Congress of

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April, 2011

Human chorionic gonadotropin (hCG)

Found in mammals and structurally related variant of fish gonadotroin.

It acts as an effective inducer of oocyte maturation in several teleost (Reviews Skoblina, 2009).

It stimulate the in vitro steridogenesis of granulosa cell and

release the MIS in the incubation medium (king et al., 1995; Sorbera et al., 1999; Hakan et al., 2005).

There are some species where ovarian follicle did not showed any response to hCG at any dose (Patino & Thomas, 1990; Amiri et al., 2001).

The diversity of the teleostean group and the reproductive adaptation unique to its various taxa, warrant a wider examination of the gonadotropin responses in a variety of species.

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Objectives

Basic aim of the research is to determine the role of hCG in the

maturation of oocyte of Barilius vagra

The study is divided in three steps

To determine the efficacy of human chorionic gonadotropin in the

induction of oocyte maturation of Barilius vagra

To investigate the effect of hCG on the secretion Free Estradiol 17β,

testosterone , progesterone, 17α-hydroxyprogesterone and 17α-20β-

dihydroxy progesterone

To examine the morphological changes in the granulosa layer and

zona radiata of oocyte in response to hCG.

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MATERIALS AND METHODS

Fish BARILIUS VAGRA (commonly found in the hill streams of Asia:

Afghanistan, Pakistan, India, Nepal, Bangladesh and Sri Lanka).

Breeding season: April - mid August

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April, 2011

Human chorionic gonadotropin (hCG)

Estradiol 17β (E2 : estra-1,3,5 (10)-triene-3,17β-diol) Testosterone (T:17β-hydroxy-4-androsten-3-one)

Progesterone (P4: pregn-4-ene-3, 20-dione)

17-α-hydroxyprogesterone (17-OHP: 17α-hydroxypregn-4-ene-3, 20-dione),

17α-20β-dihydroxypregn-4-ene-3-dione (17,20βP: 17 20β-dihydroxypregn-4-ene-3-one)

MATERIALS AND METHODSHormones

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MATERIALS AND METHODS

The culture techniques for in vitro incubation of the ovarian follicles were adapted from Upadhyaya and Haider (1986) and Zuberi et al (2002).

Solid phase extraction methodology was adapted for the extraction of free steroid hormones from cultured medium (Zuberi et al.,2002).

Free estradiol, testosterone, progesterone, 17α OH and 17α,20ßP concentrations were measured using HPLC

Morphological studies were performed with Electron microscopy

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In vitro Bioassay

To determine the efficacy of human chorionic gonadotropin in the induction of oocyte

maturation of Barilius vagra

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April, 2011

During early breeding season, mature fish anesthetized, decapitated , ovaries removed and placed in ice-cold Basic Salt Solution (BSS)

reduced to small pieces and individual ovarian follicles separated

A sub sample of the 20 largest vitellogenic ovarian follicles was treated with oocyte clearing fixative (95% ethanol, 10% formalin and acetic acid in a ratio of 95:10:5 )and observed the position of l germinal vesicle (CGV).

Mostly have central germinal vesicle.

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Vitellogenic oocyte having central germinal vesicle selected for study Samples of 30-35 ovarian follicles transferred to individual culture tubes

containing 3.0 ml fresh culture medium and hCG was added to each dish with a micro syringe to obtain concentrations of 20 IU/ml of the culture medium.

The control cultures received an equal volume of saline. The incubations for each test system were run in triplicate The replicate cultures maintained at 22 ± 0.2°C in a humidified

temperature-controlled incubator for 24, 48, 72 hr each. Upon completion of the incubations, the follicles examined under an

inverted microscope for the position of germinal vesicle (GV) and breakdown (GVBD) by treating them with egg clearing solution

The position of germinal vesicle and rate of GVBD was expressed in %age of three replicates.

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In vitro Bioassay

Results

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Multivariate analysis of variance examining the effect of hCG (20 IU/ml) at different time periods on the induction of oocyte maturation in freshwater cyprinid, Barilius vagra.

Source Position of Germinal vesicle

DF F value P value

Treatment Central 1 1954.402 .000

Migratory 1 580.276 .000

Peripheral 1 131.293 .000

GVBD 1 271.388 .000

Time Central 2 336.567 .000

Migratory 2 90.221 .000

Peripheral 2 26.873 .000

GVBD 2 48.195 .000

Treatment × Time Central 2 108.527 .000

Migratory 2 38.672 .000

Peripheral 2 4.749 .030

GVBD 2 14.606 .001

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Position of germinal vesicle in the control (BSS) and gonadotropin (hCG: 20 IU/ml) treated oocyte of Barilius vagra at different incubation period.(* = P ≤ 0.01; ** = P ≤ 0.001; *** = P ≤ 0.0001)

Relationship between time period and % position of germinal vesicle of oocyte incubated with hCG (20 IU/ml). (a) Negative linear relationship between % central germinal vesicle (% CGV) and incubation period (b) Positive linear relationship between % germinal vesicle break down (%

GVBD) and incubation period.

y = -1.0604x + 79.98

R2 = 0.892

010203040506070

20 40 60 80

Time (h)

% C

GV

y = 0.3623x + 3.3234

R2 = 0.9537

05

101520253035

20 40 60 80

Time (h)

% G

VB

D

Conclusion

Exposure of the ovarian follicles with hCG resulted in a maturation of oocyte but the response being time dependent.

Compared with the control, a substantially significant P<0.0001 smaller percentage of oocytes contained centrally placed germinal vesicle and a much larger percentage contained migratory and peripheral germinal vesicle.

In respect to GVBD, treated group differed significantly P<0.01 from the control (12.42 % vs 2.94 %) at 24 hrs.

Prolonged incubation further increased GVBD

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To investigate the synthesizing capability of oocyte in response to hCG

High Pressure Liquid Chromatography) HPLC

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Methodology

After 72 hrs of incubation, incubation media directly applied on primed Sep-Pak C18 column

Free Estradiol 17β, testosterone , progesterone, 17α-hydroxyprogesterone and 17α-20β-dihydroxy progesterone were eluted from the column with diehyl ether

Ether extract dried under nitrogen at 450C and reconstituted in 100ul methanol

20-µl aliquot of this mixture was injected on to the HPLC column for steroid analysis

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HPLC

Column:- Zorbax-ODS (4.6x 250 mm) Mobile Phase:- methanol-water Flow rate:- 1.1 ml/min. Program:- Concave exponential gradient Injection volume:- 20ul Absorbance:- 254 and 280nm Quantification of each steroid was based on peak

area calculated as peak height (cm) x peak width at half peak height (cm).

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Chromatographic separation of steroid standards (50 ng/20ul each) on Zorbax-ODS at (a) λ = 280 and (b) λ = 254. (ALDO: aldosterone, 19-OHA: 19-hydroxyandrostenedione, F: cortisol, 11B-OHA: 11β-hydroxyandrostenedione, B: corticosterone, E1: estrone, AD: androstenedione, (DOC: 11-deoxycorticosterone, 17-OHP: 17-α-hydroxyprogesterone, T: testosterone, (17,20βP: 17α-20β-dihydroxypregn-4-ene-3-dione, P4: progesterone)

a b

Steroid production from follicle-enclosed oocytes of Barilius vagra incubated in vitro for 72 hrs with and without gonadotropin (hCG; 20 IU/ml).

(T: testosterone, P4; progesterone, E2: estradiol). 17-α-hydroxyprogesterone (17-OHP) and 17,20βP: 17α-20β-dihydroxypregn-4-ene-3-dione were only found in the cultured medium of the hCG treated follicles. (*** = P ≤ 0.0001; ns = non significant)

Results

Human chorionic gonadotropin (hCG) enhanced the steroid synthesizing capability of oocyte and stimulated the production of estradiol-17ß, 17α-OHP and 17α, 20ßP at 72 hrs.

In the control incubation medium at 72 hrs both free 17α-OHP and 17α, 20ßP were below the detection limit whereas the concentration of testosterone and progesterone were statistically comparable (P=0.14) and significantly (P< 0.01) higher than estradiol-17ß.

The presence of hCG in the incubation medium stimulated the estradiol-17ß secretion ~ 6 fold while concentration of testosterone increased non significantly (8.8±0.53; P = 0.07).

To examine the morphological changes in the

granulosa layer and zona radiata of oocyte in

response to hCG.

Electron Microscopy

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ELECTRON MICROSCOPY

2-4 oocytes from both treated and control group were fixed for 2 hrs in a cold 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.2).

Post fixed in cold 1% osmium teraoxide for 1 hr. Dehydrated in acetone series. Infiltration of the follicle in 1:3, 2:2 and 3:1 resin (Durcupan):

acetone mixture. Tissues kept in absolute resin at 4°C overnight and then were

transferred to Beam capsules for 12hrs at 35°C and 45°C each and for 24hr at 60°C.

Cut semi-thin and ultra-thin sections The ultra-thin sections transferred to 150-200 mesh copper

grids, contrasted with uranyl acetate and lead citrate. The sections examined and photographed on a Joel SX-100

electron microscope.

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Fig 1. Part of a vitellogenic stage (Control) ovarian follicle showing a granulosa cell (GC) with microvilli entering pore canals (arrow head), electron dense hillock of zona radiata externa (ZRE) and thick zona radiata interna (ZRI). X 13,661

Fig 2. Granulosa cell of a vitellogenic oocyte showing nucleus (N), coated dense vesicles (arrows), mitochondria (M) coated Golgi saccules (G), Free ribosome (square). X 5,077

Fig 3. Part of a granulosa cell of a follicle (24-hrs treated). Note mitochondria (M), Golgi saccules (G) and associated vesicles (arrows), smooth endoplasmic reticulum and (SER) Rough endoplasmic reticulum, Free ribosome (square) and nucleus (N). X 42,693

RER

SER

Fig 4. Part of a granulosa cell from an ovarian follicle of the fresh water cyprinid, Barilius vagra treated 48 hours in vitro with human chorionic gonadotropin. Note vacuoles and cisternae with vesicles (arrows) and electron dense material (arrow heads) mitochondria with tubular cristae (M), Golgi saccules (G), Free ribosome (square) zona radiata externa

(ZRE) hillock (a) x 25615 ; (b) x 53,789 ; (c) x 50,567

a b

c

Fig 5. Part of granulosa cell and zona radiata (ZRE, ZRI) 48-hrs treated. Note dense material in vacuoles and cisternae, granules (arrowheads), Golgi complex (G), rough endoplasmic reticulum (RER) smooth endoplasmic reticulum (SER) and mitochondria (M) and retraction of microvilli of granulosa cell entering the pore canal (arrow). x 17,078

Morphological Changes

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24 hrs treatment

Vitellogenic follicles revealed a greater level of activity in the granulosa cells than the cells in the control follicles

Mitochondria appeared abundant and possessed a heavily coated matrix (Fig 3).

The quantity of both rough and smooth endoplasmic reticulum increased

Tiny dense core vesicles were frequently seen emanating from the saccules of the Golgi.

The cisternae of the reticulum contained electron dense

material.

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48 hrs treatment

Morphological changes become much evident Golgi saccules were associated with clusters of clear to dense

vesicles.

Dense core granules accumulated in vacuoles and cisternae of ER, where they appeared to coalesce.

Both smooth and rough ER exists.

Free ribosome become abundant.

Microvilli from the granulosa cell entering the pore canal of zona radiata also showed retraction (Fig 5).

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All the morphological changes in the granulosa cells and zona radiata in response to hCG revealed high level of metabolic and synthetic activity of the oocyte

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Conclusion

Morphologically the structural changes of zona radiata include

Decrease in reticular structure

Increase in thickness and the retraction of microvillus process (contact between microvilli of granulosa cells and pore canals zona radiata

Pakistan Congress of

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April, 2011

Conclusion

Overall result of this study reveal that in Barilius vagra hCG have a tendency to induce maturation of vitellogenic oocyt but the response is time dependent

Appearance of MIS(17,20BP) in the hCG treated incubation media revealed its role in the induction of germinal vesicle breakdown

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April, 2011

Thanks

y = -1.0604x + 79.98

R2 = 0.892

010203040506070

20 40 60 80

Time (h)

% C

GV