The MAPK signaling pathways comprise a conserved set of. ERK Signaling in the Pituitary Is Required for Female But Not Male Fertility

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1 ORIGINL RESERCH ERK Signling in the Pituitry Is Required for Femle ut Not Mle Fertility Sturt P. liss, ndrew Miller, my M. Nvrtil, JinJun Xie, Sen P. MDonough, Ptrii J. Fisher, Gry E. Lndreth, nd Mrk S. Roerson Deprtment of iomedil Sienes (S.P..,.M.,.M.N., J.X., S.P.M., P.J.F., M.S.R.), College of Veterinry Mediine, Cornell University, Ith, New York 8; nd Deprtment of Neurosienes (G.E.L.), Cse Western Reserve University Shool of Mediine, Clevelnd, Ohio 6 Mles nd femles require different ptterns of pituitry gondotropin seretion for fertility. The mehnisms underlying these gender-speifi profiles of pituitry hormone prodution re unknown; however, they re fundmentl to understnding the sexully dimorphi ontrol of reprodutive funtion t the moleulr level. Severl studies suggest tht ERK nd - re essentil modultors of hypothlmi GnRH-medited regultion of pituitry gondotropin prodution nd fertility. To test this hypothesis, we generted mie with pituitry-speifi depletion of ERK nd nd exmined rnge of physiologil prmeters inluding fertility. We find tht ERK signling is required in femles for ovultion nd fertility, wheres mle reprodutive funtion is unffeted y this signling defiieny. The effets of ERK pthwy ltion on LH iosynthesis underlie this gender-speifi phenotype, nd the moleulr mehnism involves requirement for ERK-dependent up-regultion of the trnsription ftor Egr, whih is neessry for LH expression. Together, these findings represent signifint dvne in eluidting the moleulr sis of gender-speifi regultion of the hypothlmi-pituitry-gondl xis nd sexully dimorphi ontrol of fertility. (Moleulr Endorinology : 9, 9) The MPK signling pthwys omprise onserved set of signl trnsdution modules tht re tivted in response to vriety of extrellulr stimuli (). The ERK pthwy is the most thoroughly hrterized of the MPK systems nd onsists of three-level phosphoryltion sde, whih, in its nonil form, inludes the MPK-kinse-kinse Rf-, the MPK kinses MEK nd MEK, nd the MPK s ERK (MPK) nd ERK (MPK). tivted ERKs phosphorylte multitude of trgets throughout the ell, exerting rod regultory influene over wide rnge of proesses inluding trnsription, trnsltion, ell yle regultion, ytoskeletl remodeling, nd poptosis (). ERK nd re generlly thought to serve overlpping funtions; however, it hs lso een demonstrted tht ERK nd my ply quite distint roles in some differentited ells (). In ddition, the ERK null mouse is vile nd fertile wheres the ERK null is emryoni lethl (, ). Thus, ERK nd lerly serve highly divergent funtions during development (). geneti model tht would llow for systemti nlysis of the funtions of ISSN Print ISSN Online Printed in U.S.. Copyright 9 y The Endorine Soiety doi:./me.9- Reeived Jnury, 9. epted pril 8, 9. First Pulished Online pril 6, 9 ERK nd within speifi ellulr or developmentl ontexts is highly desirle; however, the emryoni lethlity of the ERK null mouse neessittes onditionl pproh to the ltion of ERK nd hs presumly hmpered development of suh model. The pituitry glnd is omplex endorine orgn tht regultes mny spets of mmmlin homeostsis. The nterior pituitry is omposed of severl mjor hormone-produing ell types inluding thyrotropes tht produe TSH nd gondotropes tht produe FSH nd LH. TSH, LH, nd FSH re heterodimeri hormones tht hve distint -suunits ut shre ommon -suunit (glyoprotein hormone -suunit, GSU). The GSU is the first differentited ell mrker to pper during pituitry development (6). Reporter ssys suggest tht the GSU promoter my eome tivted s erly s emryoni d 9. (e9.) throughout the pituitry primordium; however, susequent expression of GSU eomes restrited to thyrotropes nd gondotropes (7 9). Thyrotropes nd gondotropes ply importnt roles in the regultion of metoli nd reprodutive funtion, respetively. revitions: DKO, doule knokout; EGR, erly growth response ftor-; GnRHR, GnRH reeptor; GSU, glyoprotein hormone -suunit; MEK, MPK kinse; qpcr, quntittive PCR. 9 mend.endojournls.org Mol Endorinol, July 9, (7):9 Downloded from mend.endojournls.org t Cse Western Reserve Univ Clevelnd Helth Siene Li on Novemer, 9

2 Mol Endorinol, July 9, (7):9 mend.endojournls.org 9 K K Control DKO Gond Pituitry Control DKO Control DKO LoxP Exon C D E F Stimultion of these ells y the hypothlmi relesing hormones TRH or GnRH leds to synthesis nd seretion of TSH y thyrotropes, nd FSH nd LH y gondotropes. The ERK pthwy is tivted in oth thyrotropes nd gondotropes fter stimultion y TRH or GnRH. Little is known regrding the importne of the ERK pthwy in thyrotropes. In ontrst, mny studies suggest tht ERK signling is ritil for the expression of severl genes essentil for the funtion of gondotropes, inluding the GSU, LH, nd regultory MPK phosphtse (MKP/DUSP) ( ). Dt supporting the importne of ERK signling for gondotrope funtion hve een generted primrily through study of the T nd L T gondotrope-derived ell lines. These ell lines hve een useful tools in the moleulr dissetion of gondotrope funtion; however, they re isolted from the omplex endorine environment of the living niml. In light of the need for n in vivo model for study of the role of ERK signling in the pituitry, we used Cre-LoxP tehnology to generte mie in whih ltion of ERK nd were trgeted to the GSU-expressing ells of the nterior pituitry. Our results demonstrte tht ERK signling is required for fertility only in the femle nd provide new insight into the mehnisms underlying sexully dimorphi regultion of reprodutive funtion. F LoxP ERK Merge 7 9 FIG.. Vlidtion of the ERK/ DKO mouse., Cre-medited reomintion t the ERK lous within the pituitry ws deteted y PCR using genomi DN from the speified tissues. For eh DN smple, the forwrd primer designted F ws pired individully with reverse primers leled, 7, nd 9 spnning the floxed genomi region s indited in the shemti. Lnes re leled with the speified reverse primer used for the retion. Moleulr weight mrker is shown in the left lne. Equivlent results were otined from mles nd femles., Cell type-speifi loss of ERK protein in the ERK/ DKO ws determined y oimmunofluoresent leling of pituitry setions from ontrol (pnels C) nd DKO (pnels D F) mle nimls using fluoresein isothioynte-onjugted ntiodies ginst ERK/ nd Texs Red-onjugted ntiodies ginst LH-. ERK ( nd D), LH ( nd E), nd simultneous visuliztion of oth wvelengths (C nd F) re shown. rrows indite representtive LH -positive ells with distint lk of ERK leling. rs, m. Results Genertion nd vlidtion of the pituitry-trgeted ERK/ doule-knokout mouse To define the physiologil importne of ERK signling in pituitry gondotropes nd thyrotropes, we egn y generting mie with onditionl, pituitry-trgeted ltion of ERK. Mie homozygous for floxed muttion t the ERK lous (ERK fl/fl ), were rossed with GSU:Cre mie, in whih Cre reominse is expressed under the regultory ontrol of.6-k frgment of the murine GSU promoter. ERK fl/fl, GSU:Cre mie were then mted to ERK null mie to generte ERK fl/fl,erk /, GSU:CRE, herefter designted DKO (doule knokout). Pituitry-speifi ERK null mles nd femles (with norml ERK) were vile nd fertile. They were orn t expeted Mendelin frequenies nd were grossly nd histologilly unremrkle t months of ge (dt not shown). In ontrst, t wk of ge, DKO nimls were represented t less thn the expeted frequeny (expeted:., oserved:.; n 7). Within litters of neontes, some DKO nimls were frequently identified either ded, or s unthrifty pups tht filed to nurse nd tht died shortly fter irth. To vlidte the fidelity of GSU:Cre-medited reomintion, we rossed GSU:Cre mles with Ros6 reporter (R6R) femles nd ssyed -gltosidse tivity in tissue lystes from oth Cre-expressing, nd nonexpressing offspring. -Gltosidse tivity ws signifintly inresed in pituitry lystes from Cre-expressing nimls s ompred with Cre-negtive ontrols (dt not shown). Differenes in -gltosidse tivity were not oserved in lystes from other tissues (lung, kidney, hert, nd rin). These dt support the pituitry-enrihed tivity of the GSU:Cre. Cre-dependent reomintion speifilly t the ERK lous ws verified y PCR from ERK fl/fl,cre nd ERK fl/fl,cre nimls using primers flnking the floxed region of the ERK lous (Fig., shemti). Produts inditing exision of the floxed region of the ERK gene were only produed from pituitry genomi DN of Cre individuls, inditing tissue-speifi suseptiility of the ERK lous to Cre-medited reomintion (Fig., left pnel). Immunohistohemil oleling of pituitry setions from dult DKO nd ontrol nimls using ntiodies ginst ERK nd LH onfirmed high level of ololiztion etween LH nd negtive ERK leling, inditing tht ERK defiieny ws reltively penetrnt within the gondotropes of DKO nimls (Fig. ). Immunohistohemil identifition of TSH within ERKdefiient, LH -negtive ells in these setions onfirmed the predition tht GSU-expressing thyrotropes were lso rendered ERK Downloded from mend.endojournls.org t Cse Western Reserve Univ Clevelnd Helth Siene Li on Novemer, 9

3 Epithelil ells (%) 9 liss et l. ERK Signling nd Fertility Mol Endorinol, July 9, (7): C Dy defiient within this line (dt not shown). Overll, these results support the fidelity of the ompound geneti deletion within the dult DKO nimls. D Control DKO FIG.. ssessment of estrous yle tivity in the ERK/ DKO mouse., Vginl ytology ws performed t -h intervls y mirosopi exmintion of Wright s stined vginl lvge effluents. One hundred ells were ounted, nd the perentge of epithelil ells t eh time point ws determined. Dt re shown for single niml; however, similr results were otined from three nimls of eh genotype., Hemtoxylin nd eosin-stined histologil setions of ovries from ontrol (pnel ) nd DKO (pnel ) nimls re shown. representtive orpus luteum is indited (CL). Mirosopi setions from ontrol (pnel C), nd DKO (pnel D) ovries were immunohistohemilly stined using n ntiody ginst ERK/. oth ontrol nd DKO nimls were ERK null; thus ll speifi leling represents ERK protein. nd, rs, m; C nd D, rs, m. inditive of estrous yle tivity were oserved in ontrol mie (Fig. ). In ontrst, vginl ytology in the DKO femles showed preponderne of polymorphonuler leukoytes with osionl sl epithelil ells over the ourse of the study, onsistent with nestrous ehvior. Histologil exmintion of vriety of tissues inluding the reprodutive trt from DKO nd ontrol mles reveled no normlities. In ontrst, ovries from DKO femles ontined folliles t vrious stges of mturtion (primry through lrge ntrl folliles) ut were speifilly devoid of lutel tissue, wheres ovries from ontrol femles were histologilly unremrkle (Fig., pnels nd ). The omined dt from ytologil nlysis of estrous yle ehvior nd histologil evidene of lk of lutel tissue within the ovry in DKO femle mie supported the preliminry onlusion tht the DKO mie were novultory. To ddress whether the ovrin lesion in DKO nimls my reflet promisuous Cre expression nd lk of ERK protein in the ovry, we exmined ERK expression in ovries from DKO nd ontrol femles immunohistohemilly. Expression of ERK ws oserved in oth DKO nd ontrol ovries, inditing tht the novultory ovrin lesion ws seondry to the primry pituitry defet (Fig., pnels C nd D). ERK/ DKO mie re euthyroid euse GSU is expressed in thyrotropes s well s in gondotropes, our model would predit fully penetrnt Cre-dependent disruption of ERK signling in the thyrotropes of DKO nimls. To evlute thyroid funtion in these nimls, we used quntittive PCR (qpcr) to mesure whole pituitry ontent of TSH mrn in DKO nd ontrol nimls. Pituitry ontent of TSH protein ws exmined y immunolot nlysis, nd T levels in serum were exmined using n ELIS. Pituitry TSH mrn, TSH protein in whole pituitry lystes or serum T onentrtions did not differ etween DKO nd ontrol mie (Fig. ). These results onfirm tht DKO nimls tht survived to dulthood were euthyroid nd effetively rule out thyroid dysfuntion s ontriuting ftor to the novultory phenotype. Phenotypi hrteriztion of the ERK/ DKO mouse DKO nimls of oth genders grew t the sme rte s Cre littermte ontrols nd were grossly indistinguishle from ontrols from irth through 8 months of ge (dt not shown). Test mtings indited tht DKO mles were fertile, wheres femles showed no evidene of estrous yle tivity nd did not mte. To further ssess estrous yle tivity, vginl ytology ws performed in ontrol nd DKO mie. Cyli ytologil hnges Expression of LH is redued in pituitries of ERK/ DKO nimls The lk of lutel tissue in the ovries of DKO femles suggests tht DKO femles were novultory nd implites gondotropin defiieny in these nimls. To test this, we used qpcr to mesure trnsript levels of the gondotropin suunits (LH, FSH, nd GSU), s well s the GnRH reeptor (GnRHR) in the pituitries of DKO nd ontrol nimls. In Downloded from mend.endojournls.org t Cse Western Reserve Univ Clevelnd Helth Siene Li on Novemer, 9

4 Reltive expression Mol Endorinol, July 9, (7):9 mend.endojournls.org p=.7 Control DKO The novultory phenotype of ERK/ DKO femles is resued y phrmologil superovultion Our histologil findings nd gene profiling dt suggest tht the novultory infertility in the DKO mie ws diret onsequene of LH defiieny. If orret, we resoned tht the ovries of DKO femles should remin ple of mounting n ovultory response to n pproprite suprovrin gondotropi stimulus. To test this, we sujeted DKO nd ontrol femles to onventionl phrmologil superovultion. Corpor lute were present in the ovries of gondotropin-treted DKO nimls leit t frequeny (numer of orpor lute per ovry) less thn tht oserved in superovulted ontrol femles (Fig. ). These results onfirm the intrinsi ility of DKO ovries to respond to n ovultory signl. TSHβ tin C ng/ml p=.89 Wild type Control DKO FIG.. Evlution of thyroid funtion in the ERK/ DKO mouse., Reltive trnsript levels of TSH from whole pituitries of femle ontrol nd DKO nimls were determined y quntittive PCR. Results were lirted to levels of TSH mrn from pituitries of rndomly yling femle wild-type mie. rs represent men SEM for five nimls of eh genotype. Mens were ompred y two-tiled t test., Levels of TSH in whole pituitry lystes from ontrol nd DKO nimls were ompred y immunolot. tin is shown s lne-loding ontrol. C, Serum levels of totl T from rndomly yling wild-type, ontrol, nd DKO femles were determined y ELIS. rs represent men SEM for three (wild type) or (ontrol nd DKO) nimls in the respetive groups. Mens for ontrol nd DKO groups were ompred y two-tiled t test. femles, LH mrn levels were signifintly lower in DKOs s ompred with rndomly yling ontrol nimls (Fig. ). In mles, LH levels were not sttistilly signifintly different etween DKO nd ontrol nimls (Fig. ). Trnsript levels of the other gondotropin suunits, s well s the GnRHR, did not differ etween DKO nd ontrol nimls for either gender (Fig. ). To ssess the effets of ERK deletion on pituitry stores of gondotropin suunits, we exmined pituitry lystes y immunolotting using ntiodies ginst LH nd FSH. LH ws less undnt in pituitries of DKO nimls of oth genders, s ompred with ontrols, nd ws essentilly undetetle in the pituitries of DKO femles (Fig. ). Levels of FSH were similr etween DKO nd ontrol nimls of oth genders (Fig. ). ERK signling is required for the response of the gondotropin suunits fter gondetomy Gondl steroids regulte the expression of the gondotropin suunit genes within the pituitry through negtive feedk inhiition of GnRH relese from the hypothlmus. ordingly, gondetomy leds to inresed expression of the -suunits of LH nd FSH through disinhiition of the hypothlmi GnRH system (, ). To determine the role of ERK signling in mediting the gondotrope response to endogenous GnRH hyperstimultion, we sujeted ontrol nd DKO mie of oth genders to gondetomy. fter 7 d, pituitry expression of the gondotropin suunits nd the GnRHR were ssyed y qpcr. fter gondetomy, signifint inreses in LH trnsript levels were oserved in ontrol mie of oth genders; however, the indutive effet of gondetomy on LH trnsript levels ws loked in DKOs (Fig. 6, nd, upper left pnels). In mles, strtion hd no signifint effet on FSH trnsript levels in either DKOs or ontrols (Fig. 6, upper right pnel). In ontrst, ovrietomy led to n pproximtely -fold inrese in FSH trnsript levels in the ontrol femles (Fig. 6, upper right pnel). In DKO femles, ovrietomy led to more modest elevtion in FSH trnsript levels, whih did not reh sttistil signifine (Fig. 6, upper right pnel). Gondetomy led to signifint inreses in GSU trnsript levels in oth ontrol mles nd femles; however, this response ws loked in DKO nimls of oth genders (Fig. 6, nd, lower left pnels). Trnsript levels of the GnRHR in mles did not differ etween either genotype or tretment, wheres in femles, sttistilly signifint ut quntittively negligile differenes were oserved in GnRHR trnsript levels etween shm-operted ontrol nimls nd ll other groups (Fig. 6, nd, lower right pnels). In prllel to the gene profiling results, serum levels of oth LH nd FSH rose signifintly fter gondetomy in ontrol nimls of oth genders (Fig. 7, nd, left pnels). seline serum FSH levels were not different etween shm-operted DKO nd ontrol nimls of either gender. In ontrst, seline serum levels of LH were signifintly lower in shm-operted DKOs s ompred with ontrols, nd signifint inreses in serum LH did not our fter gondetomy in DKO nimls of Downloded from mend.endojournls.org t Cse Western Reserve Univ Clevelnd Helth Siene Li on Novemer, 9

5 96 liss et l. ERK Signling nd Fertility Mol Endorinol, July 9, (7):9 Reltive expression Reltive expression αgsu tin Control femle DKO femle Control mle DKO mle either gender. These results support the onlusion tht ERK signling plys ritil role in mediting up-regultion of oth suunits of LH in response to GnRH stimultion in vivo. Reltive expression Reltive expression GnRHR FIG.. sl expression of gondotropin suunit nd GnRHR genes in the ERK/ DKO mouse., Whole pituitry reltive trnsript levels of the speified genes were determined y quntittive PCR. Results were lirted to orresponding trnsript levels in pituitries of rndomly yling femle wild-type mie. rs represent men SEM for five nimls of eh gender nd genotype. rs not shring ommon letter designtions represent men vlues tht re sttistilly signifintly different (P.)., Levels of LH nd FSH protein in whole pituitry lystes from ontrol nd DKO nimls were ompred y immunolotting. tin is shown s lne-loding ontrol. Given the dynmi nture of the Egr trnsriptionl response to GnRH stimultion, we onsidered tht the lk of sttistilly signifint differenes etween the shm operted nd gondetomized ontrol nimls my reflet type II error. However, power nlysis indited tht to hieve power of.8 for detetion of -fold differene in Egr trnsript levels within the ontext of this experimentl design would require smple sizes of eight nd 7 nimls per group, for mles nd femles, respetively. To irumvent this limittion, we undertook n lterntive strtegy nd mesured Egr nd LH trnsript levels y qpcr in primry pituitry ell ultures from DKO nd ontrol nimls fter stimultion with vehile or the GnRH gonist userelin (GnRH). We resoned tht this experimentl pproh would llow synhroniztion of experimentl units with respet to the onset nd durtion of the GnRH signl in mnner tht nnot e omplished within our in vivo prdigm. seline Egr trnsript levels were signifintly redued in ells from DKO femles s ompred with ontrols (Fig. 8, lower left pnel). Egr trnsripts were signifintly up-regulted fter GnRH exposure in ells from ontrol nimls of oth genders; however, up-regultion of Egr mrn ws not oserved in ells from DKO nimls of either gender (Fig. 8, left pnels). LH trnsripts were signifintly redued in ultures of vehile-treted ells from DKO nimls of oth genders, s ompred with ontrols. fter GnRH stimultion, sttistilly signifint, ut quntittively negligile, inrese in LH trnsripts ws oserved in ells from ontrol femles. Overll, the redution in GnRH-indued Egr trnsription in ERK-defiient gondotropes undersores key mehnism linking ERK signling to LH iosynthesis nd highlights the unique importne of the ERK pthwy for femle fertility. ERK-defiient gondotropes fil to up-regulte the immedite erly response gene Egr fter GnRH stimultion Erly growth response ftor- (EGR) is trnsription ftor enoded y n ERK-dependent immedite erly gene tht hs een shown to ply key role in the trnsriptionl regultion of LH (). To determine the effet of ERK ltion on the ility of gondotropes to up-regulte EGR in response to GnRH stimultion, we sujeted ontrol nd DKO mie of oth genders to gondetomy nd fter 7 d, mesured pituitry expression of Egr y qpcr. In the ontrol nimls, men Egr trnsript levels rose pproximtely -fold nd -fold fter gondetomy in mles nd femles, respetively; however, due to the high vriility in trnsript levels oserved in the gondetomized nimls, these differenes were not sttistilly different (Fig. 8). No differenes were oserved in Egr trnsript levels fter gondetomy in DKO nimls of either gender (Fig. 8). Disussion Using geneti pproh, we show here tht ltion of ERK nd in the nterior pituitry leds to infertility in femles, ut not mles. The novultory infertility in the ERK-defiient femles in this study ws ssoited with mrked redutions in pituitry levels of LH. Our studies on the pituitry response to gondetomy indite further tht ERK-defiient gondotropes re inple of trnsriptionl up-regultion of either LH or GSU fter pulstile hyperstimultion y hypothlmi GnRH within the intt endorine milieu of the living niml. Thyroid funtion remined norml in nimls with pituitry ERK defiieny, suggesting tht the funtion of the thyroid xis does not depend upon ERK signling t the level of the thyrotrope. lthough our model revels tht ERK signling is solutely required for pproprite LH iosynthesis, our dt lso suggest tht the ERK pthwy my ply prtil role in mediting Downloded from mend.endojournls.org t Cse Western Reserve Univ Clevelnd Helth Siene Li on Novemer, 9

6 Mol Endorinol, July 9, (7):9 mend.endojournls.org 97 PMSG/hCG Sline C Control trnsriptionl up-regultion of FSH in the femle. Nevertheless, our oservtions of whole pituitry nd serum FSH levels would suggest tht overll expression of FSH is lrgely ERK independent. Moreover, the presene of ntrl folliles in the ovries of the DKO femles, long with the sene of orpor lute, strongly implites LH defiieny s the primry use of infertility in these nimls nd further supports the reltive ERK independene of FSH iosynthesis. This is further orroorted y the oservtion tht the novultory phenotype ould e resued in the DKO femles y dministrtion of exogenous gondotropin reeptor lignds. Our model thus onfirms the ERK pthwy s dominnt link etween the tivtion of the GnRHR nd LH iosynthesis within the gondotrope in vivo. Gender differenes in the trnsriptionl response of the gondotropin suunits to GnRH stimultion hve een reported previously (6 9); however, the sexully dimorphi nture of the requirement for the ERK signling pthwy tht is dislosed y our model is novel oservtion tht hs not previously een ppreited. In ontrst to the reprodutive phenotype displyed within the dult DKO femles, the mjority of DKOs predited to our within this pedigree remined unounted for t wk of ge. The regulr finding of ded DKO neontes suggests tht pre- or perintl mortlity my represent distint phenotype resulting from pituitry-trgeted disruption of the ERK pthwy. Perintl mortlity hs een desried fter deletion of severl genes tht interfere with pproprite development of nterior pituitry ell lineges ( ). The pthophysiologil sis of perintl mortlity hs perhps een most lerly defined in the Prop-defiient mouse, in whih severe ongenitl hypothyroidism seondry to hypoplsi of the Pit-dependent thyrotrope linege leds to surftnt defiieny nd respirtory filure in neontl pups (). Whether ERK signling plys role in thyrotrope development through modifition of either the expression or funtion of Prop or Pit is unknown. However, s previously reported, some vriility hs een oserved D DKO FIG.. Phrmologil superovultion resues the novultory phenotype of the ERK/ DKO mouse. Control nd DKO femles were injeted ip with vehile or U pregnnt mre serum gondotropin, followed in 8 h y vehile or U of humn horioni gondotropin. fter 9 d, ovries were olleted nd exmined histologilly. Ovrin setions from vehile-treted ( nd ) nd superovulted (C nd D) ontrol ( nd C) nd DKO ( nd D) nimls re shown. rrows indite orpor lute. rs, m. in the timing of onset nd penetrne of GSU-Cre-medited trget gene reomintion within the developing pituitry (). Studies re urrently underwy to define the underlying mehnism of perintl mortlity within this model nd to lrify the role of the ERK pthwy in development of the Pit-dependent nterior pituitry ell lineges. The trnsriptionl regultion of LH hs een studied in vriety of experimentl systems (,, 6). The LH promoter ontins inding sites for severl trnsription ftors, inluding the zin-finger trnsription ftor EGR (, 7). EGR hs een shown to ply ritil role in LH trnsription nd ppers to funtion primrily s n mplifier of LH promoter tivity, ontriuting to the responsiveness of the promoter to hnges in the mplitude or pulse frequeny of GnRH stimultion (6, 8). Interestingly, despite the rod pttern of expression of EGR, the predominnt phenotype of the EGR null mouse onsists of femle novultory infertility ssoited with LH defiieny (9). The phenotype of the femle ERK DKO mouse reported here ers striking similrity to tht of the EGR null, nd the inility of ERK-defiient gondotropes to up-regulte EGR in response to GnRH stimultion indites tht ERK-dependent indution of EGR is dominnt mehnism y whih GnRHR oupny is linked to trnsription of the LH gene in vivo. However, whether the LH defiieny in the ERK DKO is due solely to filure of Egr indution, or whether the phenotype my lso reflet lk of ERK-dependent EGR phosphoryltion is not ler. For exmple, in one study, the GnRH-indued trnsriptionl tivity of onstitutively expressed Egr ws ttenuted in trnsiently trnsfeted gondotrope ells y tretment with the MEK inhiitor PD989 (). This oservtion rises the possiility tht the ERK pthwy my modulte LH gene trnsription t multiple levels involving not only gene indution ut lso diret phosphoryltion of LH trnstivtors suh s EGR. Ultimtely, full lrifition of the role of ERK tivtion in LH iosynthesis will require more omprehensive genomi nd proteomi pprohes imed t identifition of the entire omplement of ERK-dependent genes tht re expressed in the gondotrope, s well s relevnt trgets of ERK-dependent phosphoryltion. In ontrst to the femles, ERK DKO mles retined norml reprodutive funtion nd gondl histology. This oservtion provides further interesting omprison with the EGR null mouse euse EGR null mles re lso reprodutively ompetent (9). However, despite their fertility, EGR null mles did disply Leydig ell trophy wheres testiulr histology in the ERK-defiient mles in this study ws unremrkle (9). The testiulr lesion in the EGR null mle undoutedly reflets more severe impirment of LH prodution thn in the ERK DKO. Thus, despite the mesurle derese in pituitry LH protein ontent in the DKO mles in this study, prodution of LH in these nimls ws lerly suffiient to mintin norml testiulr funtion nd struture s well s fertility. The inility of gondotropes from ERK-defiient mles to up-regulte EGR in response to GnRH stimultion suggests further tht sl EGR expression is suffiient to support this level of LH prodution. Overll, our dt support model in whih the ERK pthwy funtions in oth genders s dominnt link Downloded from mend.endojournls.org t Cse Western Reserve Univ Clevelnd Helth Siene Li on Novemer, 9

7 98 liss et l. ERK Signling nd Fertility Mol Endorinol, July 9, (7):9 Reltive expression Reltive expression Reltive expression Reltive expression 6 6 αgsu Control shm Control Csx αgsu DKO shm DKO Csx Control shm Control Ovx DKO shm DKO Ovx Reltive expression Reltive expression Reltive expression Reltive expression 6 GnRHR GnRHR FIG. 6. ERK/ DKO nimls of oth genders fil to up-regulte LH fter gondetomy. Control nd DKO nimls were either strted (Csx), ovrietomized (Ovx), or shm operted (shm). fter 7 d, pituitries were olleted nd nlyzed y qpcr for reltive trnsript levels of the indited gondotropin suunit genes nd the GnRHR. Dt re shown seprtely for mles () nd femles (). Results were lirted to orresponding trnsript levels in pituitries of rndomly yling femle wild-type mie. rs represent men SEM for six mles or seven femles per group nd represent pooled results from two seprte experiments. rs not shring ommon letter designtions represent men vlues tht re sttistilly signifintly different (P.). Note the differenes in sling of the y-xis for the FSH results in the femle. etween the GnRHR nd the Egr lous. In turn, EGR serves s primry meditor of GnRH-indued up-regultion of LH. The gender differene in the requirement for ERK signling in the gondotrope would thus seem to reflet the reltively modest levels of LH tht re required to support Leydig ell funtion in the mle, s ompred with the high levels of LH tht re required for ovultion in the femle. Consistent with tht notion, trnsriptionl regultion of the gondotropin suunit genes is highly dependent upon the frequeny of the pulstile hypothlmi GnRH signl; rpid pulstility fvors LH prodution wheres slower GnRH pulsing ppers to fvor prodution of FSH (). Moreover, studies using the L T ell model indite tht the ility of the gondotrope to interpret vritions in the GnRH interpulse intervl my depend upon funtionl ERK signling module (). During the estrous yle, regulr vritions our in the frequeny of GnRH pulstility; pulstility inreses mrkedly during proestrus, eventully leding to preovultory GnRH/LH surge nd ovultion. We propose tht the ERK module plys entrl role in the ility of the gondotrope to respond to vritions in the pulse pttern of the GnRH signl, therey linking hnges in GnRH pulse frequeny with the oordinted physiologil outomes ssoited with the femle reprodutive yle. The ft tht mles hve no requirement for yli vritions in GnRH pulstility is onsistent with our oservtions tht steroidogenesis nd spermtogenesis re unffeted y disruption of the ERK signling pthwy t the level of the gondotrope. In summry, we hve shown tht ltion of the ERK signling pthwy in pituitry gondotropes leds to infertility in femle ut not mle mie. Our dt estlish n essentil role for the ERK pthwy in mediting trnsriptionl up-regultion of LH in the gondotrope fter GnRH stimultion in vivo nd lend insight into the moleulr mehnisms underlying sexully dimorphi ontrol of reprodutive funtion. Mterils nd Methods nimls ERK null (ERK / ) nd ERK floxed (ERK fl/fl ) mie hve een desried previously (, ). GSU:CRE mie were purhsed from Jkson Lortories (r Hror, ME). Swiss Wester mie used s wild-type ontrols were otined from Toni Frms (Germntown, NY). Ros 6 reporter (R6R) mie were generously provided y Dr. Mihel Kotlikoff (Cornell University, Ith, NY). nimls were hndled in ompline with the Cornell University Institutionl niml Cre nd Use Committee. For ssessment of fertility, femles were individully housed with mles of proven fertility nd exmined dily for vginl plugs. Upon identifition of plug, femles were isolted nd oserved for pregnny. Femles in whih vginl plugs were not identified, or tht filed to develop pregnny fter identifition of plug, were reintrodued to new mles, for totl of d of reeding opportunity. fter the reeding hllenge, femles were isolted for d nd then killed nd exmined for pregnny post mortem. For superovultion, mie were injeted ip with either IU pregnnt mre serum gondotropin or vehile, followed in 8 h y injetion of IU of either humn horioni gondotropin or vehile. fter 7 h, mie were killed nd ovries were olleted for histologil exmintion. Ovrietomy nd strtion were performed vi ventrl midline eliotomy under isoflurne nesthesi using stndrd septi onditions. Genotyping Genomi DN ws isolted from til snips ( mm), or n equivlent quntity of other tissues s indited, using ommeril kit (Dnesy; Downloded from mend.endojournls.org t Cse Western Reserve Univ Clevelnd Helth Siene Li on Novemer, 9

8 Mol Endorinol, July 9, (7):9 mend.endojournls.org 99 ng/ml 6 Control shm Control Csx DKO shm DKO Csx ng/ml Reltive expression Control shm Control gondetomy DKO shm DKO gondetomy Reltive expression ng/ml 6 Control shm Control Ovx DKO shm DKO Ovx ng/ml QIGEN, Vleni, C) s per the mnufturer s instrutions. Routine genotyping of nimls ws performed y PCR s previously desried (). PCR for onfirmtion of reomintion t the ERK lous ws performed using primer F: -GCCCTCCCCCTG- ; primer : -GCTGCCTGCTGGGCTGC- ; primer 7: -GCTCTTTCCTCCCTGCCTGC- ; nd primer 9: - GCCGCTGCTCCCTTGCGC-. The identities of PCR produts were onfirmed y diret nuleotide sequening for ll founder nimls. Histology, immunohistohemistry, nd immunofluoresent leling For histologil exmintion, tissues were fixed in % formlin, emedded in prffin, setioned t m, stined with hemtoxylin nd eosin using stndrd histologil tehnique, nd exmined y light mirosopy. In some studies, ovries were serilly setioned, nd every fourth setion ws exmined mirosopilly for identifition of lutel tissue. For ERK immunohistohemistry, setions were deprffinized in xylene, rehydrted through EtOH dilution series to distilled H. ntigen retrievl ws performed y oiling the slides in. M Citrte uffer (ph 6.). Setions were wshed in PS nd loked with % norml rit serum/% nonft dry milk in PS for min t room temperture. Setions were then inuted in got nti-erk primry ntiody (Snt Cruz iotehnology, In., Snt Cruz, C) diluted :6 in PS/ sein (Vetor Lortories, In., urlingme, C), forht7 C. Setions were further wshed in PS, nd inuted t room temperture for min with iotinylted rit-ntigot IgG (Invitrogen, Crlsd, C). Setions were then treted with C regent nd diminoenzidine s per the mnufturer s reommendtions (Vetor). For immunofluoresent oleling of ERK nd LH, - m pituitry setions were deprffinized nd rehydrted s desried ove. ntigen retrievl nd inution in nti-erk primry ntiody were lso performed s ove, sustituting norml got IgG t equivlent onentrtion (mirogrms/ml) s negtive ontrol. fter inution with iotinylted rit-ntigot IgG nd further wshing, setions 6 FIG. 7. Serum gondotropin levels do not inrese in DKO nimls of either gender fter gondetomy. Control nd DKO nimls were either strted (Csx), ovrietomized (Ovx), or shm operted (shm). fter 7 d, nimls were euthnized, nd serum levels of LH nd FSH were determined y ELIS. Dt re shown seprtely for mles () nd femles (). rs represent men SEM for six mles or seven femles per group nd represent pooled results from two seprte experiments. rs not shring ommon letter designtions represent men vlues tht re sttistilly signifintly different (P.). Reltive expression Reltive expression Egr Control vehile Control GnRH DKO vehile DKO GnRH Egr d Reltive expression Reltive expression were inuted with Streptvidin lex Fluor 88 (Invitrogen) for min t room temperture in the drk. Stined slides were wshed further with PS nd stored in distilled wter overnight t C. On d, setions were loked with F Frgment got ntirit IgG H L (Jkson ImmunoReserh Lortories, West Grove, P) diluted : in PS for min t 7 C, nd reloked for min with % got serum/ sein (Vetor) in PS. Rit nti-lh primry ntiody (Ntionl Hormone nd Peptide Progrm; NIDDK) ws reonstituted in PS t onentrtion of g/ l, nd pplied t : dilution forht7c, sustituting norml rit IgG (Vetor) t n equivlent onentrtion (mirogrms/ml) s negtive ontrol. LH ws deteted with Texs Red got-ntirit IgG H&L (Vetor) t :8 dilution in PS for FIG. 8. ERK-defiient gondotropes fil to up-regulte the immedite erly gene Egr in response to GnRH stimultion., Control nd DKO nimls of oth genders were either gondetomized (gondetomy) or shm operted (shm). fter 7 d, pituitries were olleted nd nlyzed y qpcr for reltive trnsript levels of Egr. Dt re shown seprtely for mles (left pnel) nd femles (right pnel). Results were lirted to orresponding trnsript levels in pituitries of rndomly yling femle wild-type mie. rs represent men SEM for six nimls per group nd represent pooled results from two seprte experiments for eh gender. rs not shring ommon letter designtions represent men vlues tht re sttistilly signifintly different (P.). Pituitries from ontrol nd DKO nimls were dispersed into primry ulture (two pituitries per well) nd treted for min with vehile or with nm of the GnRH gonist userelin (GnRH). Reltive trnsript levels of Egr were mesured in eh smple y qpcr. rs represent men SEM for six wells representing pooled results from two seprte experiments. Dt re shown seprtely for mles (left) nd femles (right). For these experiments, the expression level of the vehiletreted ontrol nimls ws ritrry ssigned s the lirtor. Downloded from mend.endojournls.org t Cse Western Reserve Univ Clevelnd Helth Siene Li on Novemer, 9

9 liss et l. ERK Signling nd Fertility Mol Endorinol, July 9, (7):9 min t room temperture in the drk. Slides were wshed nd mounted in Vetshield,6-dimidino--phenylindole (Vetor). Imges were otined on Nikon E epifluoresene mirosope using the pproprite filters. Vginl ytology Vginl lvge ws performed with l PS, nd the effluent ws used to mke routine ytologil smers. Slides were fixed in methnol nd stined with Wright s stin. Slides were exmined y light mirosopy, nd epithelil ells nd leukoytes were differentited on the sis of ell morphology. Serum hormone mesurements Immeditely fter euthnsi, mie were exsnguinted y rdi punture. lood smples were llowed to lot t room temperture for h nd were then entrifuged t 8 rpm for min. Serum superntnts were olleted nd stored t 8 C until ssyed. Totl serum T onentrtions were determined y ELIS using ommeril kit ording to the mnufturer s instrutions (lph Dignostis, Sn ntonio, TX) with smples run in duplite. Mesurements of LH nd FSH were performed y the University of Virgini Center for Reserh in Reprodution Lignd ssy nd nlysis Core fility with smples run in singlet. Immunolotting Tissues were homogenized in lysis uffer ontining mm Tris- HCl (ph 8.), mm NCl, % glyerol, % Nonidet P-,.% sodium dodeyl sulfte,.% deoxyholte, mm EDT, mm sodium vndte,. mm phenylmethysulfonylfluoride, mm enzmidine. Lystes were lered y entrifugtion, nd protein onentrtions were determined y rdford ssy. Protein smples were oiled for min in sodium dodeyl sulfte lod uffer, resolved y SDS-PGE, nd trnsferred to polyvinylidine difluoride memrnes y eletrolotting. Memrnes were loked with % nonft dry milk in TST ( mm Tris- HCl, ph 7.; mm NCl;.% Tween ) nd then inuted with speified ntiser (nti-lh, FSH, nd TSH from the Ntionl Hormone nd Peptide Progrm; NIDDK; nd ntitin nd horserdish peroxidse-onjugted seondry ntiodies from Snt Cruz). Protein nds were visulized using enhned hemiluminesene ording to the mnufturer s instrutions (PerkinElmer, oston, M). Pituitry ell primry ulture Mie were euthnized nd pituitries were pled in DMEM ontining % fetl ovine serum on ie. Tissues were digested with gittion t 7 C for min in DMEM ontining. mg/ml eh of ollgense nd hyluronidse (Sigm Chemil Co.; St Louis, MO). fter tritiurtion, tissue remnnts were llowed to settle y grvity. The superntnt ws removed nd the tissue pellet ws resuspended in DMEM ontining. mg/ml eh of the sme enzymes nd digested for n dditionl min. fter seond round of tritiurtion nd sedimenttion, the superntnts were omined. Cells were wshed one in DMEM ontining % fetl ovine serum, nd plted in like medium on -mm dimeter poly-l-lysine oted dishes t density of two pituitry equivlents per well. Cells were mintined t 7 C in % CO for h efore tretment. RN isoltion nd quntittive PCR fter the indited tretments, totl RN ws isolted from ultured ells or whole pituitries using the RNesy kit (QIGEN) ording to the mnufturer s instrutions. Reverse trnsription ws rried out in l retion volumes using the High Cpity DN rhive Kit (pplied iosystems, Foster City, C). Tqmn primer-proe sets for mouse LH, GSU, FSH, GnRHR, TSH, nd Egr, were purhsed ommerilly (pplied iosystems). mplifition ws performed under stndrd onditions using the I Prism 7 Sequene Detetion System. Trnsript levels were normlized to orresponding levels of -tin nd were lirted to orresponding trnsript levels represented in pooled DN stok derived from wild-type femle mie, or to levels of the ontrol group, s indited. Dt nlysis Quntittive PCR nd ELIS results were nlyzed y t test or one-wy NOV. Post ho tests were performed with onferroni ll-pirwise omprisons. ll differenes were onsidered signifint t P.. knowledgments We thnk Dr. Tim O rien (Cornell University, Ith, NY) nd his ollegues for their generous help nd regents during the ourse of these studies. ddress ll orrespondene nd requests for reprints to: Dr. Mrk S. Roerson, Deprtment of iomedil Sienes, T-8 Veterinry Reserh Tower, College of Veterinry Mediine, Cornell University, Ith, New York 8. E-mil: msr@ornell.edu. This work ws supported y Ntionl Institutes of Helth/Ntionl Institute of Child Helth nd Humn Development (NICHD) Grnt RHD7 (to M.S.R.) nd NICHD (SCCPRR) Grnt U- HD89, University of Virgini, Center for Reserh in Reprodution, Lignd ssy nd nlysis Core. Dislosure Summry: The uthors hve nothing to dislose. Referenes. Person G, Roinson F, eers Gison T, Xu E, Krndikr M, ermn K, Co MH Mitogen-tivted protein (MP) kinse pthwys: regultion nd physiologil funtions. Endor Rev : 8. Mzzuhelli C, Vntggito C, Cimei, Fsno S, Pkhotin P, Krezel W, Welzl H, Wolfer DP, Pgès G, Vlverde O, Mrowsky, Porrzzo, Orn PC, Mldondo R, Ehrengruer MU, Cestri V, Lipp HP, Chpmn PF, Pouysségur J, rmill R Knokout of ERK MP kinse enhnes synpti plstiity in the stritum nd filittes stritl-medited lerning nd memory. Neuron :87 8. Nekrsov T, Shive C, Go Y, Kwmur K, Gurdi R, Lndreth G, Forsthuer TG ERK-defiient mie show norml T ell effetor funtion nd re highly suseptile to experimentl utoimmune enephlomyelitis. J Immunol 7:7 8. Yo Y, Li W, Wu J, Germnn U, Su MS, Kuid K, ouher DM Extrellulr signl-regulted kinse is neessry for mesoderm differentition. Pro Ntl d Si US : Newern J, Zhong J, Wikrmsinghe SR, Li X, Wu Y, Smuels I, Cherosky N, Krlo JC, O Loughlin, Wikenheiser J, Grgesh M, Doughmn YQ, Chrron J, Ginty DD, Wtne M, Sitt SC, Snider WD, Lndreth GE 8 Mouse nd humn phenotypes indite ritil onserved role for ERK signling in neurl rest development. Pro Ntl d Si : Jpón M, Ruinstein M, Low MJ 99 In situ hyridiztion nlysis of nterior pituitry hormone gene expression during fetl mouse development. J Histohem Cytohem :7 7. Kendll SK, Gordon DF, irkmeier TS, Petrey D, Srpur VD, O She KS, Wood WM, Lloyd RV, Ridgwy EC, Cmper S 99 Enhner-medited high level expression of mouse pituitry glyoprotein hormone -suunit trnsgene in thyrotropes, gondotropes, nd developing pituitry glnd. Mol Endorinol 8: 8. Pope C, MNeilly JR, Coutts S, Millr M, nderson R, MNeilly S 6 Gondotrope nd thyrotrope development in the humn nd mouse nterior pituitry glnd. Dev iol 97: urrows HL, irkmeier TS, Sesholtz F, Cmper S 996 Trgeted ltion of ells in the pituitry primordi of trnsgeni mie. Mol Endorinol : Liu F, ustin D, Mellon PL, Olefsky JM, Wester NJ GnRH tivtes ERK/ leding to the indution of -fos nd LH protein expression in L T ells. Mol Endorinol 6:9. Roerson MS, Misr-Press, Lurne ME, Stork PJ, Murer R 99 role for mitogen-tivted protein kinse in mediting tivtion of the glyoprotein hormone -suunit promoter y gondotropin-relesing hormone. Mol Cell iol : 9 Downloded from mend.endojournls.org t Cse Western Reserve Univ Clevelnd Helth Siene Li on Novemer, 9

10 Mol Endorinol, July 9, (7):9 mend.endojournls.org. Zhng T, Mulvney JM, Roerson MS tivtion of mitogen-tivted protein kinse phosphtse y gondotropin-relesing hormone. Mol Cell Endorinol 7: Lindzey J, Jyes FL, Ytes MM, Couse JF, Korh KS 6 The i-modl effets of estrdiol on gondotropin synthesis nd seretion in femle mie re dependent on estrogen reeptor-. J Endorinol 9:9 7. Lindzey J, Wetsel WC, Couse JF, Stoker T, Cooper R, Korh KS 998 Effets of strtion nd hroni steroid tretments on hypothlmi gondotropinrelesing hormone ontent nd pituitry gondotropins in mle wild-type nd estrogen reeptor- knokout mie. Endorinology 9:9. Dorn C, Ou Q, Svren J, Crwford P, Sdovsky Y 999 tivtion of luteinizing hormone gene y gondotropin-relesing hormone requires the synergy of erly growth response- nd steroidogeni ftor-. J iol Chem 7: mdor-noguez D, Zimmermn J, Venle S, Drlington G Genderspeifi ltertions in gene expression nd loss of liver sexul dimorphism in the long-lived mes dwrf mie. iohem iophys Res Commun :86 7. Cin P, iserni, Ttngelo L, Tiveron C, Sirroni F, Ottorini L, Mggi 7 novel peroxisome prolifertor-tivted reeptor responsive element-luiferse reporter mouse revels gender speifiity of peroxisome prolifertor-tivted reeptor tivity in liver. Mol Endorinol :88 8. Nogueirs R, Gllego R, Gulillo O, Cminos JE, Grí-Cllero T, Csnuev FF, Diéguez C Resistin is expressed in different rt tissues nd is regulted in tissue- nd gender-speifi mnner. FES Lett 8: 7 9. Snhez D, Vllée, Figrell C, ez N, Guy-Crotte O Sexul dimorphism of pnreti gene expression in norml mie. Pnres :7. Li H, Zeitler PS, Vlerius MT, Smll K, Potter SS 996 Gsh-, n orphn Hox gene, is required for norml pituitry development. EMO J :7 7. Retzmn LT, Wrd R, Cmper S Lhx nd Prop re required for ell survivl nd expnsion of the pituitry primordi. Development 9:9 9. Sheng HZ, Zhdnov, Mosinger Jr, Fujii T, ertuzzi S, Grinerg, Lee EJ, Hung SP, Mhon K, Westphl H 996 Speifition of pituitry ell lineges y the LIM homeoox gene Lhx. Siene 7: 7. Nsonkin IO, Wrd RD, Retzmn LT, Sesholtz F, Sunders TL, Gillespie PJ, Cmper S Pituitry hypoplsi nd respirtory distress syndrome in Prop knokout mie. Hum Mol Genet :77 7. Cushmn LJ, urrows HL, Sesholtz F, Lewndoski M, Muzyzk N, Cmper S Cre-medited reomintion in the pituitry glnd. Genesis 8:67 7. Keri R, Wolfe MW, Sunders TL, nderson I, Kendll SK, Wgner T, Yeung J, Gorski J, Nett TM, Cmper S 99 The proximl promoter of the ovine luteinizing hormone -suunit gene onfers gondotrope-speifi expression nd regultion y gondotropin-relesing hormone, testosterone, nd 7 -estrdiol in trnsgeni mie. Mol Endorinol 8: Lwson M, Tsutsumi R, Zhng H, Tlukdr I, utler K, Sntos SJ, Mellon PL, Wester NJ 7 Pulse sensitivity of the luteinizing hormone promoter is determined y negtive feedk loop involving erly growth response- nd Ngfi- inding protein nd. Mol Endorinol : Jing Q, Jeong KH, Horton CD, Hlvorson LM Pituitry homeoox (Pitx) stimultes rt LH gene expression vi two funtionl DN-regultory regions. J Mol Endorinol : 8 8. Tremly JJ, Drouin J 999 Egr- is downstrem effetor of GnRH nd synergizes y diret intertion with Ptx nd SF- to enhne luteinizing hormone gene trnsription. Mol Cell iol 9: Lee SL, Sdovsky Y, Swirnoff H, Polish J, God P, Gvrilin G, Milrndt J 996 Luteinizing hormone defiieny nd femle infertility in mie lking the trnsription ftor NGFI- (Egr-). Siene 7:9. Mudsley S, Nor Z, onfil D, Dvidson L, Krli D, Pwson J, Lrder R, Pope C, Nelson N, Millr RP, rown P 7 Proline-rih tyrosine kinse medites gondotropin-relesing hormone signling to speifi extrellulrly regulted kinse-sensitive trnsriptionl lous in the luteinizing hormone -suunit gene. Mol Endorinol :6. édérrts GY, Kiser U Differentil regultion of gondotropin suunit gene promoter tivity y pulstile gondotropin-relesing hormone (GnRH) in perifused L T ells: role of GnRH reeptor onentrtion. Endorinology :8 8. Knski H, ederrts GY, Km KY, Xu S, Kiser U Gondotropinrelesing hormone pulse frequeny-dependent tivtion of extrellulr signl-regulted kinse pthwys in perifused L T ells. Endorinology 6:. Pgès G, Guérin S, Grll D, onino F, Smith, njuere F, uerger P, Pouysségur J 999 Defetive thymoyte mturtion in p MP kinse (Erk ) knokout mie. Siene 86:7 77. Smuels IS, Krlo JC, Fruzzi N, Pikering K, Herrup K, Swett JD, Sitt SC, Lndreth GE 8 Deletion of ERK mitogen-tivted protein kinse identifies its key roles in ortil neurogenesis nd ognitive funtion. J Neurosi 8: Downloded from mend.endojournls.org t Cse Western Reserve Univ Clevelnd Helth Siene Li on Novemer, 9