Influence of FSH on in vitro growth, steroidogenesis and DNA synthesis of buffalo (Bubalus bubalis) ovarian preantral follicles

Transcription

1 Anim. Reprod., v.10, n.1, p.32-40, Jn./Mr Influene of FSH on in vitro growth, steroidogenesis nd DNA synthesis of ufflo (Bulus ulis) ovrin prentrl folliles G. Tmilmni, V.P. Vrshney, P.K. Duey, M.C. Pthk, G. Tru Shrm 1 Reprodutive Physiology Lortory, Physiology nd Climtology Division, Indin Veterinry Reserh Institute, Iztngr , Indi. Astrt The present study ws designed to exmine the effet of different doses of FSH on the growth, steroidogenesis nd DNA synthesis of two different sizes of ufflo ovrin prentrl folliles (PFs) during in vitro ulture. Bufflo ovries were olleted from lol ttoir nd PFs of smll (<150 µm) nd lrge (>150 µm) sizes were isolted through the mirodissetion method nd ultured in TCM-199 supplemented with 10% FBS, 1% ITS nd 30 ng/ml EGF (ontrol group) t 38.5 C in 5% CO 2 with mximum humidity. The ddition of three different doses of FSH (0.5, 1.0 nd 2.0 µg/ml) in the ontrol medium served s the tretment groups. The ulture medium ws replenished every third dy nd spent ulture medium ws stored t -30 C for steroid hormone nlysis. The results showed tht the supplementtion of FSH in the ulture medium stimulted (P < 0.05) folliulr growth, steroidogenesis nd DNA synthesis ompred to the ontrol. The ddition of FSH t 1.0 µg/ml in the ulture medium stimulted etter (P < 0.05) folliulr growth, DNA synthesis nd steroidogenesis ompred to 0.5 µg/ml FSH, wheres higher dose of FSH (2.0 µg/ml) inhiited folliulr growth in oth sizes of folliles. In onlusion, the present study demonstrted tht FSH plys key role during the development of ufflo ovrin prentrl folliles, s it stimulted in vitro survivl, growth rte, steroidogenesis nd DNA synthesis of ufflo prentrl folliles t n optiml dose of 1.0 µg/ml. Keywords: ufflo, DNA synthesis, FSH, prentrl folliles, steroidogenesis. Introdution The development of ulture systems to promote the growth of prentrl folliles (PFs) up to the Grfin follile stge nd to quire meiotilly ompetent ooytes would ertinly e useful to redue genertion intervl, onserve geneti resoures nd understnd folliulogenesis. Severl in vivo nd in vitro studies hve shown tht FSH filitted the development of folliles t prentrl stges in humns (Roy nd Trey, 1993), ows (Wndji et l., 1996; Gutierrez et l., 2000), gots (Mtos et l., 2007) nd pigs (Mo et l., 2002). Sudo et l. (2007) reported tht the in vitro growth nd survivl of ovine PFs depend on the presene of physiologil onentrtions of gondotropins. DNA synthesis in sheep prentrl folliulr ells is lso indued y FSH, s evidened y their ility to inorporte romodeoxyuridine (BrdU; Hemmlini et l., 2003). The mturtion of ooytes from in vitro ultured PFs up to the metphse II stge ws hieved with n inrese in DNA synthesis in sheep prentrl folliles when ultured in medium ontining gondotropins nd growth ftors (Hemmlini et l., 2003; Tmilmni et l., 2005). The inution of intt folliles with FSH nd susequent mesurement of progesterone nd estrdiol in the spent ulture medium fter the ulture period n estlish the funtionl hnges in the in vitro ultured PFs (Kishi nd Greenwld, 1999). Severl ulture systems hve een developed with FSH filitting the indution nd mintenne of estrdiol seretion in mouse (Nyudu nd Osorn, 1992), sheep (Cmpell et l., 1996) nd ow grnulos ells (Gutierrez et l., 1997). The role of FSH in domesti ruminnts during the development of lrge nd smll prentrl folliles is detle. We nd Armstrong (1998) showed tht prentrl folliles ould grow up to ntrl folliles (2 to 4 mm in dimeter) under in vitro ulture onditions either in the sene or presene of very low onentrtions of gondotropins. Similr studies were lso reported in mie (Nyudu nd Osorn, 1992), rts (Cmp et l., 1991) nd sheep (Cmpell et l., 1995) whih further strengthened the generl ssumption tht PFs re not gondotropin dependent, while other studies in mie (Edwrd et l., 1977), gots (Rossetto et l., 2009; Lim-Verde et l., 2010) ts (Sint-Dizer et l., 2007), sheep (Dufour et l., 1979) nd ttle (Evns nd Fortune, 1997) lerly demonstrted tht the development of PFs re gondotropin dependent nd tht FSH supplementtion is essentil to mintin folliulr growth nd integrity. Sriv et l. (2011) reported tht the ddition of inresed onentrtions of FSH (sequentil medium) hd signifint impt on the in vitro development of prine prentrl folliles. Previously, our lortory lso demonstrted the loliztion of FSH reeptors (Tru Shrm et l., 2011) nd the expression of FSH reeptor genes in smll nd 1 Corresponding uthor: gts553@gmil.om Phone: +91(581) Reeived: Septemer 28, 2011 Aepted: Deemer 27, 2012

2 Tmilmni et l. Influene of FSH on growth of ufflo prentrl folliles. lrge ufflo PFs (Kurvil et l., 2010). However, n pproprite onentrtion of FSH, time nd stge of PFs during in vitro development in ufflo needs to e known, long with the impt of FSH on DNA synthesis. Therefore, this study ws designed to investigte the miniml effetive dose of FSH in order to otin n idel omintion of follile growth, survivl nd steroidogenesis in in vitro developing prentrl folliles. Chemils Mterils nd Methods All hemils used in this study were purhsed from Sigm Chemil Co. (St Louis, MO, USA) unless otherwise indited. Ovries Bufflo ovries from rndom stges of the estrous yle were olleted from lol ttoir immeditely fter slughter nd trnsported to the lortory within 2 h. During trnsit, they were mintined t C in 0.9% norml sline supplemented with ntiiotis. In the lortory, the ovries were freed from ligments nd rinsed well in prewrmed phosphte-uffered sline (PBS) supplemented with ntiiotis (75 mg/l peniillin-g, 50 mg/l streptomyin sulfte). Isoltion nd seletion of PFs Under sterile onditions, fine ortil slies (~ mm) were ut from the ovrin surfe using surgil lde nd pled in the follile olletion medium supplemented with sodium pyruvte (2 mm), glutmine (2 mm), ovine serum lumin (BSA, 3 mg/ml) nd ntiiotis (75 mg/l peniillin-g, 50 mg/l streptomyin sulfte) t room temperture. PFs ( µm dimeter, folliles/experimentl dy) were isolted y the mirodissetion method s desried y Tru Shrm et l. (2009). Helthy PFs seleted for in vitro ulture were hrterized sed on grnulos ell lyers nd visile, entrlly loted ooyte. Bsed on their dimeter nd lyer(s) of grnulos ells, seleted folliles were lssified s smll (<150 µm in dimeter, one lyer of flt grnulos ells) nd lrge ( µm in dimeter, two to three lyers of grnulos ells) PFs. In vitro ulture of PFs The si ulture medium ws HEPES uffered (5 mm) tissue ulture medium-199 (TCM-199) supplemented with sodium ironte (26 mm). All the seleted PFs were ultured with or without FSH to determine the most suitle medium. The ontrol medium onsisted of the si ulture medium supplemented with 10% fetl ovine serum (FBS), 1% ITS liquid medium (1 mg/ml insulin, 0.55 mg/ml trnsferrin nd 0.5 µg/ml sodium selenite) nd epiderml growth ftor (30 ng/ml EGF). The tretments onsisted of dding 0.5, 1.0 or 2.0 µg/ml FSH (Sigm, humn pituitry, reominnt) in the ontrol medium. Seleted PFs of oth sizes were pled in 70 µl droplets of the respetive ulture medium (4-5 PFs/group) in 35 mm plsti tissue ulture dish (Nun, Denmrk) nd overlid with 2 ml of emryo tested lightweight minerl oil. These ulture dishes were pled in humidified inution hmer (Thermo Form, USA) t 38 ± 1 C in mximum humidified tmosphere hving 5% CO 2 in ir for 10 dys. Hlf of the medium ws replenished y n equl volume of fresh medium every third dy. The repled medium ws stored t -30 C for steroid nlysis. Morphologil evlution of in vitro ultured PFs At the end of the ulture period, PFs from ll the ove mentioned tretments were olleted to evlute survivl, growth, DNA synthesis nd steroid prodution (estrdiol, progesterone). The in vitro growth of PFs ws monitored y mesurement of PF dimeter using Imge J 1.33U softwre (Ntionl Institutes of Helth, Bethesd, MD, USA) sed on lirted oulr mirometer t 0 nd 10 dys of ulture. As eentri movement of the ooyte within the PFs is ssoited with growth nd ntrum formtion, this ws onsidered to e n inditor of in vitro development of PFs through exmintion under n inverted mirosope (Olympus CKX 41, Jpn). The viility of in vitro grown PFs ws evluted using the 0.05% (w/v) trypn lue (Sigm) dye exlusion test for 5 to 10 min. Folliles exhiiting drk lue stin were onsidered s ded nd were disrded. Mesurement of totl DNA The totl DNA ontent of individul freshly isolted (dy 0) nd in vitro ultured folliles (dy 10) in different tretment groups ws mesured. Totl DNA from the PFs ws isolted using the QIAmp DNA miro kit (Qigen In., Germny) s per the mnufturer s protool nd quntified (ng/µl) diretly using Nno-drop 1000 UV visile spetrophotometer (Thermo Sientifi, USA). Mesurement of steroids Estrdiol nd progesterone were estimted in the spent ulture medium olleted on dys 2, 4, 6 nd 8 of ulture using ommerilly ville rdioimmunossy kits (Immunoteh, Czeh Repuli). Assys for steroids were rried out s per the mnufturer s protool using duplite smples. The Anim. Reprod., v.10, n.1, p.32-40, Jn./Mr

3 Tmilmni et l. Influene of FSH on growth of ufflo prentrl folliles. nlytil sensitivities of the progesterone nd estrdiol kits were 0.02 pg/ml nd 4.5 pg/ml, respetively. Inter nd intr ssy vrition oeffiients for progesterone were 8.5 nd 4.5% nd for estrdiol were 11.2 nd 12.1%, respetively. Sttistil nlyses Dt olleted from 18 to 20 replites on different dys of ulture were expressed s men ± stndrd error of the men (SEM). Sttistil signifine ws determined using SPSS softwre for Windows, version 7.5 (SPSS GmH Softwre, Munih, Germny) y nlysis of vrine (ANOVA) followed y Dunn s post-ho multiple omprison test for proportion. A proility of P < 0.05 ws onsidered to e sttistilly signifint. Progesterone nd estrdiol levels were sujeted to sttistil nlysis over the ulture period t 2, 4, 6 nd 8 dys. These dt were nlyzed y ANOVA nd tretments were further ompred y Dunn s multiple rnge test. Results Influene of FSH on growth of smll (<150 μm) nd lrge ( μm) sizes ufflo PFs Supplementtion of the ulture medium with FSH t different onentrtions influened the growth, survivl nd mintenne of the rhiteture of PFs ompred to the ontrol. The ddition of FSH (1.0 μg/ml) in the ontrol medium signifintly inresed the growth, survivility nd ntrum formtion of smll nd lrge PFs ompred to other doses of FSH on dy 10 of ulture (Fig. 1). On dy 10 of ulture in the 1.0 μg/ml FSH group, n inrese in dimeter up to nd 234.1% ws notied for smll nd lrge PFs, respetively (Tles 1 nd 2). At the end of ulture period, nerly 70% of folliles survived from oth the groups s onfirmed y the trypn lue dye test. Unstined folliles were lssified s vile nd fully stined folliles s ded (Fig. 2). o o o d e f A A Figure 1. In vitro growth nd development of ufflo prentrl folliles: ( & ) smll nd lrge prentrl follile (dy 0) showing entrlly loted ooyte (o), respetively, ( & d) growth of follile during ulture, (e & f) ntrum formtion (A) in smll nd lrge ultured prentrl folliles fter 10 dys of ulture, respetively (originl mgnifition of photomirogrphs ws 200X). Tle 1. Influene of different onentrtions of FSH on in vitro growth of smll ufflo prentrl folliles. Tretment Follile size on Dy 0 (μm) Men ± SEM Follile size on Dy 10 (μm) Men ± SEM Totl inrese in dimeter (%) Control ± ± FSH 0.5 μg/ml ± ± FSH 1.0 μg/ml ± ± FSH 2.0 μg/ml ± ± ,, Within olumns, mens with different supersripts were signifintly different (P < 0.05). 34 Anim. Reprod., v.10, n.1, p.32-40, Jn./Mr. 2013

4 Tmilmni et l. Influene of FSH on growth of ufflo prentrl folliles. Tle 2. Influene of different onentrtions of FSH on in vitro growth of lrge ufflo prentrl folliles. Tretment Follile size on Dy 0 (μm) Men ± SEM Follile size on Dy 10 (μm) Men ± SEM Totl inrese in dimeter (%) Control ± ± FSH 0.5 μg/ml ± ± FSH 1.0 μg/ml ± ± FSH 2.0 μg/ml ± ± , Within olumns, mens with different supersripts were signifintly different (P < 0.05). Figure 2. Photomirogrphs of live nd ded prentrl folliles: () follile showing drk, lue olor, onsidered s ded: () follile not stined, onsidered s live (originl mgnifition of photomirogrphs ws 200X). Influene of FSH on estrdiol seretion y smll nd lrge sizes ufflo PFs Irrespetive of the FSH onentrtion, folliles ultured in medium ontining FSH produed higher onentrtion of estrdiol ompred to the ontrol. Smll size PFs ultured with 1.0 μg/ml FSH produed more (P < 0.05) estrdiol throughout the ulture ompred to the 0.5 nd 2.0 μg/ml FSH groups. FSH 2.0 μg/ml tretment used n inhiitory (P < 0.05) effet on estrdiol seretion throughout the ulture period (Fig. 3). Lrge sized PFs showed no signifint differene in estrdiol onentrtions etween the 0.5 nd 1.0 μg/ml FSH groups throughout the ulture period (Fig. 4). The supplementtion of FSH (1.0 μg/ml) in the ulture medium produed more (P < 0.05) estrdiol ompred to the ontrol nd 2.0 µg/ml FSH groups. Estrdiol levels in the ontrol medium on dys 6 nd 8 in smll nd lrge sizes PFs were elow the sensitivity limit of the ssy (4.5 pg/ml). This oservtion further supported tht n optiml dose of FSH enhnes the seretion of estrdiol. Control FSH 0.05 μg/ml Estrdiol umultion (pg/ml) FSH 1.0 μg/ml FSH 2.0 μg/ml 0 Dy 2 Dy 4 Dy 6 Dy 8 Dys of ulture Figure 3. Influene of different onentrtions of FSH on estrdiol seretion y smll ufflo prentrl folliles ultured in vitro. Within eh dy, rs with different supersripts were signifintly different (P < 0.05). Anim. Reprod., v.10, n.1, p.32-40, Jn./Mr

5 Tmilmni et l. Influene of FSH on growth of ufflo prentrl folliles. Control FSH 0.05 μg/ml Estrdiol umultion (pg/ml) FSH 1.0 μg/ml FSH 2.0 μg/ml 0 Dy 2 Dy 4 Dy 6 Dy 8 Dys of ulture Figure 4. Influene of different onentrtions of FSH on estrdiol seretion y lrge ufflo prentrl folliles ultured in vitro. Within eh dy, rs with different supersripts were signifintly different (P < 0.05). Influene of FSH on progesterone seretion y smll nd lrge sizes ufflo PFs In vitro seretion of progesterone in the ulture medium ws gretly influened y the presene of FSH ompred to the ontrol. PFs treted with 0.5 nd 1.0 μg/ml FSH produed higher (P < 0.05) onentrtions of progesterone ompred to the ontrol nd 2.0 μg/ml FSH groups on dys 4, 6 nd 8 of ulture for smll PFs (Fig. 5). In lrge size PFs, progesterone onentrtions were signifintly different mong different tretment groups, exept on dy 2 of ulture. On dy 8 of ulture, 1.0 μg/ml FSH produed more (P < 0.05) progesterone ompred to other groups. However, on dys 4 nd 6 of ulture, 0.5 nd 1.0 μg/ml FSH indued higher (P < 0.05) seretion of progesterone ompred to 2.0 μg/ml FSH (Fig. 6). Control FSH 0.05 μg/ml Progesterone umultion (pg/ml) FSH 1.0 μg/ml FSH 2.0 μg/ml 0 Dy 2 Dy 4 Dy 6 Dy 8 Dys of ulture Figure 5. Influene of different onentrtions of FSH on progesterone seretion y smll ufflo prentrl folliles ultured in vitro. Within eh dy, rs with different supersripts were signifintly different (P < 0.05). 36 Anim. Reprod., v.10, n.1, p.32-40, Jn./Mr. 2013

6 Tmilmni et l. Influene of FSH on growth of ufflo prentrl folliles. Control FSH 0.05 μg/ml Progesterone umultion (pg/ml) FSH 1.0 μg/ml FSH 2.0 μg/ml 0 Dy 2 Dy 4 Dy 6 Dy 8 Dys of ulture Figure 6. Influene of different onentrtions of FSH on progesterone seretion y lrge ufflo prentrl folliles ultured in vitro. Within eh dy, rs with different supersripts were signifintly different (P < 0.05). Influene of FSH on totl DNA ontent of smll (<150 μm) nd lrge ( μm) sizes ufflo PFs The DNA ontent of PFs in ll the treted nd ontrol groups inresed with growth. The ddition of 1.0 μg/ml FSH in the ulture medium signifintly (P < 0.05) inresed the totl DNA ontent (912 ± 144 ng) in smll PFs ompred to other tretment groups on dy 10 of ulture. However, the totl DNA ontent of n individul PF ultured in the ontrol group (380 ± 22 ng) did not differ signifintly from the PFs ultured with 2.0 μg/ml FSH (418 ± 42 ng; Tle 3). In the se of lrge size PFs, 0.5 nd 1.0 μg/ml FSH produed signifintly (P < 0.05) higher mounts of DNA ontent (2884 ± 615 nd 6139 ± 1166 ng) ompred to the ontrol nd 2.0 μg/ml FSH (1290 ± 170 nd 1407 ± 117 ng) groups on dy 10 of ulture (Tle 4). Tle 3. Influene of different onentrtions of FSH on totl DNA ontent of smll ufflo prentrl folliles. Tretment Totl DNA on dy 0 (ng) Men ± SEM Totl DNA on dy 10 (ng) Men ± SEM Control 327 ± ± 22 FSH 0.5 μg/ml 327 ± ± 69 FSH 1.0 μg/ml 327 ± ± 144 FSH 2.0 μg/ml 327 ± ± 42,, Within olumns, mens with different supersripts were signifintly different (P < 0.05). Tle 4. Influene of different onentrtions of FSH on totl DNA ontent of lrge ufflo prentrl folliles. Tretment Totl DNA on Dy 0 (ng) Men ± SEM Totl DNA on Dy 10 (ng) Men ± SEM Control 1134 ± ± 170 FSH 0.5 μg/ml 1134 ± ± 615 FSH 1.0 μg/ml 1134 ± ± 1166 FSH 2.0 μg/ml 1134 ± ± 117,, Within olumns, mens with different supersripts were signifintly different (P < 0.05). Disussion The widespred implementtion of dvned reprodutive tehnologies ould provide greter ess to the femle germ plsm. The lk of supply of fertilizle ooytes for in vitro mnipultion is mjor limiting ftor of these tehniques in frm nimls (Vn den Hurk et l., 2000; Kne et l., 2003; Vn den Hurk nd Sntos, 2009). The isoltion, ulture nd growth of the most undnt folliulr stges in the ovry, nmely Anim. Reprod., v.10, n.1, p.32-40, Jn./Mr

7 Tmilmni et l. Influene of FSH on growth of ufflo prentrl folliles. the primordil nd prentrl folliles, up to the Grfin follile stge nd susequent ovultion in vitro n serve s n dditionl soure of fertilizle ooytes to produe emryos. Follile stimulting hormone is known to hve divergent effets on folliulr growth nd differentition in mmmls oth in vivo nd in vitro (Zeleznik, 1993). Most of the studies hve shown tht FSH is involved in the growth nd development of ttle (Gutierrez et l., 2000; Itoh et l., 2002), got (Mtos et l., 2007; Sriv et l., 2011) nd ufflo (Tru Shrm et l., 2011) prentrl folliles. While mny studies deling with in vitro systems for follile growth indite the growth promoting effets of FSH, it is still unler whether or not exposure to different gondotropins influenes steroidogenesis, growth nd ooyte qulity for further emryoni development. In the present study, the sensitivity of the erly prentrl follile stges to FSH ws lerly oserved throughout the ulture period. The supplementtion of FSH (1.0 μg/ml) in the ulture medium supported follile survivl, growth, ntrum formtion nd enhned DNA synthesis nd estrdiol nd progesterone seretion in oth sizes of ufflo prentrl folliles. Our results suggest dose dependent tion for FSH (0.5 nd 1.0 μg/ml vs. 2.0 μg/ml) on grnulos ell prolifertion in prentrl folliles. The differene in the dose of FSH in the present study nd in other studies whih support the est development of PFs my lso e due to the differene in the soure of FSH nd/or ulture medium. Furthermore, when FSH ws omitted from the ulture (ontrol), follile growth, survivl nd steroidogenesis were extremely low nd norml development ws retrded. These results lerly suggest tht FSH promotes grnulos ell prolifertion in the erly prentrl phse nd lso show tht n optiml essentil dose of FSH is required to llow for norml folliulogenesis. Sriv et l. (2011) reported tht inresed onentrtions of FSH hve signifint impt on the in vitro development of prine prentrl folliles. Ceoni et l. (1999) reported tht ntrl vities were formed in sheep prentrl folliles with high sereted levels of E2 in serum-ontining medium with 1.0 μg/ml FSH in low-oxygen environment (5% O2). The dt presented here onfirm tht the presene of FSH during the prentrl phse ould e importnt to ensure ritil numer of grnulos ells to llow finl survivl nd growth. The ddition of LH in the presene of FSH might hve hd enefiil effet on growth y enhning ntrum formtion nd supporting the development of erly ntrl folliles. However, the role of LH during the development of prentrl folliles hs not yet een reported. Therefore, in the present study, LH ws not inluded euse severl studies hve indited tht LH is involved minly in the development of dvned stged ntrl folliles. Shnmugm et l. (2010) demonstrted serum-free ulture system for ufflo grnulos ells nd reported stimultory effet of FSH, ut not LH, on steroid hormone prodution y ufflo grnulos ells. Roy nd Greenwld (1988) lso reported tht LH suppressed FSH-indued DNA synthesis in hmster prentrl folliles. Further reserh is required to understnd the mehnism y whih LH influenes FSH nd whether or not it stimultes romtse tivity nd DNA synthesis in ufflo prentrl folliulr grnulos ells. The totl DNA ontent in the ultured PFs signifintly inresed ompred to freshly isolted PFs. The inrementl dimeter of the folliles indeed orresponded to n inrese in ell numer nd hene inresed the DNA ontent. Similr results were lso reported in mie (Heise et l., 2005), rts (Rowghni et l., 2004) nd gots (Rossetto et l., 2009). These results suggest tht FSH ts diretly s primry stimulus t the level of smll primry nd seondry folliles to regulte DNA synthesis nd perhps the growth nd differentition of grnulos ells. In the present study, FSH-indued folliulr DNA synthesis in dose-dependent mnner suggests tht FSH indues grnulos ell prolifertion, with AMP s one of the possile intrellulr meditors of FSH tion in inititing DNA replition. Estrogen nd progesterone re well known endorine nd intrfolliulr utorine mitogeni ompounds (Tonett nd Dizereg, 1989). Lim-Verde et l. (2010) reported tht the intertion etween estrdiol nd FSH mintins ultrstruturl integrity nd stimultes tivtion nd further growth of ultured prine prentrl folliles. In our study, FSH (1.0 μg/ml) indued estrdiol nd progesterone prodution in ll the ulture groups of oth follile sizes throughout the ulture period, wheres higher mount of FSH (2.0 μg/ml) inhiited this tion. Regrdless of the FSH onentrtion, folliles ultured in medium ontining FSH produed higher onentrtions of progesterone nd estrdiol in omprison with those ultured without FSH. FSH lso indued signifintly higher expression of mrna for P450 romtse enzymes (Silv nd Christopher, 2000). FSH infusion in the physiologil rnge ppers to e the stimulus to initite the expression of mrna for P450 romtse in grnulos ells. It is lso possile tht FSH modultes romtse tivity t two levels: trnsription nd trnsltion/enzyme tivity. It ws oserved tht estrdiol nd progesterone seretion y smll size prentrl folliulr grnulos ells re less sensitive thn in lrge prentrl folliles with regrd to the stimultory effets of FSH. The mehnism(s) y whih ufflo prentrl folliulr grnulos ells from lrge folliles re le to respond in more sensitive wy to FSH thn smll folliles nd lso the down regultion of steroid seretion fter the tive period requires further study. It n e onluded tht FSH plys key role during the development of ufflo ovrin prentrl folliles s it stimulted in vitro survivl, growth rte, steroidogenesis nd DNA synthesis t n optiml dose of 1.0 µg/ml FSH. 38 Anim. Reprod., v.10, n.1, p.32-40, Jn./Mr. 2013

8 Tmilmni et l. Influene of FSH on growth of ufflo prentrl folliles. Aknowledgments The study ws finnilly supported y The Deprtment of Biotehnology, Ministry of Siene nd Tehnology, Government of Indi. The uthors thnk the Diretor of the Indin Veterinry Reserh Institute. Referenes Cmp TA, Rhl JO, Myo KE Cellulr loliztion nd hormonl regultion of follile stimulting hormone nd luteinizing hormone reeptor messenger RNAs in the rt ovry. Mol Endorinol, 5: Cmpell BK, Srmuzzi RJ, We R Control of ntrl follile development nd seletion in sheep nd ttle. J Reprod Fertil, 49: Cmpell BK, Srmuzzi RJ, We R Indution nd mintenne of oestrdiol nd immunoretive inhiin prodution with FSH y ovine grnulos ells ultured in serum-free medi. J Reprod Fertil, 106:7-16. Ceoni S, Broni B, Coi M, Mttioli M In vitro development of sheep prentrl folliles. Biol Reprod, 60: Dufour J, Chill LP, Muléon P Short- nd long-term effets of hypophysetomy nd unilterl ovrietomy on ovrin folliulr popultions in sheep. J Reprod Fertil 57: Edwrd RG, Fowler RE, Gore-Lngton RE, Gosden RG, Jones EC, Redhed C, Steptoe PC Norml nd norml folliulr growth in mouse, rt nd humn ovries. J Reprod Fertil, 51: Evns AC, Fortune JE Seletion of the dominnt follile in ttle ours in the sene of differenes in the expression of messenger rionulei id for gondotropin reeptors. Endorinology, 138: Gutierrez CG, Cmpell BK, We R Development of long-term ovine grnulos ellulture system: indution nd mintenne of estrdiol prodution, response to follile stimulting hormone, nd morphologil hrteristis. Biol Reprod, 56: Gutierrez CG, Rlph JH, Telfer EE, Wilmut I, We R Growth nd ntrl formtion of ovine prentrl folliles in long-term ulture in vitro. Biol Reprod, 62: Heise M, Koepsel R, Russel AJ, MGee EA Clium lginte miroenpsultion of ovrin folliles impts FSH delivery nd follile morphology. Reprod Biol Endorinol, 3: Hemmlini NC, Ro BS, Tmilmni G, Amrnth D, Vgdevi R, Nidu KS, Reddy KK, Ro VH Influene of trnsforming growth ftor-α, insulin-like growth ftor-ii, epiderml growth ftor or follile stimulting hormone on in vitro development of prentrl folliles in sheep. Smll Rumin Res, 50: Itoh T, Khi M, Ae H, Sendi Y, Hoshi H Growth, ntrum formtion, nd estrdiol prodution of ovine prentrl folliles ultured in serum free medium. Biol Reprod, 67: Kne MT A review of in vitro gmete mturtion nd emryo ulture nd potentil impt on future niml iotehnology. Anim Reprod Si, 79: Kishi H, Greenwld GS In vitro steroidogenesis y dissoited rt folliles, primry to ntrl, efore nd fter injetion of equine horioni gondotropin. Biol Reprod, 61: Kurvil A, Singh G, Kumr GS, Shrm GT Cloning nd sequening of FSH reeptor gene from ufflo prentrl folliles. Eur J Biol Sienes, 2: Lim-Verde IB, Mtos MH, Sriv MV, Bruno JB, Tenório SB, Mrtins FS, Rossetto R, Cunh LD, Nme KP, Báo SN, Cmpello CC, Figueiredo JR Intertion etween estrdiol nd follilestimulting hormone promotes in vitro survivl nd development of prine prentrl folliles. Cells Tissues Orgns, 191: Mo J, Wu G, Smith MF, MCuley TC, Cntley TC, Prther RS, Didion BA, Dy BN Effets of ulture medium, serum type, nd vrious onentrtions of follile stimulting hormone, on porine prentrl follile development nd ntrum formtion in vitro. Biol Reprod, 67: Mtos MH, Lim-Verde IB, Luque MC, Mi JE Jr, Silv JR, Celestino JJ, Mrtins FS, Báo SN, Lui CM, Figueiredo JR Essentil role of follile stimulting hormone in the mintenne of prine prentrl follile viility in vitro. Zygote, 15: Nyudu PL, Osorn SM Ftors influening the rte of prentrl nd ntrl growth of mouse ovrin folliles in vitro. J Reprod Fertil, 95: Rossetto R, Lim-Verde IB, Mtos MH, Sriv MV, Mrtins FS, Fustino LR, Arújo VR, Silv CM, Nme KP, Bo SN, Cmpello CC, Figueiredo JR, Blume H Intertion etween sori id nd follile-stimulting hormone mintins folliulr viility fter long-term in vitro ulture of prine prentrl folliles. Domest Anim Endorinol, 37: Rowghni NM, Heise MK, MKeel D, MGee EA, Koepsel RR, Russell AJ Mintenne of morphology nd growth of ovrin folliles in suspension ulture. Tissue Eng, 10: Roy SK, Greenwld GS In vitro effets of follile-stimulting hormone, luteinizing hormone, nd proltin on folliulr deoxyrionulei id synthesis in the hmster. Endorinology, 122: Roy SK, Trey BJ Isoltion nd long-term ulture of humn prentrl folliles. Fertil Steril, 59: Sint-Dizier M, Mlndin E, Thoumire S, Remv B, Chstnt-millrd S Expression of follile stimulting hormone nd luteinizing hormone reeptors Anim. Reprod., v.10, n.1, p.32-40, Jn./Mr

9 Tmilmni et l. Influene of FSH on growth of ufflo prentrl folliles. during folliulr growth in the domesti t ovry. Mol Reprod Dev, 74: Sriv MV, Celestino JJ, Arújo VR, Chves RN, Almeid AP, Lim-Verde IB, Durte AB, Silv GM, Mrtins FS, Bruno JB, Mtos MH, Cmpello CC, Silv JR, Figueiredo JR Expression of follilestimulting hormone reeptor (FSHR) in got ovrin folliles nd the impt of sequentil ulture medium on in vitro development of prine prentrl folliles. Zygote, 19: Shnmugm M, Pndit S, Plt P Effets of FSH nd LH on steroid prodution y ufflo (Bulus ulis) grnulos ells ultured in vitro under serumfree onditions. Reprod Domest Anim, 45: Silv JM, Christopher A Effet of follilestimulting hormone on steroid seretion nd messenger rionulei ids enoding ytohromes P450 romtse nd holesterol side-hin levge in ovine grnulos ells in vitro. Biol Reprod, 62: Sudo N, Shimizu T, Kwshim C, Kneko E, Tetsuk M, Miymoto A Insulin-like growth ftor-i (IGF-I) system during follile development in the ovine ovry: reltionship mong IGF-I, type 1 IGF reeptor (IGFR-1) nd pregnny-ssoited plsm protein-a (PAPP-A). Mol Cell Endorinol, 264: Tmilmni G, Ro BS, Vgdevi R, Amrnth D, Nik BR, Muthro M, Ro VH Nuler mturtion of ovine ooytes from sheep prentrl folliles ultured in vitro. Smll Rumin Res, 60: Tru Shrm G, Pwn KD, Meur SK Effet of different mehnil isoltion tehniques on developmentl ompetene nd survivl of ufflo ovrin prentrl folliles. Livest Si, 123: Tru Shrm G, Pwn KD, Kumr GS Loliztion nd expression of follile stimulting hormone reeptor gene in ufflo prentrl folliles. Reprod Domest Anim, 46: Tonett SA, Dizereg GS Intrgondl regultion of folliulr mturtion. Endor Rev, 10: Vn den Hurk R, Air R, Telfer EE, Bevers MM Primte nd ovine immture ooytes nd folliles s soures of fertilizle ooytes. Hum Reprod Updte, 6: Vn den Hurk R, Sntos R Development of fresh nd ryopreserved erly-stge ovrin folliles, with speil ttention to ruminnts. Anim Reprod, 6: Wndji SA, Eppig JJ, Fortune JE FSH nd growth ftors ffet the growth nd endorine funtion in vitro of grnulos ells of ovine prentrl folliles. Theriogenology, 45: We R, Armstrong DG Control of ovrin funtion; effet of lol intertions nd environmentl influenes on folliulr turnover in ttle: review. Livest Si, 53: Zeleznik AJ Dynmis of primte folliulr growth: physiologi perspetive. In: Adshi EY, Leung PCK (Ed.). The Ovry. New York, NY: Rven Press. pp Anim. Reprod., v.10, n.1, p.32-40, Jn./Mr. 2013