Oocytes determine cumulus cell lineage in mouse ovarian follicles

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1 133 Reserh tile Ooytes determine umulus ell linege in mouse ovrin folliles Frniso J. Diz, Kren Wigglesworth nd John J. Eppig* The Jkson Lortory, 6 Min Street, Br Hror, ME 469, USA *Author for orrespondene (e-mil: john.eppig@jx.org) Aepted 13 Ferury 27 Journl of Cell Siene 12, Pulished y The Compny of Biologists 27 doi:1.1242/js.968 JCS epress online pulition dte 27 Mrh 27 Journl of Cell Siene Summry The two prinipl funtions of ovrin folliles re developmentl nd endorine. The umulus ells surrounding the ooyte re speilized to serve the development of the ooyte nd steroidogenesis is prinipl role of murl grnulos ells tht line the follile wll. The findings in this report demonstrte tht ooytetomy or tretment with n inhiitor of SMAD2/3 tivtion results in deresed umulus mrker mrna trnsript levels nd llows to indue murl mrker trnsripts in umulus ells. In ddition, SMAD2/3 signling is involved in enling umulus expnsion nd EGF-indued inreses in Introdution Ovrin Grfin folliles re omprised of somti ells tht exhiit oth endorine nd developmentl funtions. The endorine funtions, inluding steroidogenesis, re rried out primrily y the murl grnulos ells (Fig. 1). The umulus ells, whih re losely ssoited with the ooyte, promote ooyte growth nd developmentl ompetene through omplex i-diretionl intertions with the ooyte. In ft, ooyte stimultion of umulus ell funtion is ruil for its own susequent development (Eppig, 21; Hussein et l., 26; Su et l., 24). To fulfil oth endorine nd developmentl funtions, follile growth must involve the oordinted development of oth umulus nd murl grnulos ells, s defiieny in either hormone prodution or development of ooyte ompetene would impir fertility. Although grnulos ells express mny mrna trnsripts nd proteins in ommon, the murl nd umulus ells eh express suset of different trnsripts, with mrkedly different stedy stte levels (Fig. 1). Prentrl grnulos ells re the ommon preursors of oth murl nd umulus ells. Lrge prentrl folliles (seondry folliles) develop into ntrl folliles (tertiry folliles) fter forming fluid-filled ntrum under stimultion of follile stimulting hormone (). Antrum formtion physilly seprtes the prentrl grnulos ells into murl ell omprtment long the follile wll nd umulus ell omprtment surrounding the ooyte (Fig. 1). The murl ells produe inresing mounts of estrogen, whih eventully initites the surge of luteinizing hormone (LH) from the pituitry resulting in ooyte mturtion, expnsion of the umulus oophorus nd ovultion. The umulus ells promote ooyte growth nd developmentl ompetene (Eppig, 21; Hussein et l., 26; Su et l., 24). A defining feture of umulus ells is their ility to Ptx3, Ptgs2 nd Hs2 mrna levels. By ontrst, follilestimulting hormone () stimulted expression of murl trnsripts, ut suppressed levels of umulus trnsripts. Thus, nd ooyte-stimulted SMAD2/3 signling estlish opposing grdients of influene in the follile. These speify the murl nd umulus grnulos ell phenotypes tht re pivotl for pproprite endorine funtion nd ooyte development. Key words: Ovrin follile, Ooytes,, SMAD2/3 undergo expnsion following the LH surge. Cumulus expnsion involves the prodution of muified mtrix y the umulus ells tht is neessry for ovultion nd therefore required for fertility (Chen et l., 1993). The mehnisms responsile for differentition of prentrl grnulos ells into murl nd umulus ells re not fully defined, ut involve ftors sereted y ooytes nd produed y the pituitry. is essentil, s folliles fil to develop eyond the erly ntrl stge in mie lking (Kumr et l., 1997) or reeptors (Dierih et l., 1998). stimultes expression of (luteinizing hormone reeptor) mrna in murl ells (Fig. 1). mrna is key mrker of murl ell differentition (Lei et l., 21). Both umulus nd murl ells express reeptors (Rihrds nd Midgley, 1976); however, ooyte suppression of in umulus ells restrits mrna to murl ells (Eppig et l., 1997). In ddition to suppressing mrna, prrine ftors sereted y ooytes hve profound effets on follile growth nd development oth efore nd fter the LH surge. For exmple, sene of ooyte-derived growth nd differentition ftor 9 (GDF9) results in filure to progress eyond the erly prentrl stges of follile development (Dong et l., 1996; Elvin et l., 1999). Lk of one morphogeneti protein 15 (BMP15), nother ooyte-sereted protein, leds to defetive umulus ell differentition nd umulus expnsion (Su et l., 24; Yn et l., 21; Yoshino et l., 26). Ooyte-derived ftors re lso required to enle umulus expnsion fter the preovultory LH-surge (Buione et l., 199; Diz et l., 26). The mjor impt tht ooytes hve in direting follile development ws demonstrted y reomining mid-growth stge ooytes from lrge prentrl folliles with folliulr somti ells from neworn ovries. The resulting re-ggregted ovry not only forms folliles

2 Ooytes promote umulus phenotype 1331 Pre-LH surge Post- LH surge Prolifertion & Differentition Cumulus Sl383 Murl Cyp111 LH Surge Hs2 Ptgs2 Ptx3 Tnfip6 Prentrl Follile Antrl Follile Cumulus Expnsion Reltive Levels of Sl383 mrna Sl383 Reltive Levels of mrna Cumulus Mrker Trnsripts * * Murl Murl Reltive Levels of mrna * Murl Reltive Levels of mrna Murl Mrker Trnsripts Cyp *.5 * *.2 Reltive Levels of Cyp111 mrna Murl Murl Murl Reltive Levels of mrna Journl of Cell Siene Fig. 1. Model depiting folliulr growth nd umulus expnsion. Prentrl folliles under the stimultion of gondotropins nd ooyte ftors develop into ntrl folliles. The formtion of the ntrum seprtes the grnulos ells into murl nd umulus ells. Murl ells (green) express, Cyp111 nd trnsripts more highly thn umulus ells (red), wheres Sl383, nd trnsripts re highly expressed in umulus ells. At ovultion, the LH surge indues umulus expnsion with ssoited inreses in expnsion-relted trnsripts (Hs2, Ptgs2, Ptx3 nd Tnfip6). Rel-time PCR nlysis of murl nd umulus mrker trnsripts in freshly isolted s nd murl ells is lso shown. Trnsripts were normlized to Rpl19 mrna. *Indites signifint differene y Student s t-test P<.5. when trnsplnted to reipient mie, ut the hronologilly more dvned ooytes tully elerte the development of the neworn somti ells (Eppig et l., 22). These oservtions provided strong evidene tht ooyte-derived ftors re importnt meditors of follile growth, umulus ell differentition nd umulus expnsion. Ooyte ftors nd signl from opposite omprtments. The ooyte signls emnte from entrl folliulr lotion, wheres signls rrive from outside the follile. This leds to grdient of expression of murl nd umulus trnsripts. Levels of the murl mrker trnsript re highest ner the sl lmin nd lowest in the umulus ells (Meduri et l., 1992) (Fig. 1). Conversely, expression of mrna, whih is stimulted y ooytes (Slmon et l., 24) is highest in the umulus ells (Brends et l., 1995) (Fig. 1). Ooyte-sereted prrine ftors re lso required to enle inreses in Hs2, Ptgs2, Ptx3 nd Tnfip6 trnsripts during umulus expnsion (Diz et l., 26). Eh of these trnsripts is solutely required for umulus expnsion s the phenotype of null muttions in the Ptgs2, Ptx3 or Tnfip6 genes or inhiition of HAS2 tivity severely ompromises umulus expnsion (Chen et l., 1993; Fulop et l., 23; Ohsner et l., 23; Ohsner et l., 23; Vrni et l., 22). The signling pthwys tivted y ooytes nd required for umulus ell funtion remin lrgely unler. However, pthwys tivted y TGF -relted proteins, suh s GDF9 nd BMP15, re proly ruil for mediting the effet of ooytes on grnulos ells. BMP15 nd GDF9 tivte SMAD1/5/8 nd SMAD2/3 signling pthwys, respetively, in grnulos ells (Moore et l., 23; Roh et l., 23). Signling through either SMAD1/5/8 or SMAD2/3 pthwys requires tht these reeptor-regulted SMADs ind the ommon SMAD, SMAD4. Conditionl deletion of the Smd4 gene in grnulos ells of prentrl folliles leds to severe defets in susequent follile development nd differentition owing to lk of SMAD4-dependent signling (Pngs et l., 26). Moreover, mie defiient in Smd3 show inresed rtes of ell deth nd norml ell differentition (Tomi et l., 24). Thus, SMAD signling is ruil for proper follile development. More reently, the BMP reeptor type II (BMPR2) extrellulr domin nd n inhiitor of SMAD2/3 tivtion,, were used to lok ooyte-stimulted prolifertion of murl nd umulus ells (Gilhrist et l., 26). However, the ext TGF -relted signling pthwys tivted in umulus ells y ooytes tht led to umulus ell differentition nd expnsion remin undefined. The work reported here tests the hypothesis tht ooyte-stimulted SMAD2/3 signling medites the ility of ooytes to stimulte the umulus ell phenotype during ntrl follile development. Results Ooyte regultion of SMAD2 tivtion nd loliztion during follile development A strong psmad2 signl ws deteted y immunofluoresene stining of tissue setions in prentrl grnulos ells of smll nd lrge prentrl folliles (Fig. 2A,B). In ntrl folliles, psmad2 lolized more strongly to umulus ells thn to murl ells (Fig. 2C). Surprisingly, tretment with hcg in vivo resulted in deresed psmad2 levels y 8 hours (Fig. 2D). No psmad2 signl ws deteted in ovry setions inuted with seondry ntiody lone or

3 1332 Journl of Cell Siene 12 (8) when the psmad2 ntiody ws preinuted with SMAD2 phosphopeptide (pser465/467, Am) (Fig. 2E nd dt not shown). To verify the results oserved in tissue setions, we ompred psmad2 levels in freshly isolted umulus-ooyte omplexes (s) nd murl ells y western lot nd found tht levels of totl SMAD2 were similr in murl nd s, ut levels of phosphorylted SMAD2 were higher in s ompred with murl ells (Fig. 2F). The levels of psmad2 oserved in umulus ells were stimulted y the ooyte. Levels of psmad2 deteted y western lot were high in untreted s (Fig. 3). Removl of the ooyte gretly redued the psmad2 signl to n lmost undetetle level, whih ws restored upon o-ulture with ooytes. suppressed psmad2 in s nd OOX ells o-ultured with ooytes (Fig. 3). Thus, the ooyte potently stimultes psmad2 in umulus ells. Journl of Cell Siene Speifiity of inhiitors The effet of nd on tivtion of vrious signling moleules in ultured s ws determined to ssess the speifiity of the inhiitors. loked SMAD2 nd SMAD3 tivtion in s, ut hd no effet on totl SMAD2 levels or levels of psmad1/5/8 (Fig. 4A). By ontrst, loked psmad3 tivtion without ffeting totl SMAD2, psmad2 or psmad1/5/8 levels (Fig. 4A). The effiy of ws determined y dose-response experiment where s were treted with onentrtions of rnging from.1 to 1 M (Fig. 4B). The minimum dose tht effetively loked psmad2 levels in s ws 3 M (Fig. 4B). Although nd re highly speifi for inhiiting the kinse tivity of ALK-4, ALK-5 nd ALK-7, the possiility exists tht these moleules ould hve effets on other untested ellulr kinses or even other non-kinse proteins. Suppression of murl trnsripts y ooytes Trnsripts enoding the LH reeptor,, the steroidogeni Fig. 2. Immunololiztion of psmad2 in prentrl (A), trnsitionl prentrl-ntrl (B) nd ntrl folliles efore (C) nd fter (D) 8 hours hcg (5 IU) tretment in vivo. (E) No stining ws oserved when the primry ntiody ws pre-inuted with psmad2 phosphopeptide. rows indite umulus-ooyte omplexes. Brs, 5 m. (F) Western lot immunostined for psmad2, totl SMAD2 nd ACTB in freshly isolted s nd murl ells from ntrl folliles. Fig. 3. Ooytes stimulte psmad2 in umulus ells. Western lot immunostined for psmad2 nd ACTB in s ultured in medi lone or with (1 M) nd in OOX ells ultured lone, OOX ells o-ultured with nd o-ultured OOX ells treted with (1 M) for 15 hours. Grphil representtion of reltive pixel intensity normlized to ACTB pixel intensity is lso shown.,p<.5, signifint differenes.

4 Ooytes promote umulus phenotype 1333 Journl of Cell Siene Fig. 4. Speifiity of inhiitors. (A) Western lot of lystes olleted from s treted with medi only, (1 M) or (2 M) for 4 hours nd immunostined for psmad2, totl SMAD2, psmad3, psmad1/5/8 nd ACTB proteins. (B) Western lot immunostined for psmad2 nd ACTB of s treted with inresing onentrtion of (.1 M to 1 M) for 4 hours. enzyme P45 side hin levge, Cyp111, nd the immune moleule were higher in murl thn in umulus ells s mesured y rel-time PCR (Fig. 1). We used these trnsripts s mrkers of the pre-lh-surge murl ell phenotype. Ooytederived ftors suppress expression of mrna in umulus ells (Eppig et l., 1998; Eppig et l., 1997). We hypothesized tht ooytes might suppress, Cyp111 nd through tivtion of the TGF signling pthwy. To test this ide, the effets of ooyte extirption (OOX), (1 ng/ml), (n inhiitor of SMAD2 nd SMAD3 tivtion, 1 M) nd ( SMAD3-speifi inhiitor, 2 M) on expression of murl trnsripts in nd murl ells were mesured. ws required to mintin expression of murl ell mrkers in vitro (Fig. 5A,B). Fully-grown, germinl vesile (GV) stge ooytes () potently suppressed the stedy-stte levels of the three murl trnsripts tht hd een mintined y (Fig. 5A,B). The suppression of murl trnsripts y ooytes in murl ells ws loked y tretment with, ut not (SMAD3 inhiitor) suggesting tht SMAD3 signling ws not involved (Fig. 5B). In s, we hypothesized tht ooytes would lso suppress murl trnsripts through TGF signling pthwys. Removl of the ooyte (OOX) llowed to stimulte inresed expression of murl trnsripts in umulus ells (Fig. 6A). Co-ulture with ooytes prevented -indued inreses of murl trnsripts, ut tretment with, ut not, loked the ility of ooytes to suppress murl trnsript indution y (Fig. 6B,C), inditing tht ooyte suppression of murl trnsripts in umulus ells is likely vi psmad2. Ooytes promote umulus trnsripts Ooyte-derived ftors promote expression of Sl383 (Eppig Reltive Levels of mrna Reltive Levels of Cyp111 mrna Reltive Levels of mrna A. Murl Control Control Cyp111 Control Control Reltive Levels of mrna et l., 25) nd mrna (Slmon et l., 24) in umulus ells. These trnsripts long with (ndrogen reeptor) mrna re more highly expressed in s thn murl ells (Fig. 1). We hypothesized tht ooytes my promote expression of the umulus trnsripts Sl383, nd though the TGF signling pthwy. To test this ide, the effets of ooyte extirption (OOX), (1 ng/ml), (n inhiitor of SMAD2 nd SMAD3 tivtion, 1 M) nd ( SMAD3-speifi inhiitor, 2 M) on expression of umulus trnsripts ws tested. OOX resulted in derese in umulus mrkers (Sl383, nd mrna), whih were restored y ooyte o-ulture (Fig. 7A) inditing tht ooyte ftors stimulte these trnsripts. ut not (Fig. 7B,C) used derese in umulus mrker trnsript levels. Surprisingly, ompletely suppressed Sl383 nd deresed trnsript levels y ~5%, even in the presene of ooytes (Fig. 7B). Murl ells express lower levels of Sl383, nd mrnas (Fig. 1). Sine ooytes n stimulte psmad2 levels in murl ells (Gilhrist et l., 26) Reltive Levels of Cyp111 mrna Reltive Levels of mrna B. Murl Control Control Control Control Control Control Control Control Cyp111 Fig. 5. Regultion of murl trnsripts, Cyp111 nd. Effet of (1 M) (A) nd (2 M) (B) on levels of murl trnsripts in murl ells ultured lone or with s (2 ooytes/ l) for 12 hours. Trnsripts were mesured y rel-time PCR nd normlized to Rpl19 mrna. P<.5, signifint differenes.

5 1334 Journl of Cell Siene 12 (8) Journl of Cell Siene Reltive Levels of mrna Reltive Levels of mrna A. -OOX Reltive Levels of Cyp111 mrna Ctrl Ctrl OOX OOX Ctrl Ctrl Cyp111 Ctrl Ctrl OOX OOX OOX OOX B. - Reltive Levels of mrna Reltive Levels of Cyp111 mrna Reltive Levels of mrna Cyp111 nd psmad2 ppers importnt for expression of umulus trnsripts (Fig. 7B), we hypothesized tht ooytes would stimulte umulus trnsripts in murl ells. Levels of Sl383 nd were not indued y ooytes in murl ells. However, ooytes did stimulte trnsript levels in murl ells, whih ws loked y, ut not (Fig. 8A,B). Cumulus expnsion requires SMAD signling Cumulus expnsion requires one or more ooyte-sereted ftors (Vnderhyden et l., 199). nd were used to test the effet of loking oth SMAD2 nd SMAD3 or SMAD3 only on umulus expnsion. As expeted, expnsion ws stimulted y EGF tretment in vitro (Fig. 9A). Tretment with ompletely loked umulus expnsion (Fig. 9A). By ontrst, expnsion ws not severely ffeted y tretment, ut some ells did tth to the ulture dish suggesting tht expnsion ws not ompletely norml (white rrows Fig. 9A). The minimum effetive dose of tht loks expnsion ws 1-3 M (Fig. 9B) nd is similr to the minimum dose tht loks psmad2 levels in (Fig. 4B). Effet of nd on expnsion-relted trnsripts To etter define the moleulr effets of nd on umulus expnsion, n nlysis of umulus-expnsionrelted trnsripts t severl time points during umulus expnsion ws undertken. EGF stimulted expression of Hs2, Ptgs2, Ptx3 nd Tnfip6 mrna in s with the C. - Reltive Levels of mrna Reltive Levels of Cyp111 mrna Reltive Levels of mrna Cyp111 Fig. 6. (A) Effet of OOX nd (1 ng/ml) on levels of murl trnsripts in umulus ells treted for 12 hours. Effet of (1 M) (B) or (2 M) (C) lone or in omintion with (1 ng/ml) on levels of murl trnsripts in s ultured for 15 hours. Trnsripts were mesured y rel-time PCR nd normlized to Rpl19 mrna. P<.5, signifint differenes. highest level mesured ourring 6 hours fter EGF tretment (Fig. 9C). The effet of the inhiitors on EGF-indued expression of expnsion-relted trnsripts ws dependent on the speifi trnsript nd on the time fter EGF tretment. Levels of Ptgs2 mrna were not ffeted y, wheres tretment with suppressed levels of Ptgs2 mrna t 8 nd 1 hours (Fig. 9C). Both nd redued levels of Ptx3 mrna y 75-95% (Fig. 9C). Levels of Tnfip6 mrna were not ffeted y either or t 6 or 8 hours fter EGF, ut y 1 hours, oth inhiitors suppressed Tnfip6 mrna levels (Fig. 9C). Levels of Hs2 mrna were not ffeted y, ut were ompletely suppressed y t ll times exmined (Fig. 9C). Thus, regultion of expnsion trnsripts is likely ontrolled through omplited network of pthwys whih inludes psmad2 nd psmad3 signling. Intertion of MAPK nd SMAD signling pthwys Cumulus expnsion requires oth tivtion of MAPK3/1 nd MAPK14 nd ooyte-stimulted SMAD2/3 signling. n inhiit MAPK14 tivtion in ell-free ssy system (Inmn et l., 22). Therefore, we nlyzed the effet of on phosphoryltion of MAPK3/1 nd MAPK14 y EGF nd the effet of EGF on psmad2 levels in order to egin to identify t wht level the MAPK nd SMAD signling pthwys intert. EGF stimulted MAPK3/1 nd MAPK14 phosphoryltion in s (Fig. 1A). tretment ompletely loked psmad2 levels in s, ut lk of SMAD2 phosphoryltion did not prevent MAPK3/1 or

6 Ooytes promote umulus phenotype 1335 A. Murl- B. Murl- Journl of Cell Siene Reltive Levels of Sl383 mrna Reltive Levels of mrna A. -OOX Reltive Levels of mrna OOX OOX Sl383 OOX OOX OOX OOX B. - Reltive Levels of Sl383 mrna Reltive Levels of mrna Reltive Levels of mrna Sl383 C. - Sl383 Fig. 7. Regultion of umulus trnsripts (Sl383,, nd ) y ooytes. (A) Effet of OOX nd ooyte o-ulture on umulus trnsript levels in s nd OOX umulus ells ultured in vitro for 12 hours. Effet of (1 M) nd (1 ng/ml) (B) or (2 M) (C) on umulus trnsript levels in s ultured for 15 hours. Trnsripts were mesured y rel-time PCR nd normlized to Rpl19 mrna. P<.5, signifint differenes. Reltive Levels of Sl383 mrna Reltive Levels of mrna Reltive Levels of mrna Reltive Levels of Sl383 mrna Reltive Levels of mrna Reltive Levels of mrna Sl383 Control Control Control Reltive Levels of Sl383 mrna Reltive Levels of mrna Reltive Levels of mrna Sl383 Control SiS3 Control SiS3 Ctrl Fig. 8. Effet of (1 M) (A) nd (2 M) (B) on expression of umulus trnsripts in murl grnulos ells ultured lone or with s (2 ooytes/ l) for 15 hours. Trnsripts were mesured y rel-time PCR nd normlized to Rpl19 mrna. P<.5, signifint differenes. MAK14 tivtion 4 hours fter EGF tretment (Fig. 1A). Similr to in vivo hcg-treted folliles (Fig. 2E), levels of psmad2 egn to derese signifintly (P>.5) y 8 hours with further derese y 12 hours fter EGF tretment in vitro (Fig. 1B). The levels of totl SMAD2 did not hnge during EGF stimultion inditing tht the derese in SMAD2 tivtion oserved t 8 nd 12 hours ws due to deresed psmad2 tivtion nd not to loss of SMAD2 protein. Thus, tivtion of MAPK is independent of SMAD2/3 tivtion during umulus expnsion. However, tivtion of MAPK pthwys led to deresed psmad2 signling during umulus expnsion. Disussion Evidene is presented here tht ooyte-stimulted SMAD signling is ruil for defining the differentition nd funtion of umulus ells. Moreover, ooyte-sereted ftors oppose the tion of, whih would, in the sene of the ooyte, promote the expression in umulus ells of hrteristis more typil of murl grnulos ells. Solule ooyte-derived ftors strongly promoted phosphoryltion of SMAD2 in umulus ells. Ativted SMAD2 promoted expression of mrker umulus trnsripts nd suppressed expression of murl trnsripts in umulus ells, nd together with psmad3, enled umulus expnsion. The SMAD3-speifi inhiitor,, hd no effet on expression of murl or umulus mrker trnsripts, ut did lok inreses in Ptx3 nd Tnfip6 mrna, two trnsripts indued y EGF during umulus expnsion, suggesting tht SMAD2 nd SMAD3 hve prtilly divergent effets on umulus ell funtion. By ontrst, stimulted expression of murl mrker trnsripts nd suppressed the expression of some umulus mrker trnsripts. In the presene of ooytes, ws unle to indue murl trnsripts, ut did suppress umulus trnsripts in vitro. Thus, we present model (Fig. 11) where ooyte-stimulted psmads nd estlish opposing grdients of influene tht define the umulus nd murl grnulos ell phenotypes, respetively. These mehnisms re importnt for promoting optiml endorine nd developmentl funtions in the ovrin follile. Murl nd umulus ells oth originte from prentrl

7 1336 Journl of Cell Siene 12 (8) Journl of Cell Siene grnulos ells. The divergent funtions (endorine vs developmentl) of these two lineges develop within the ontext of follile nd thus the speifition of the umulus vs murl ell fte must depend in prt on the lol miroenvironment. In the ntrl follile, the ooyte is emerging s entrl regultor of umulus ell funtion. We hypothesized tht SMAD2/3 signling pthwys might e involved in speifying the umulus ell phenotype euse of two reent reports showing: (1) n inhiitor of SMAD2/3 tivtion,, loks the ility of the ooyte to stimulte umulus ell prolifertion (Gilhrist et l., 26) nd (2) deletion of the Smd4 gene in grnulos ells results in defetive umulus ell differentition (Pngs et l., 26). In SMAD4-defiient grnulos ells, tivted forms of SMAD2/3 nd SMAD1/5/8 would e unle to lter trget gene trnsription euse of the sene of the o-smad, SMAD4, effetively lting signling through these pthwys. As first step in nlyzing SMAD2/3 signling in the follile, tivted SMAD2 ws lolized in tissue setions y immunofluoresene nd immunolot nlyses. Strong psmad2 immunostining ws evident in grnulos ells of prentrl folliles. However, in lrge ntrl folliles, 48 hours fter egc tretment, psmad2 levels were muh higher in umulus ells ompred with murl ells, similr results were otined y immunolotting. The pttern of psmad2 stining suggested tht the ooyte is responsile for tivting this pthwy in umulus ells. Ooytetomy ompletely lted psmad2 levels in umulus ells, whih were restored upon ooyte o-ulture showing tht ooytes stimulte psmad2 signling in umulus ells. These oservtions re onsistent with role of SMAD2/3 signling in umulus ell funtion. The TGF signling pthwys re emerging s key regultor of ovrin funtion. In this report, two inhiitors of TGF -tivin-gdf9 signling, nd were used. The tivin-like kinse (ALK) inhiitor,, speifilly inhiits the tivity of ALK4, ALK5 nd ALK7 without effet on other ellulr kinses in living ells (Inmn et l., 22; Lping et l., 22) (Figs 4, 1). ALK4 nd ALK5 re the type 1 reeptors involved in GDF9 nd tivin signling. When tivted y lignd, ALK4 nd ALK5 pir with speifi type II reeptor, resulting in phosphoryltion of SMAD2 nd SMAD3 signling moleules (Moore et l., 23; Roh et l., 23). speifilly loks SMAD3 tivity y inhiiting ALK5- medited phosphoryltion of SMAD3 nd ssoition with Fig. 9. Involvement of SMAD signling in umulus expnsion. (A) Effet of medium only, (1 M) or (2 M) on umulus expnsion in s treted with EGF (1 ng/ml) for 15 hours. White rrows indite umulus ells tthed to the ulture dish in treted with. (B) Expnsion of s treted with EGF (1 ng/ml) lone or with 1 M or.1 M for 15 hours. (C) Effet of (1 M) or (2 M) on expression of umulus expnsion trnsripts in s treted with EGF (1 ng/ml) for 6, 8 nd 1 hours. Trnsripts were mesured y rel-time PCR nd normlized to Rpl19 mrna. P<.5, signifint differenes. SMAD4 (Jinnin et l., 25). In s, is speifi inhiitor of SMAD2 nd SMAD3 tivtion, wheres speifilly loks SMAD3 tivtion. Neither inhiitor hd ny effet on totl SMAD2 levels or on phosphoryltion of the relted SMAD1/5/8 pthwy. Inresing evidene supports diret role of the ooyte in promoting umulus ell funtion efore the LH surge. Ooytes re known to promote glyolysis (Sugiur et l., 25),

8 Ooytes promote umulus phenotype 1337 Journl of Cell Siene Fig. 1. (A) Western lot immunostined for psmad2, pmapk14, pmapk3/1 nd ACTB in s treted with EGF (1 ng/ml) lone or together with (1 M) for 4 hours. Grphil representtions of psmad2, MAPK14 nd MAPK3/1 pixel intensity normlized to ACTB re shown elow representtive western lot (n=3). (B) Western lot immunostined for psmad2, totl SMAD2 nd ACTB in s treted with EGF (1 ng/ml) for, 4, 8 nd 12 hours. Grphil representtions of psmad2, totl SMAD2, MAPK14 nd MAPK3/1 pixel intensity, normlized to ACTB, re shown elow representtive western lot (n=4). Note tht eh imge shown is of the sme lot sequentilly stined for the proteins indited. P<.5, signifint differenes. prolifertion (Gilhrist et l., 26; Vnderhyden et l., 1992), mino id trnsport (Eppig et l., 25) nd ell survivl (Hussein et l., 25), nd to suppress inpproprite expression of murl trnsripts suh s in umulus ells (Eppig et l., 1997). Results presented in this study onfirm nd extend these oservtions to inlude ooyte regultion of other umulus nd murl trnsripts efore the LH surge. For exmple, we show tht ooytes promote expression of three umulus trnsripts, Sl383, nd. Eh of these trnsripts hs importnt funtions. Expression of SLC38A3 in umulus ells proly medites, t lest in prt, the trnsport of speifi mino ids from the umulus ells to the ooyte in support of ooyte growth (Eppig et l., 25). Expression of AR in the follile is required for full fertility euse null mie re severely sufertile nd show defets in umulus ell morphology nd differentition (Hu et l., 26). In ddition, ndrogens ugment the ility of ooytes to stimulte ellulr prolifertion (Hikey et l., 25). AMH in erly ntrl folliles ntgonizes -indued follile growth (Durlinger et l., 21; Durlinger et l., 1999). Moreover, we show tht tretment with, ut not, resulted in redued umulus mrker trnsript levels. These oservtions suggest tht SMAD2 nd SMAD3 hve divergent funtions in the regultion of the stedy-stte levels of these trnsripts. Surprisingly, suppressed Sl383 nd mrna levels in ultured s nd is likely to e one reson why Sl383 nd re not expressed in murl ells. The murl ell phenotype is produed to gret extent y stimultion of trnsripts involved in steroidogenesis (Cyp111), ovultion () nd immune funtion (). However, ooytes potently suppressed murl trnsripts in murl ells even in the presene of. Beuse ooyte ftors nd form opposing onentrtion grdients in the follile, grdient of trnsript levels is oserved in ntrl folliles, where grnulos ells frther wy from the ooyte express higher levels of murl trnsripts (Eppig et l., 22; Lei et l., 21). Cumulus ells re losely ssoited with the ooyte nd therefore it is not surprising tht expression of murl trnsripts is low in umulus ells euse of the suppressive tion of ooyte-derived ftors. This helps to explin why did not promote expression of the murl mrker trnsripts in ultured s even though umulus ells roustly express reeptors (Rihrds nd Midgley, 1976). However, is le to stimulte murl trnsript expression in umulus ells in the sene of ooytes (OOX) or in s treted with, ut not inditing tht SMAD2 tivity is required for suppression of murl trnsripts y ooyte ftors. Tht suppressed umulus mrker trnsripts nd stimultes murl trnsripts in vitro, suggests tht ooytes must ontinuously overome these two potentil effets of on umulus ells efore the LH surge in vivo. It is remrkle tht ooyte-stimulted SMAD2 signling promotes elevted levels of umulus mrker trnsripts nd suppresses murl trnsripts in umulus ells, while only suppressing murl trnsripts in murl ells. These oservtions might reflet reruitment of different otivtors or o-repressors to the promoter regions of these genes. However, further studies re needed to define how ommon signl n generte oth positive nd negtive responses in the sme ell type. In ddition to regulting the levels of umulus trnsripts efore the LH surge, ooyte-sereted ftors lso modify

9 1338 Journl of Cell Siene 12 (8) Journl of Cell Siene umulus ell funtion fter the LH surge y enling umulus expnsion. The proess of umulus expnsion requires the presene of umulus expnsion enling ftors (CEEFs) sereted y the ooyte. The nture of the CEEFs hs remined elusive, ut ould inlude GDF9 (Elvin et l., 1999; Gui nd Joye, 25) nd/or BMP15 (Su et l., 24; Yoshino et l., 26) sine oth reominnt proteins stimulte expnsion. Here, we demonstrted tht ompletely loks EGF-indued expnsion, wheres the effet of is more sutle, expnsion still ours in treted s, ut mny ells from tthments to the ulture dish, inditing n norml expnsion. Cumulus expnsion is dependent on inreses in trnsript levels of Hs2, Ptx3, Ptgs2 nd Tnfip6 (Chen et l., 1993; Fulop et l., 23; Ohsner et l., 23; Ohsner et l., 23; Vrni et l., 22). The potentil requirement of psmad2 nd psmad3 in driving the inresed levels of these trnsripts in umulus ells ws unknown. Here we demonstrte omplex pttern of trnsript regultion during umulus expnsion. One trnsript, Tnfip6, ws lrgely unffeted y either inhiitor during EGF-indued expnsion. At the other extreme, EGF-indued inrese in Ptx3 mrna ws suppressed y oth inhiitors t ll time points exmined. Ptgs2 mrna levels were not suppressed t 6 hours, ut y 8-1 hours,, ut not, suppressed levels of this trnsript. Finlly, Hs2 ws suppressed y, ut not y. These results indite tht the ooyte-enled inrese in Ptx3 requires tivtion of oth SMAD2 nd SMAD3, wheres SMAD2 tivtion is suffiient for enling inreses in Ptgs2 nd Hs2 mrna. However, the CEEF tivity responsile for enling inreses in Tnfip6 mrna signl through pthwys tht re not utely dependent on either psmad2 or psmad3. This suggests the possile existene of one or more CEEF ftors sereted y the ooyte. The ftor(s) responsile for psmad2/3 tivtion in umulus ells re not known. Cndidte moleules inlude ooyte-derived GDF9 or TGFB1 or the grnulos-ell-derived tivin (Knight nd Glister, 26). GDF9 is n ttrtive ndidte euse mie lking this protein do not form ntrl folliles (Dong et l., 1996), rgdf9 tivtes SMAD2 in grnulos ells in vitro (Roh et l., 23) nd RNAi inhiition of GDF9 prodution in ooytes rogtes umulus expnsion (Gui nd Joye, 25), whih we now show is dependent on psmad2/3. Thus, GDF9 might hve roles during oth umulus ell differentition nd umulus expnsion. However, not ll the dt support role of GDF9 during expnsion, sine GDF9-neutrlizing ntiody did not lok umulus expnsion enled y ooytes (Drgovi et l., 25). TGFB1 is nother possile ndidte sine it is expressed y the ooyte nd tivtes the SMAD2/3-signling pthwy. Mie with null muttions in the Tgf1 gene hve redued fertility (Ingmn et l., 26) suggesting possile involvement in follile development. However, TGFB1-neutrlizing ntiody does not lok umulus expnsion enled y ooytes (Slustri et l., 199). The possile involvement of tivin in umulus ell differentition or umulus expnsion hs not een investigted. Regrdless of whih lignd is responsile for SMAD2/3 tivtion in umulus ells, ooyte-stimulted psmad2/3 in umulus ells is prt of differentition mehnism required to speify the umulus ell phenotype. Communition etween the umulus ells nd ooyte is ruil for susequent fertility. Disruption of these signling Opposing effets of nd SMAD2/3 signling psmad2 Murl Cells Cyp111 Sl383 Cumulus Cells Sl383 Cyp111 OSFs Fig. 11. Summry model depiting the opposing grdients of ooytestimulted SMAD2 nd signling in the ntrl follile tht result in divergent expression of murl nd umulus trnsripts. Cumulus ells (red) form under the influene of the ooyte sereted ftors (OSPs), wheres murl ells (green) form under the influene of. Grnulos ells loted in etween ooyte nd signls exhiit intermedite levels of murl nd umulus trnsripts (light green or pink) depending on their proximity to the sl lmin or the ooyte. pthwys etween ooytes nd umulus ells leds to poor developmentl potentil or even infertility. Thus, resolving how umulus ells nd ooytes signl eh other is key to understnding the mehnisms responsile for fundmentl proess in iology, the prodution of mture femle gmete. Fig. 11 summrizes our working model nd highlights the ruil role of ooyte-stimulted SMAD2/3 signling in determining spets of the umulus ell phenotype, inluding expression of umulus trnsripts, suppression of murl trnsripts efore the LH surge nd promotion of umulus expnsion fter the LH surge. Two opposing rdil grdients within the follile re reted y signling from outside the follile nd ooyte-stimulted SMAD2 signling from within the follile. Together, they speify hrteristis nd funtions of the murl nd umulus ell omprtments within the follile to foster n optiml miroenvironment for proper endorine (murl) nd developmentl (umulus-ooyte omplex) funtions. Mterils nd Methods Animls Femle B6SJLF1 mie (Mus musulus) were produed nd rised in the reserh olony of the investigtors. Ovries were olleted from mie on dy 22, 48 hours fter I.P. injetion of 5 IU ecg (Ntionl Hormone nd Peptide Progrm, NIDDK). For some experiments, ovries from 12-dy-old nimls were used for nlysis of prentrl grnulos ells. Animls were mintined ording to the Guide for the Cre nd Use of Lortory Animls (Institute for Lerning nd Animl Reserh). Isoltion nd ulture of ooytes nd grnulos ells s nd fully grown denuded ooytes t the GV (germinl vesile) stge were olleted from ntrl folliles. Following olletion, lumps of murl grnulos ells from pproximtely one primed ovry were eqully divided into four groups. Murl grnulos ells, s (25 for mrna or 5 for protein), OOX omplexes (25 or 5) nd o-ultures of murl or OOX omplexes with fully grown ooytes (s 2 ooytes/ l) were ultured in ironte-uffered MEM- (Life

10 Ooytes promote umulus phenotype 1339 Journl of Cell Siene Tehnologies, Grnd Islnd, NY) with Erles slts, supplemented with 75 mg/l peniillin G, 5 mg/l streptomyin sulfte, 1 M milrinone (to prevent GVB in s),.23 mm pyruvte, nd 3 mg/ml rystllized lyophilized ovine serum lumen., murl ells or OOX omplexes were treted for one or more of the following time points, 4, 6, 8, 12 or 15 hours with ontrol medium, (1 ng/ml) or EGF (1 ng/ml). Some ultures were pre-inuted with (.1 to 1 M, Cliohem) or (2 M, Cliohem) to lok oth SMAD2 nd SMAD3 () or SMAD3 () tivtion for 1 hour efore eginning tretment. Expnsion ws stimulted in medi (MEM- ) ontining EGF 1 ng/ml) nd 5% FBS. Smples were immeditely frozen in liquid nitrogen nd stored t 7 C until nlyzed for protein or mrna levels. All regents were purhsed from Sigm Chemil Compny (St Louis, MO) unless otherwise noted. All experiments were repeted t lest three times. Quntifition of RNA trnsripts Totl RNA ws isolted from frozen smples nd reverse trnsried into DNA s desried previously (Diz et l., 26). Quntifition of murl nd umulus nd expnsion-relted trnsripts ws onduted using gene-speifi primers s desried previously using Rpl19 s n internl ontrol (Diz et l., 26). Only one produt of the pproprite size ws identified for eh set of primers nd ll mplifition produts were sequened to onfirm speifiity. All experiments were repeted three to four times nd vlues shown re the men ± s.e.m. Immunolotting Smples were prepred from 5 s or OOX omplexes ultured lone or with s (2 ooytes/ l). Some smples were pretreted with (1 M) or (2 M) for 1 hour. Groups were treted with medium only (ontrol), (1 ng/ml) or EGF (1 ng/ml) for 1, 4 or 15 hour(s). Smples were simultneously dentured y oiling in 1 loding uffer for 5 minutes followed y quenhing on ie for 5 minutes. Proteins were seprted on 1% SDS PAGE gel nd trnsferred to PVDF memrne. Memrnes were loked in 1 loking uffer (Odyssey Bloking uffer, Lior Biosiene, Linoln, NE) for 1 hour with shking t room temperture followed y inution with speifi nti-pmapk3/1 ntiody (1:2, Sigm), nti-pmapk14 ntiody (1:1, Cell Signling Tehnology, Dnvers, MA), nti-psmad2 ntiody (1:1, Invitrogen), nti-totl SMAD2 ntiody (1:2, Cell Signling Tehnology, Dnvers, MA), nti-psmad1/5/8 ntiody (1:1, Cell Signling Tehnology, Dnvers, MA), nti-psmad3 ntiody (1:1, Invitrogen) or -tin (ACTB) ntiody (1:6, Sigm) diluted in loking uffer with.1% Tween-2 for 2-12 hours t room temperture. Following inution, lots were wshed three times for 1 minutes eh with wsh uffer (PBS,.1% Tween-2). Fluoresently leled seondry ntiodies (IRDye TM 8 nti-mouse or nti-rit, Roklnd Immunohemils, Gilertsville, PA) were diluted t 1:5 nd inuted with the lots for 1 hour t room temperture. Blots were wshed s ove with n dditionl finl wsh in PBS without Tween-2. Detetion ws omplished with n infrred snner (Lior Biosiene, Linoln, NE). Representtive lots of three to four independent experiments re shown in Figs 2-4 nd 1. Immunofluoresene Ovries from 12-dy-old mie, 22-dy-old mie primed with ecg (44 hours) nd 22-dy-old mie primed with ecg plus hcg (5 IU, 8 hours) were fixed overnight in 4% prformldehyde nd emedded in prffin wx. Ovrin setions were dewxed in xylene (2 5 minutes) followed y inution for 5 minutes in eh of the following: xylene:ethnol (1:1), 1% ethnol (2 ), 95% ethnol, 85% ethnol, 75% ethnol nd ddh 2 O. Slides were then inuted in 1 ntigen retrievl solution (DkoCytomtion Retrievl solution) t 95 C for 25 minutes followed y wshing with ddh 2 O (2 ) for 1 minutes nd PBS (3 ) for 5 minutes. Slides were then inuted in loking uffer (PBS, ph 7.4, 3% BSA, 1% got serum nd.5% Triton X-1) for 1 hour t room temperture followed y inution with ntipsmad2 (1:4, Invitrogen) diluted in loking uffer for 12 hours t 4 C. Slides were then wshed three times for 15 minutes with wsh uffer (PBS.1% Triton- X 1), followed y inution with nti-rit IgG Alex Fluor 594 onjugte (1:15, Moleulr Proes) for 1 hour t room temperture, ounterstined with DAPI nd mounted with nti-fde solution (Slow-fde, Moleulr Proes). Slides were imged using in n Olympus BX6 upright fluoresent mirosope onneted to 3CCD mer nd omputer. Sttistil nlyses Results from rel-time PCR nd immunolot experiments were nlyzed y twowy ANOVA followed y Tukeys HSD post-ho test or Dunnetts HSD (Fig. 1B) if positive F-test ws deteted. Results in Fig. 1 were nlyzed y Student s t-test. The JMP 6. sttistil nlysis softwre ws used for ll nlysis (SAS, Cry, NC). P<.5 ws onsidered sttistilly signifint. We thnk Susn Akermn, Roert Burgess nd Mry Ann Hndel for their helpful suggestions in prepring this mnusript. This work ws supported y grnt HD23839 from the NICHD (J.J.E.). Referenes Brends, W. M., Uilenroek, J. T. J., Krmer, P., Hoogerrugge, J. W., Vn Leeuwen, E. C. M., Themmen, A. P. N. nd Grootegoed, J. A. (1995). Anti- Mullerin hormone nd nti-mullerin hormone type II reeptor messenger rionulei id expression in rt ovries during postntl development, the estrous yle, nd gondotropin-indued follile growth. Endorinology 136, Buione, R., Vnderhyden, B. C., Cron, P. J. nd Eppig, J. J. (199). -indued expnsion of the mouse umulus oophorus in vitro is dependent upon speifi ftor(s) sereted y the ooyte. Dev. Biol. 138, Chen, L., Russell, P. T. nd Lrsen, W. J. (1993). Funtionl signifine of umulus expnsion in the mouse: roles for the preovultory synthesis of hyluroni id within the umulus mss. Mol. Reprod. Dev. 34, Diz, F. J., O Brien, M. J., Wigglesworth, K. nd Eppig, J. J. (26). The prentrl grnulos ell to umulus ell trnsition in the mouse ovry: development of ompetene to undergo expnsion. Dev. Biol. 299, Dierih, A., Sirm, M. R., Mono, L., Fimi, G. M., Gnsmuller, A., LeMeur, M. nd SssoneCorsi, P. (1998). Impiring follile-stimulting hormone () signling in vivo: Trgeted disruption of the reeptor leds to errnt gmetogenesis nd hormonl imlne. Pro. Ntl. Ad. Si. USA 95, Dong, J. W., Alertini, D. F., Nishimori, K., Kumr, T. R., Lu, N. F. nd Mtzuk, M. M. (1996). Growth differentition ftor-9 is required during erly ovrin folliulogenesis. Nture 383, Drgovi, R. A., Ritter, L. J., Shulz, S. J., Amto, F., mstrong, D. T. nd Gilhrist, R. B. (25). Role of ooyte-sereted growth differentition ftor 9 in the regultion of mouse umulus expnsion. Endorinology 146, Durlinger, A. L. L., Krmer, P., Krels, B., de Jong, F. H., Uilenroek, J. T. J., Grootegoed, J. A. nd Themmen, A. P. N. (1999). Control of primordil follile reruitment y nti-mullerin hormone in the mouse ovry. Endorinology 14, Durlinger, A. L. L., Gruijters, M. J. G., Krmer, P., Krels, B., Kumr, T. R., Mtzuk, M. M., Rose, U. M., de Jong, F. H., Uilenroek, J. T. J., Grootegoed, J. A. et l. (21). Anti-Mullerin hormone ttenutes the effets of on follile development in the mouse ovry. Endorinology 142, Elvin, J. A., Yn, C. N., Wng, P., Nishimori, K. nd Mtzuk, M. M. (1999). Moleulr hrteriztion of the follile defets in the growth differentition ftor 9-defiient ovry. Mol. Endorinol. 13, Eppig, J. J. (21). Ooyte ontrol of ovrin folliulr development nd funtion in mmmls. Reprodution 122, Eppig, J. J., Wigglesworth, K., Pendol, F. L. nd Hiro, Y. (1997). Murine ooytes suppress expression of luteinizing hormone reeptor messenger rionulei id y grnulos ells. Biol. Reprod. 56, Eppig, J. J., Pendol, F. L. nd Wigglesworth, K. (1998). Mouse ooytes suppress AMP-indued expression of LH reeptor messenger RNA y grnulos ells in vitro. Mol. Reprod. Dev. 49, Eppig, J. J., Wigglesworth, K. nd Pendol, F. L. (22). The mmmlin ooyte orhestrtes the rte of ovrin folliulr development. Pro. Ntl. Ad. Si. USA 99, Eppig, J. J., Pendol, F. L., Wigglesworth, K. nd Pendol, J. K. (25). Mouse ooytes regulte metoli oopertivity etween grnulos ells nd ooytes: mino id trnsport. Biol. Reprod. 73, Fulop, C., Sznto, S., Mukhopdhyy, D., Brdos, T., Kmth, R. V., Rugg, M. S., Dy, A. J., Slustri, A., Hsll, V. C., Glnt, T. T. et l. (23). Impired umulus muifition nd femle sterility in tumor nerosis ftor-indued protein-6 defiient mie. Development 13, Gilhrist, R. B., Ritter, L. J., Myllym, S., Kivo-Oj, N., Drgovi, R. A., Hikey, T. E., Ritvos, O. nd Mottershed, D. G. (26). Moleulr sis of ooyte-prrine signlling tht promotes grnulos ell prolifertion. J. Cell Si. 119, Gui, L.-M. nd Joye, I. M. (25). RNA interferene evidene tht growth differentition ftor-9 medites ooyte regultion of umulus expnsion in mie. Biol. Reprod. 72, Hikey, T. E., Mrroo, D. L., Amto, F., Ritter, L. J., Normn, R. J., Gilhrist, R. B. nd mstrong, T. D. (25). Androgens ugment the mitogeni effets of ooytesereted ftors nd growth differentition ftor 9 on porine grnulos ells. Biol. Reprod. 73, Hu, Y. C., Wng, P. H., Yeh, S., Wng, R. S., Xie, C., Xu, Q., Zhou, X., Cho, H. T., Tsi, M. Y. nd Chng, C. (26). Sufertility nd defetive folliulogenesis in femle mie lking ndrogen reeptor. Pro. Ntl. Ad. Si. USA 11, Hussein, T. S., Froilnd, D. A., Amto, F., Thompson, J. G. nd Gilhrist, R. B. (25). Ooytes prevent umulus ell poptosis y mintining morphogeni prrine grdient of one morphogeneti proteins. J. Cell Si. 118, Hussein, T. S., Thompson, J. G. nd Gilhrist, R. B. (26). Ooyte-sereted ftors enhne ooyte developmentl ompetene. Dev. Biol. 296, Ingmn, W. V., Roker, R. L., Woittiez, K. nd Roertson, S. A. (26). Null muttion in trnsforming growth ftor 1 disrupts ovrin funtion nd uses ooyte inompetene nd erly emryo rrest. Endorinology 147, Inmn, G. J., Niolás, F. J., Cllhn, J. F., Hrling, J. D., Gster, L. M., Reith, A. D., Lping, N. J. nd Hill, C. S. (22). SB is potent nd speifi inhiitor of trnsforming growth ftor-superfmily type I tivin reeptor-like kinse (ALK) reeptors ALK4, ALK5, nd ALK7. Mol. Phrmol. 62, Jinnin, M., Ihn, H. nd Tmki, K. (25). Chrteriztion of, novel speifi inhiitor of smd3, nd its effet on trnsforming growth ftor-1-indued extrellulr mtrix expression. Mol. Phrmol. 69,

11 134 Journl of Cell Siene 12 (8) Journl of Cell Siene Knight, P. G. nd Glister, C. (26). TGF- superfmily memers nd ovrin follile development. Reprodution 132, Kumr, T. R., Wng, Y., Lu, N. F. nd Mtzuk, M. M. (1997). Follile stimulting hormone is required for ovrin follile mturtion ut not mle fertility. Nt. Genet. 15, Lping, N. J., Grygielko, E., Mthur, A., Butter, S., Bomerger, J., Tweed, C., Mrtin, W., Fornwld, J., Lehr, R., Hrling, J. et l. (22). Inhiition of trnsforming growth ftor (TGF)-et1-indued extrellulr mtrix with novel inhiitor of the TGF-et type I reeptor kinse tivity: SB Mol. Phrmol. 62, Lei, Z. M., Mishr, S., Zou, W., Xu, B., Foltz, M., Li, X. nd Ro, C. V. (21). Trgeted disruption of luteinizing hormone/humn horioni gondotropin reeptor gene. Mol. Endorinol. 15, Meduri, G., Vuhi-Luuthi, M. T., Jolivet, A. nd Milgrom, E. (1992). New funtionl zontion in the ovry s shown y immunohistohemistry of luteinizing hormone reeptor. Endorinology 131, Moore, R. K., Otsuk, F. nd Shimski, S. (23). Moleulr sis of one morphogeneti protein-15 signling in grnulos ells. J. Biol. Chem. 278, Ohsner, S. A., Dy, A. J., Rugg, M. S., Breyer, R. M., Gomer, R. H. nd Rihrds, J. S. (23). Disrupted funtion of tumor nerosis ftor-lph-stimulted gene 6 loks umulus ell-ooyte omplex expnsion. Endorinology 144, Ohsner, S. A., Russell, D. L., Dy, A. J., Breyer, R. M. nd Rihrds, J. S. (23). Deresed expression of tumor nerosis ftor-lph-stimulted gene 6 in umulus ells of the ylooxygense-2 nd EP2 null mie. Endorinology 144, Pngs, S. A., Li, X., Roertson, E. J. nd Mtzuk, M. M. (26). Premture luteiniztion nd umulus ell defets in ovrin-speifi smd4 knokout mie. Mol. Endorinol. 2, Rihrds, J. S. nd Midgley, J. A. R. (1976). Protein hormone tion: key to understnding ovrin folliulr nd lutel ell development. Biol. Reprod. 14, Roh, J. S., Bondestm, J., Mzerourg, S., Kivo-Oj, N., Groome, N., Ritvos, O. nd Hsueh, A. J. W. (23). Growth differentition ftor-9 stimultes inhiin prodution nd tivtes Smd2 in ultured rt grnulos ells. Endorinology 144, Slmon, N. A., Hndyside, A. H. nd Joye, I. M. (24). Ooyte regultion of nti- Mullerin hormone expression in grnulos ells during ovrin follile development in mie. Dev. Biol. 266, Slustri, A., Ulisse, S., Yngishit, M. nd Hsll, V. C. (199). Hyluroni id synthesis y murl grnulos ells nd umulus ells in vitro is seletively stimulted y ftor produed y ooytes nd y trnsforming growth ftor. J. Biol. Chem. 265, Su, Y. Q., Wu, X., O Brien, M. J., Pendol, F. L., Denegre, J. A., Mtzuk, M. M. nd Eppig, J. J. (24). Synergisti roles of BMP15 nd GDF9 in the development nd funtion of the ooyte-umulus ell omplex in mie: geneti evidene for n ooytegrnulos ell regultory loop. Dev. Biol. 276, Sugiur, K., Pendol, F. L. nd Eppig, J. J. (25). Ooyte ontrol of metoli oopertivity etween ooytes nd ompnion grnulos ells: energy metolism. Dev. Biol. 279, 2-3. Tomi, D., Miller, K. P., Kenny, H. A., Woodruff, T. K., Hoyer, P. nd Flws, J. A. (24). Ovrin follile development requires Smd3. Mol. Endorinol. 18, Vnderhyden, B. C., Cron, P. J., Buione, R. nd Eppig, J. J. (199). Developmentl pttern of the seretion of umulus-expnsion enling ftor y mouse ooytes nd the role of ooytes in promoting grnulos ell differentition. Dev. Biol. 14, Vnderhyden, B. C., Telfer, E. E. nd Eppig, J. J. (1992). Mouse ooytes promote prolifertion of grnulos ells from prentrl nd ntrl folliles in vitro. Biol. Reprod. 46, Vrni, S., Elvin, J. A., Yn, C., DeMyo, J., DeMyo, F. J., Horton, H. F., Byrne, M. C. nd Mtzuk, M. M. (22). Knokout of pentrxin 3, downstrem trget of growth differentition ftor-9, uses femle sufertility. Mol. Endorinol. 16, Yn, C., Wng, P., DeMyo, J., Elvin, J. A., Crino, C., Prsd, S. V., Skinner, S. S., Dunr, B. S., Due, J. L., Celeste, A. J. et l. (21). Synergisti roles of one morphogeneti protein 15 nd growth differentition ftor 9 in ovrin funtion. Mol. Endorinol. 15, Yoshino, O., MMhon, H. E., Shrm, S. nd Shimski, S. (26). A unique preovultory expression pttern plys key role in the physiologil funtions of BMP- 15 in the mouse. Pro. Ntl. Ad. Si. USA 13,