Effect of Human chorionic Gonadotrpin (hCG) on invitro oocyte maturation in freshwater cyprinid,Barilius vagraPakistanCong...
Amina zuberi & Samina JalaliDepartment of Animal sciencesFaculty of biological sciencesQuaid-i-Azam UniversityPakistanCong...
 Like other vertebrates, final oocyte maturation in teleostsoccurs prior to ovulation It consists of the migration and b...
Cont. Morphological and cytoplasmic changes in the follicular componentduring oocyte maturation include the increase num...
 Several studies have determined the effectiveness of variousgonadotropin and pituitary preparations to induce oocytematu...
Gonadotropin induction of maturationIt is a two step process Making the oocyte sensitive to maturation inducingsteroids (...
Human chorionic gonadotropin (hCG) Found in mammals and structurally related variant of fishgonadotroin. It acts as an e...
Objectives Basic aim of the research is to determine the role of hCG in thematuration of oocyte of Barilius vagra The st...
MATERIALS AND METHODS FishBARILIUS VAGRA(commonly found in the hill streams of Asia:Afghanistan, Pakistan, India, Nepal,B...
 Human chorionic gonadotropin (hCG) Estradiol 17β (E2 : estra-1,3,5 (10)-triene-3,17β-diol) Testosterone (T:17β-hydroxy...
MATERIALS AND METHODS The culture techniques for in vitro incubation of the ovarianfollicles were adapted from Upadhyaya ...
In vitro BioassayTo determine the efficacy of human chorionicgonadotropin in the induction of oocytematuration of Barilius...
 During early breeding season, mature fish anesthetized,decapitated , ovaries removed and placed in ice-cold Basic SaltSo...
 Vitellogenic oocyte having central germinal vesicle selected for study Samples of 30-35 ovarian follicles transferred t...
In vitro BioassayResultsPakistanCongress ofZoologyApril, 2011
Multivariate analysis of variance examining the effect of hCG (20IU/ml) at different time periods on the induction of oocy...
Position of germinal vesicle in the control (BSS) and gonadotropin(hCG: 20 IU/ml) treated oocyte of Barilius vagra at diff...
Relationship between time period and % position of germinal vesicle ofoocyte incubated with hCG (20 IU/ml). (a) Negative l...
Conclusion Exposure of the ovarian follicles with hCG resulted in a maturation ofoocyte but the response being time depen...
To investigate the synthesizing capability ofoocyte in response to hCGHigh Pressure LiquidChromatography) HPLCPakistanCong...
Methodology After 72 hrs of incubation, incubation media directly applied onprimed Sep-Pak C18column Free Estradiol 17β,...
HPLC Column:- Zorbax-ODS (4.6x 250 mm) Mobile Phase:- methanol-water Flow rate:- 1.1 ml/min. Program:- Concave exponen...
Chromatographic separation of steroid standards (50 ng/20ul each) on Zorbax-ODS at (a) λ = 280 and (b) λ = 254. (ALDO: ald...
Steroid production from follicle-enclosed oocytes ofBarilius vagra incubated in vitro for 72 hrs with andwithout gonadotro...
Results Human chorionic gonadotropin (hCG) enhanced the steroidsynthesizing capability of oocyte and stimulated the produ...
To examine the morphological changes in thegranulosa layer and zona radiata of oocyte inresponse to hCG.Electron Microscop...
ELECTRON MICROSCOPY 2-4 oocytes from both treated and control group were fixed for 2hrs in a cold 2.5% glutaraldehyde in ...
Fig 1. Part of a vitellogenic stage (Control) ovarian follicle showing agranulosa cell (GC) with microvilli entering pore ...
Fig 2. Granulosa cell of a vitellogenic oocyte showing nucleus(N), coated dense vesicles (arrows), mitochondria (M) coated...
Fig 3. Part of a granulosa cell of a follicle (24-hrs treated). Notemitochondria (M), Golgi saccules (G) and associated ve...
Fig 4. Part of a granulosa cell from an ovarian follicle of the fresh watercyprinid, Barilius vagra treated 48 hours in vi...
Fig 5. Part of granulosa cell and zona radiata (ZRE, ZRI) 48-hrs treated.Note dense material in vacuoles and cisternae, gr...
Morphological ChangesPakistanCongress ofZoologyApril, 2011
24 hrs treatment Vitellogenic follicles revealed a greater level of activity in thegranulosa cells than the cells in the ...
48 hrs treatment Morphological changes become much evident Golgi saccules were associated with clusters of clear to dens...
 All the morphological changes in thegranulosa cells and zona radiata in responseto hCG revealed high level of metabolic ...
Conclusion Morphologically the structural changes of zona radiatainclude Decrease in reticular structure Increase in th...
Conclusion Overall result of this study reveal that in Bariliusvagra hCG have a tendency to induce maturation ofvitelloge...
Thanks
y = -1.0604x + 79.98R2= 0.89201020304050607020 40 60 80Time (h)%CGV
Effect of human chorionic gonadotrpin (h cg)
Effect of human chorionic gonadotrpin (h cg)
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Effect of human chorionic gonadotrpin (h cg)

  1. 1. Effect of Human chorionic Gonadotrpin (hCG) on invitro oocyte maturation in freshwater cyprinid,Barilius vagraPakistanCongress ofZoologyApril, 2011
  2. 2. Amina zuberi & Samina JalaliDepartment of Animal sciencesFaculty of biological sciencesQuaid-i-Azam UniversityPakistanCongress ofZoologyApril, 2011
  3. 3.  Like other vertebrates, final oocyte maturation in teleostsoccurs prior to ovulation It consists of the migration and breakdown of the germinalvesicle (GVBD), chromosome condensation, and formationof the first polar body. Terminates in ovulationPakistanCongress ofZoologyApril, 2011Introduction
  4. 4. Cont. Morphological and cytoplasmic changes in the follicular componentduring oocyte maturation include the increase number of organelles including mitochondria, Golgibodies and endoplasmic reticulum and free ribosomes Follicular somatic cells cytodifferentiation that cooperate in theproduction of steroidal mediators (MIS) (Kayaba et al., 2001;Srijunngam et al., 2005; Unal et al 2005). Retraction of microvillar process (contact between microvilli ofgranulosa cells and pore canals of zona radiata (Kayaba et al., 2001)PakistanCongress ofZoologyApril, 2011
  5. 5.  Several studies have determined the effectiveness of variousgonadotropin and pituitary preparations to induce oocytematuration in vivo (Haniffa et al.,2000 ; Miwa et al., 2001; Rehman etal.,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 etal.,2001) in many species.PakistanCongress ofZoologyApril, 2011Cont.
  6. 6. Gonadotropin induction of maturationIt is a two step process Making the oocyte sensitive to maturation inducingsteroids (MIS) Induce MIS synthesis in follicle cells surrounding theoocyte In majority of teleost 17, 20ßP appeared as the most potentMIH, whereas a number of other ovarian steroids includingestradiol, corticosteroids and deoxycorticosteroids havealso been found to have equal and in some cases evengreater potency as MIS in vitro in several species.PakistanCongress ofZoologyApril, 2011
  7. 7. Human chorionic gonadotropin (hCG) Found in mammals and structurally related variant of fishgonadotroin. It acts as an effective inducer of oocyte maturation in severalteleost (Reviews Skoblina, 2009). It stimulate the in vitro steridogenesis of granulosa cell andrelease 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 anyresponse to hCG at any dose (Patino & Thomas, 1990; Amiri et al.,2001). The diversity of the teleostean group and the reproductiveadaptation unique to its various taxa, warrant a wider examinationof the gonadotropin responses in a variety of species.PakistanCongress ofZoologyApril, 2011
  8. 8. Objectives Basic aim of the research is to determine the role of hCG in thematuration of oocyte of Barilius vagra The study is divided in three steps To determine the efficacy of human chorionic gonadotropin in theinduction 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 andzona radiata of oocyte in response to hCG.PakistanCongress ofZoologyApril, 2011
  9. 9. MATERIALS AND METHODS FishBARILIUS VAGRA(commonly found in the hill streams of Asia:Afghanistan, Pakistan, India, Nepal,Bangladesh and Sri Lanka).Breeding season: April - mid AugustZoologicalSocietyApril, 2011
  10. 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 METHODSHormonesPakistanCongress ofZoologyApril, 2011
  11. 11. MATERIALS AND METHODS The culture techniques for in vitro incubation of the ovarianfollicles were adapted from Upadhyaya and Haider (1986) andZuberi et al (2002). Solid phase extraction methodology was adapted for theextraction of free steroid hormones from cultured medium(Zuberi et al.,2002). Free estradiol, testosterone, progesterone, 17α OH and17α,20ßP concentrations were measured using HPLC Morphological studies were performed with Electron microscopyPakistanCongress ofZoologyApril, 2011
  12. 12. In vitro BioassayTo determine the efficacy of human chorionicgonadotropin in the induction of oocytematuration of Barilius vagraPakistanCongress ofZoologyApril, 2011
  13. 13.  During early breeding season, mature fish anesthetized,decapitated , ovaries removed and placed in ice-cold Basic SaltSolution (BSS) reduced to small pieces and individual ovarian follicles separated A sub sample of the 20 largest vitellogenic ovarian follicles wastreated with oocyte clearing fixative (95% ethanol, 10% formalinand acetic acid in a ratio of 95:10:5 )and observed the position ofl germinal vesicle (CGV). Mostly have central germinal vesicle.PakistanCongress ofZoologyApril, 2011
  14. 14.  Vitellogenic oocyte having central germinal vesicle selected for study Samples of 30-35 ovarian follicles transferred to individual culture tubescontaining 3.0 ml fresh culture medium and hCG was added to each dishwith a micro syringe to obtain concentrations of 20 IU/ml of the culturemedium. 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 humidifiedtemperature-controlled incubator for 24, 48, 72 hr each. Upon completion of the incubations, the follicles examined under aninverted microscope for the position of germinal vesicle (GV) andbreakdown (GVBD) by treating them with egg clearing solution The position of germinal vesicle and rate of GVBD was expressed in %ageof three replicates.PakistanCongress ofZoologyApril, 2011
  15. 15. In vitro BioassayResultsPakistanCongress ofZoologyApril, 2011
  16. 16. Multivariate analysis of variance examining the effect of hCG (20IU/ml) at different time periods on the induction of oocytematuration in freshwater cyprinid, Barilius vagra.Source Position ofGerminal vesicleDF F value P valueTreatment Central 1 1954.402 .000Migratory 1 580.276 .000Peripheral 1 131.293 .000GVBD 1 271.388 .000Time Central 2 336.567 .000Migratory 2 90.221 .000Peripheral 2 26.873 .000GVBD 2 48.195 .000Treatment × Time Central 2 108.527 .000Migratory 2 38.672 .000Peripheral 2 4.749 .030GVBD 2 14.606 .001PakistanCongress ofZoologyApril, 2011
  17. 17. Position of germinal vesicle in the control (BSS) and gonadotropin(hCG: 20 IU/ml) treated oocyte of Barilius vagra at differentincubation period.(* = P ≤ 0.01; ** = P ≤ 0.001; *** = P ≤ 0.0001)
  18. 18. Relationship between time period and % position of germinal vesicle ofoocyte incubated with hCG (20 IU/ml). (a) Negative linear relationshipbetween % central germinal vesicle (% CGV) and incubation period (b)Positive linear relationship between % germinal vesicle break down (%GVBD) and incubation period.y = -1.0604x + 79.98R2= 0.89201020304050607020 40 60 80Time (h)%CGVy = 0.3623x + 3.3234R2= 0.95370510152025303520 40 60 80Time (h)%GVBD
  19. 19. Conclusion Exposure of the ovarian follicles with hCG resulted in a maturation ofoocyte but the response being time dependent. Compared with the control, a substantially significant P<0.0001smaller percentage of oocytes contained centrally placed germinalvesicle and a much larger percentage contained migratory andperipheral germinal vesicle. In respect to GVBD, treated group differed significantly P<0.01 fromthe control (12.42 % vs 2.94 %) at 24 hrs. Prolonged incubation further increased GVBDPakistanCongress ofZoologyApril, 2011
  20. 20. To investigate the synthesizing capability ofoocyte in response to hCGHigh Pressure LiquidChromatography) HPLCPakistanCongress ofZoologyApril, 2011
  21. 21. Methodology After 72 hrs of incubation, incubation media directly applied onprimed Sep-Pak C18column Free Estradiol 17β, testosterone , progesterone, 17α-hydroxyprogesterone and 17α-20β-dihydroxy progesterone wereeluted from the column with diehyl ether Ether extract dried under nitrogen at 450C and reconstituted in 100ulmethanol 20-µl aliquot of this mixture was injected on to the HPLC column forsteroid analysisPakistanCongress ofZoologyApril, 2011
  22. 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 peakarea calculated as peak height (cm) x peak widthat half peak height (cm).PakistanCongress ofZoologyApril, 2011
  23. 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. 24. Steroid production from follicle-enclosed oocytes ofBarilius vagra incubated in vitro for 72 hrs with andwithout 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 culturedmedium of the hCG treated follicles. (*** = P ≤ 0.0001; ns = nonsignificant)
  25. 25. Results Human chorionic gonadotropin (hCG) enhanced the steroidsynthesizing capability of oocyte and stimulated the production ofestradiol-17ß, 17α-OHP and 17α, 20ßP at 72 hrs. In the control incubation medium at 72 hrs both free 17α-OHP and17α, 20ßP were below the detection limit whereas the concentration oftestosterone 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 theestradiol-17ß secretion ~ 6 fold while concentration of testosteroneincreased non significantly (8.8±0.53; P = 0.07).
  26. 26. To examine the morphological changes in thegranulosa layer and zona radiata of oocyte inresponse to hCG.Electron MicroscopyPakistanCongress ofZoologyApril, 2011
  27. 27. ELECTRON MICROSCOPY 2-4 oocytes from both treated and control group were fixed for 2hrs in a cold 2.5% glutaraldehyde in 0.1M phosphate buffer (pH7.2). 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 weretransferred to Beam capsules for 12hrs at 35°C and 45°C eachand for 24hr at 60°C. Cut semi-thin and ultra-thin sections The ultra-thin sections transferred to 150-200 mesh coppergrids, contrasted with uranyl acetate and lead citrate. The sections examined and photographed on a Joel SX-100electron microscope.PakistanCongress ofZoologyApril, 2011
  28. 28. Fig 1. Part of a vitellogenic stage (Control) ovarian follicle showing agranulosa cell (GC) with microvilli entering pore canals (arrow head),electron dense hillock of zona radiata externa (ZRE) and thick zonaradiata interna (ZRI). X 13,661
  29. 29. Fig 2. Granulosa cell of a vitellogenic oocyte showing nucleus(N), coated dense vesicles (arrows), mitochondria (M) coatedGolgi saccules (G), Free ribosome (square). X 5,077
  30. 30. Fig 3. Part of a granulosa cell of a follicle (24-hrs treated). Notemitochondria (M), Golgi saccules (G) and associated vesicles(arrows), smooth endoplasmic reticulum and (SER) Roughendoplasmic reticulum, Free ribosome (square) and nucleus (N). X42,693RERSER
  31. 31. Fig 4. Part of a granulosa cell from an ovarian follicle of the fresh watercyprinid, Barilius vagra treated 48 hours in vitro with human chorionicgonadotropin. Note vacuoles and cisternae with vesicles (arrows) andelectron 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 50,567a bc
  32. 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) smoothendoplasmic reticulum (SER) and mitochondria (M) and retraction ofmicrovilli of granulosa cell entering the pore canal (arrow). x 17,078
  33. 33. Morphological ChangesPakistanCongress ofZoologyApril, 2011
  34. 34. 24 hrs treatment Vitellogenic follicles revealed a greater level of activity in thegranulosa cells than the cells in the control follicles Mitochondria appeared abundant and possessed a heavilycoated matrix (Fig 3). The quantity of both rough and smooth endoplasmic reticulumincreased Tiny dense core vesicles were frequently seen emanating fromthe saccules of the Golgi. The cisternae of the reticulum contained electron densematerial.PakistanCongress ofZoologyApril, 2011
  35. 35. 48 hrs treatment Morphological changes become much evident Golgi saccules were associated with clusters of clear to densevesicles. Dense core granules accumulated in vacuoles and cisternae ofER, 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 zonaradiata also showed retraction (Fig 5).PakistanCongress ofZoologyApril, 2011
  36. 36.  All the morphological changes in thegranulosa cells and zona radiata in responseto hCG revealed high level of metabolic andsynthetic activity of the oocytePakistanCongress ofZoologyApril, 2011
  37. 37. Conclusion Morphologically the structural changes of zona radiatainclude Decrease in reticular structure Increase in thickness and the retraction of microvillusprocess (contact between microvilli of granulosa cells andpore canals zona radiataPakistanCongress ofZoologyApril, 2011
  38. 38. Conclusion Overall result of this study reveal that in Bariliusvagra hCG have a tendency to induce maturation ofvitellogenic oocyt but the response is timedependent Appearance of MIS(17,20BP) in the hCG treatedincubation media revealed its role in the induction ofgerminal vesicle breakdownPakistanCongress ofZoologyApril, 2011
  39. 39. Thanks
  40. 40. y = -1.0604x + 79.98R2= 0.89201020304050607020 40 60 80Time (h)%CGV

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