1. Effect of Human chorionic Gonadotrpin (hCG) on in
vitro oocyte maturation in freshwater cyprinid,
Barilius vagra
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2. Amina zuberi & Samina Jalali
Department of Animal sciences
Faculty of biological sciences
Quaid-i-Azam University
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3.  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
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Introduction

4. 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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5.  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.

6. 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.
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7. 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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8. 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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9. 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

10.  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 METHODS
Hormones
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11. 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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12. In vitro Bioassay
To determine the efficacy of human chorionic
gonadotropin in the induction of oocyte
maturation of Barilius vagra
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13.  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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14.  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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15. In vitro Bioassay
Results
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16. 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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17. 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)

18. 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
0
10
20
30
40
50
60
70
20 40 60 80
Time (h)
%CGV
y = 0.3623x + 3.3234
R
2
= 0.9537
0
5
10
15
20
25
30
35
20 40 60 80
Time (h)
%GVBD

19. 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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20. To investigate the synthesizing capability of
oocyte in response to hCG
High Pressure Liquid
Chromatography) HPLC
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21. Methodology
 After 72 hrs of incubation, incubation media directly applied on
primed Sep-Pak C18column
 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 450
C 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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22. 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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23. 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

24. 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)

25. 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).

26. To examine the morphological changes in the
granulosa layer and zona radiata of oocyte in
response to hCG.
Electron Microscopy
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27. 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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28. 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

29. 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

30. 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

31. 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

32. 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

33. Morphological Changes
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34. 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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35. 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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36.  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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37. 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
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38. 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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39. Thanks

40. y = -1.0604x + 79.98
R
2
= 0.892
0
10
20
30
40
50
60
70
20 40 60 80
Time (h)
%CGV