Nelly Pitteloud, Andrew A. Dwyer, Suzzunne DeCruz, Hang Lee, Paul A. Boepple, William F. Crowley, Jr., and Frances J. Hayes

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1 ORIGINAL Endocrine ARTICLE Cre Inhiition of Luteinizing Hormone Secretion y Testosterone in Men Requires Aromtiztion for Its Pituitry But Not Its Hypothlmic Effects: Evidence from the Tndem Study of Norml nd Gondotropin- Relesing Hormone-Deficient Men Nelly Pitteloud, Andrew A. Dwyer, Suzzunne DeCruz, Hng Lee, Pul A. Boepple, Willim F. Crowley, Jr., nd Frnces J. Hyes Reproductive Endocrine Unit of Deprtment of Medicine (N.P., A.A.D., S.D., P.A.B., W.F.C., F.J.H.) nd Deprtment of Biosttistics nd Generl Clinicl Reserch Center (H.L.), Msschusetts Generl Hospitl, Boston, Msschusetts 1 Context: Studies on the regultion of LH secretion y sex steroids in men re conflicting. Ojective: Our ims were to determine the reltive contriutions of testosterone (T) nd estrdiol (E ) to LH regultion nd loclize their sites of negtive feedck. Design: This ws prospective study with three rms. Setting: The study ws conducted t Generl Clinicl Reserch Center. Ptients or Other Prticipnts: Twenty-two norml (NL) men nd 11 men with GnRH deficiency due to idiopthic hypogondotropic hypogondism (IHH) prticipted. Intervention: Medicl cstrtion nd inhiition of romtse were chieved using high-dose ketoconzole (KC) for 7 d with 1) no sex steroid dd-ck; ) T ennthte 1 mg im strting on d ; or 3) E ptch 37. g/d strting on d. Blood smpling ws performed every min for 1 h t seline, overnight on d 3 nd d 6 7. Min Outcome Mesures: Men LH levels, LH pulse mplitude, nd GnRH pulse frequency were ssessed t seline, d 3, nd d 6 7. Results: In NL men, KC cused 3-fold increse in men LH on d 3, which ws stle on d 6 7 with no dd-ck. Addition of T reduced LH levels ( to IU/liter, P.) y slowing GnRH pulse frequency (13.3. to pulses/1 h, P.). LH mplitude incresed ( to IU/liter, P.). E dd-ck suppressed LH levels (36..6 to 19.. IU/liter, P.), y slowing GnRH pulse frequency (11.. to.6. pulses/1 h, P.) nd hd no impct on LH pulse mplitude. In IHH men, restoring norml T levels cused no suppression of men LH levels or LH mplitude. E dd-ck normlized men LH levels nd decresed LH mplitude from to 1 1. IU/liter (P.). Conclusions: 1) T nd E hve independent effects on LH. ) Inhiition of LH y T requires romtiztion for its pituitry, ut not hypothlmic effects. 3) E negtive feedck on LH occurs t the hypothlmus. (J Clin Endocrinol Met 93: 7 791, ) 1-97X//$1./ Printed in U.S.A. Copyright y The Endocrine Society doi:.1/jc.7-16 Received Septemer 6, 7. Accepted Decemer, 7. First Pulished Online Decemer 11, 7 Arevitions: BL, Bseline; CV, coefficient of vrition; E, estrdiol; FAS, free -suunit; IHH, idiopthic hypogondotropic hypogondism; KC, ketoconzole; NL men, norml men; T, testosterone. 7 jcem.endojournls.org J Clin Endocrinol Met. Mrch, 93(3):7 791 Downloded from y guest on 6 August 1

2 J Clin Endocrinol Met, Mrch, 93(3):7 791 jcem.endojournls.org 7 Under physiologicl conditions, serum LH levels in men re kept within tight rnge, reflecting the lnce etween stimultion y GnRH nd inhiition y gondl sex steroids. However, questions remin out the precise mechnisms of the sex steroid component to LH regultion, specificlly the reltive contriutions of testosterone (T) nd estrdiol (E ), the degree to which romtiztion to E is importnt in mediting T s effects, nd the respective sites of feedck of these hormones. Previous work y our group (1, ) nd others (3) suggested tht T nd E were eqully effective t suppressing endogenous LH levels in helthy men (Tle 1). However, the estrogen replcement regimens in these studies resulted in suprphysiologicl E levels. Similrly, some investigtors hve reported tht romtiztion is needed for T negtive feedck t the pituitry (), wheres others hve suggested tht T s restrint of LH secretion is lrgely conditionl on its romtiztion t oth hypothlmic nd pituitry levels (3). In contrst, other studies provide evidence for n E -independent effect of T y demonstrting elevted LH levels in ptients with ndrogen insensitivity () nd in helthy men fter dministrtion of nti-ndrogens (6 ). Similrly, previous work from our group showed tht the increse in LH levels in helthy men in response to selective E suppression with n romtse inhiitor is only one third of tht seen when oth T nd E re suppressed to cstrte levels (9). A numer of issues hve hindered clinicl investigtion in this field nd contriute to the discrepncies etween studies. First, use of phrmcologicl sex steroid regimens precludes conclusions out the reltive importnce of T nd E for LH regultion under norml physiologicl conditions. Second, the romtse inhiitor used in some studies, testolctone, ws susequently shown to hve dditionl ntindrogenic ctivity (). Third, in the intct mle, gondotropin secretion reflects the integrted response of oth the hypothlmus nd pituitry. Some investigtors hve ssumed tht ny chnge in LH pulse frequency reflects n exclusively hypothlmic site of negtive feedck nd, conversely, tht ll chnges in LH pulse mplitude re determinedsolelyyltertionsinpituitrysensitivitytognrh.however, there is ctully liner reltionship etween the mount of GnRHsecretedperpulsendthemplitudeoftheensuingpituitryLH response (11, 1). Therefore, it follows tht ny chnge in LH mplitude could, in fct, reflect either pituitry nd/or hypothlmic effect. In the humn, it is not possile to mesure GnRH to help distinguishhypothlmicfrompituitryeffectsecuseofitsconfinement to the hypophysel-portl circultion nd short hlf-life. Accurte locliztion of the sites of T nd E feedck in the humn thus requires complementry model in which the dose nd frequency of GnRH cn e experimentlly controlled. Men with idiopthic hypogondotropic hypogondism (IHH), who lck endogenous hypothlmic GnRH secretion nd whose pituitry-gondl xis cn e normlized with long-term pulstile GnRH replcement (13), provide such model (Fig. 1). Becuse the dose nd frequency of exogenous GnRH therpy cn e experimentlly controlled, this model in effect represents hypothlmic clmp. Therefore, ny effects of ltering gondl steroid levels on gondotropin secretion in such GnRH-deficient men cn only reflect pituitry site of ction. In contrst, in norml men with n intct hypothlmic-pituitry gondl xis, gondl steroids cn modulte gondotropin secretion y pituitry nd/or hypothlmic effects (Fig. 1). Thus, using the tndem study of these two humn models, hypothlmic site of ction of sex steroids cn e inferred whenever there is difference in the gondotropin responses of norml nd GnRH-deficient men to ltertions in their sex steroid milieu. Thus, the ims of this study were to 1) determine the reltive contriutions of T nd E to LH regultion in men, ) ssess the degree to which romtiztion medites T s effects on LH, nd 3) loclize the precise sites of T nd E negtive feedck. Sujects nd Methods Sujects Helthy norml men (NL men) Thirty-one helthy NL men (ge yr) were enrolled in the study. All study sujects met the following criteri: 1) norml puertl development, sexul function, nd generl helth; ) norml physicl exmintion including testiculr volume of t lest 1 ml; 3) norml renl nd heptic function; ) norml serum levels of T, E, LH, FSH, TSH, nd prolctin; nd ) norml semen nlysis ccording to World Helth Orgniztion criteri (). GnRH-deficient men Thirteen men (3. 9. yr) with congenitl IHH prticipted in the study. The dignosis of IHH ws sed on the following criteri: 1) filure to go through puerty y ge 1 yr, ) serum T less thn or equl to ng/dl (3. nmol/liter) in ssocition with inppropritely low gondotropin levels, 3) sence of norml endogenous gondotropin pulstions during 1- to -h period of frequent lood smpling, ) otherwise norml reserve testing of nterior pituitry function, nd ) norml hypothlmic-pituitry region y mgnetic resonnce imging. At the time of study prticiption, ll sujects hd normlized their serum T, LH, nd FSH for t lest 3 months with pulstile GnRH therpy delivered t -h intervls (13), which they continued to receive throughout the study. The study ws pproved y the Humn Reserch Committee t Msschusetts Generl Hospitl, nd ll sujects provided written informed consent efore the initition of ny study relted procedures. Study design Sujects were studied using n ltion nd replcement model. A detiled seline (BL) neuroendocrine evlution ws performed in the presence of norml sex steroids. It ws repeted on d 3 of cute sex steroid ltion nd gin on d 6 7 with or without selective sex steroid dd-ck from d 7. Eleven NL nd three GnRH-deficient men were withdrwn from the study ecuse of dverse events (norml liver function tests, skin rsh, or nuse). Therefore, NL nd 11 GnRHdeficient sujects completed the protocol. One NL volunteer prticipted in two rms nd five GnRH-deficient men prticipted in more thn one rm of the study. In ech cse, the intervl etween consecutive studies ws t lest 3 months. All sujects were dmitted to the Generl Clinicl Reserch Center t Msschusetts Generl Hospitl t 1 h, nd commencing t h, lood ws drwn every min for 1 h for their BL evlution. Medicl cstrtion nd inhiition of romtse ctivity were then induced using high-dose ketoconzole (KC), 1 g loding dose followed y mg four times dily for 7 d. KC is potent inhiitor of C 17 lyse, the rtelimiting step in ndrogen iosynthesis (1), nd we (9, 16) nd others (3) hve previously shown tht this regimen suppresses T levels to the cstrte rnge within h. At high doses, KC lso inhiits oth cortisol iosynthesis nd romtse ctivity (17, 1). For this reson, we gve study prticipnts dexmethsone (. mg twice dily) for the durtion Downloded from y guest on 6 August 1

3 76 Pitteloud et l. Differentil Regultion of LH y Sex Steroids in Men J Clin Endocrinol Met, Mrch, 93(3):7 791 TABLE 1. Summry of key studies on sex steroid regultion of LH secretion in men Ref. Steroid regimen Sujects Men LH LH mp LH frequency Finkelstein et l. (1) T 1 mg/d d Norml men GnRH-deficient men % % EC Finkelstein et l. () E 9 g/d d Norml men GnRH-deficient men % % EC Schnorr et l. (3) KC with T ptch 7. mg/d, KC with E ptch g/d, KC with T ptch plus AI Norml men 1 3-fold fter KC; to BL with T or E No chnge fter T plus AI Not ssessed Not ssessed Bgtell et l. () Snten et l. () T 1 mg/d 3d E 9 g/d 3d DHT g/d 3d E 3. g/h 6h T 6 g/h 6h AI, Aromtse inhiitor; EC, experimentlly controlled. GnRH-deficient men % % no chnge Norml men 1% 1% Not ssessed 1 EC No of the study, hving first shown tht this dose does not suppress gondotropin or testosterone secretion in men (9). After completing the BL evlution, sujects prticipted in one or more of the following regimens. 1) Sex steroid ltion lone (T, E ) Nine NL nd five GnRH-deficient men received KC for 7dnd underwent repet overnight frequent-smpling studies on d 3 nd d 6 7. ) Sex steroid ltion with T dd-ck (T, E ) Six NL nd GnRH-deficient men received the sme KC regimen ut were replced with T for d 7 dministered s single im injection of T ennthte 1 mg fter completing the second frequentsmpling study. The fct tht KC inhiits romtse ment tht concomitnt dministrtion of n romtse inhiitor ws not necessry to prevent endogenous E levels from incresing in response to T dd-ck. FIG. 1. Schemtic of the hypothlmic-pituitry gondl xis in norml nd GnRH-deficient men. In norml men, T nd E cn exert negtive feedck t oth the hypothlmus nd pituitry. In contrst, men with congenitl GnRH deficiency lck endogenous GnRH secretion. However, their pituitry-gondl xis cn e normlized with pulstile GnRH therpy. Becuse the dose nd frequency of exogenous GnRH re experimentlly controlled, this model represents hypothlmic clmp. Therefore, ny effect on gondotropin secretion of ltering gondl steroids in such GnRH-deficient men cn reflect only pituitry site of ction. Thus, hypothlmic site of ction of sex steroids cn e inferred whenever there is difference in the gondotropin responses of norml nd GnRH-deficient men to ltertions in their sex steroid milieu. 3) Sex steroid ltion with E dd-ck (T, E ) Seven NL nd seven IHH men received KC for 7 d with selective dd-ck of E on d 7 dministered s trnsderml ptch t dose of 37. g/d. All smples were ssyed for LH nd free -suunit (FAS). T nd E levels were determined from study pool composed of equl liquots of ech smple drwn t -min intervls. For every frequent-smpling study, ech of the -min LH nd FAS results ws verged to give men, nd this single men vlue from ech suject ws then used for sttisticl nlysis. Pulstile LH nd FAS secretion were nlyzed using modified Snten nd Brdin (19) method s previously vlidted y our group (). Becuse we hve shown FAS to e etter mrker of GnRH secretion t fst pulse frequencies, FAS ws used to nlyze pulse frequency (). Hormone ssys Serum LH concentrtions were determined y microprticle enzyme immunossy using n utomted Aott AxSYM system (Aott Lortories, Chicgo, IL). The Second Interntionl Reference Preprtion ws used s the reference stndrd. The ssy sensitivity for LH ws 1.6 miu/ml with n intrssy coefficient of vrition (CV) of less thn 7% nd n interssy CV of less thn 7.%. FAS concentrtions were determined y previously descried monoclonl ntiody RIA, using highly purified -suunit of humn chorionic gondotropin s stndrd. The crossrectivity of LH in the FAS ssy ws.7%, comptile with the known contmintion of the humn LH stndrd with FAS (1). Serum T concentrtions were mesured using the DPC Cot-A-Count RIA kit (Dignostics Products Corp., Los Angeles, CA), which hs n intr- nd interssy CV of less thn %. E ws mesured y RIA using hexne ethylcette extrction nd LH- chromtogrphy (Endocrine Sciences, Clss Hills, CA). This E ssy hs sensitivity of pg/ml (1 pmol/liter) nd, sed on mle serum pool, hs n intrssy CV of.9% nd n interssy CV of 1%; the norml rnge for dult men in this ssy is 3 pg/ml. Sttisticl methods Dt re presented s men SEM unless otherwise stted. Respective pir-wise comprisons etween BL, d 3, nd d 6 7 were performed Downloded from y guest on 6 August 1

4 J Clin Endocrinol Met, Mrch, 93(3):7 791 jcem.endojournls.org 77 A Helthy B C Helthy D T (ng/dl) T (ng/dl) E (pg/ml) E (pg/ml) No ddck T ddck E ddck n = 9 n = 6 n = 6 n = 9 n = 6 n = n = using two tiled t tests. When ssy results were elow the level of detection, the level of detection ws used for sttisticl nlysis, nd P vlue. ws considered sttisticlly significnt. n = n = BL D3- D6-7 BL D3- D6-7 BL D3- D6-7 T E FIG.. Serum T nd E levels in helthy mle volunteers (A nd C) nd GnRH-deficient men on GnRH therpy (B nd D) during medicl cstrtion for 7 d with either no sex steroid dd-ck or replcement with T or E from d 7. Vlues represent the pool of smples drwn every min for 1 h., Significntly different from BL, P.;, significntly different from BL, P.. n = 6 restored T levels on d 6 7 (7 99 ng/dl) to BL vlues (Fig. A) without ny significnt increse in E levels (11 pg/ml) (Fig. C). Similrly, E dministrtion restored E levels on d 6 7 to the norml rnge (3 6 pg/ml), lthough levels were slightly higher thn BL concentrtions of 1 pg/ml (P.) (Fig. C). In NL men receiving no sex steroid ddck, men LH levels incresed from 9..9 to 6..1 IU/liter on d 3 (P.) nd remined stle t.. on d 6 7 (Fig. 3A). After T replcement, men LH levels decresed from to IU/liter (P.), such tht levels on d 6 7 were not significntly different from BL (Fig. 3A). E dd-ck cused LH levels to decrese from to 19.. IU/liter (P.); however, vlues on d 6 7 remined significntly higher thn BL concentrtions of. 1 3 IU/liter (P.) (Fig. 3A). In NL sujects receiving no sex steroid dd-ck, GnRH pulse frequency incresed from.1. pulses/1 h t BL to.6. on d 3 (P.) nd.3.9 on d 6 7 (P.) (Fig. A). Administrtion of T slowed GnRH pulse frequency to tht seen t BL (Fig. A). E dministrtion lso slowed pulse frequency, ut the numer of pulses on d 6 7 remined significntly higher thn BL (.6. vs...9, P.) (Fig. A). In sujects receiving no sex steroid ddck, LH pulse mplitude on d 3 ws unchnged from tht t BL (6.6. vs IU/liter, respectively), ut levels on d 6 7 were significntly lower thn d 3 (.9.7 vs IU/liter, P.) (Fig. C). Administrtion of T ws ssocited with n increse in LH mplitude from IU/liter on d 3 to IU/liter on d 6 7 (P.) (Fig. C). No significnt chnges in LH pulse mplitude were seen with E dd-ck ( on d3 vs IU/liter on d 6 7) (Fig. C). Results Norml men The KC regimen employed ws successful in creting the desired sex steroid milieu (Fig., A nd C). In ll three study rms, T levels decresed from the physiologicl rnge t BL to cstrte levels on d 3 of KC dministrtion (P.) (Fig. A). In sujects receiving no sex steroid dd-ck, T nd E levels on d 6 7 remined suppressed t 37 ng/dl, P. (Fig. A) nd 9 1 pg/ml (P.) (Fig. C), respectively. T dd-ck GnRH-deficient men As in the NL men, the desired hormonl milieu ws chieved in the GnRH-deficient sujects with sustined suppression of T (Fig. B) nd E levels (Fig. D) in those receiving no dd-ck. Restortion of physiologicl T levels on d 6 7 (7 9 ng/dl) with T replcement (Fig. B) occurred without significnt increse in E levels (Fig. D). Similrly, E dministrtion led to normliztion of E levels on d 6 7 (3.. IU/liter) (Fig. D). In GnRH-deficient men who received no dd-ck, men LH levels incresed from..9 to..9 IU/liter (P Downloded from y guest on 6 August 1

5 7 Pitteloud et l. Differentil Regultion of LH y Sex Steroids in Men J Clin Endocrinol Met, Mrch, 93(3):7 791 A Helthy B.) on d 3, nd remined stle t IU/liter on d 6 7 (Fig. 3B). In striking contrst to the response of the NL men, dministrtion of T to the GnRH-deficient sujects cused no suppression of either men LH levels (Fig. 3B) or LH pulse mplitude (Fig. D) on d 6 7 when their GnRH pulse frequency ws mintined constnt t h (Fig. B). E replcement, on the other hnd, normlized men LH levels (Fig. 3B) nd LH pulse mplitude on d 6 7 (Fig. D). Discussion Men LH (IU/L) Men LH (IU/L) n = 9 n = 6 n = 6 n = No ddck T ddck E ddck n = Our tndem study of norml nd GnRH-deficient men provides unique insights to the understnding of LH regultion in the humn mle. Using the experimentl model of iochemicl cstrtion nd selective sex steroid replcement, our dt demonstrte tht T nd E hve independent effects on LH secretion. The demonstrtion tht T cn slow GnRH pulse frequency in norml men while E levels remin suppressed indictes tht T feedck t the hypothlmus cn occur vi direct ndrogen effect tht does not require romtiztion. In contrst, the filure of T dd-ck to suppress either men LH levels or LH mplitude in the GnRH-deficient men who re mintined t constnt dose nd frequency of GnRH dministrtion indictes tht T s negtive feedck t the pituitry is medited y romtiztion to E. These dt lso confirm previous studies indicting tht E hs dul sites of negtive feedck (, 3). However, the demonstrtion tht E lowers LH levels in the intct mle y slowing pulse frequency without ny effect on pulse mplitude dds to our c BL D3- D6-7 BL D3- D6-7 BL D3- D6-7 T E FIG. 3. Men LH levels in helthy mle volunteers (A) nd GnRH-deficient men therpy (B) during medicl cstrtion for 7 d with either no sex steroid dd-ck or replcement with T or E from d 7. Vlues represent the men of smples drwn every min for 1 h., Significnt difference from BL, P.;, significnt difference from BL, P.; c, significnt difference etween d 3 nd d 6 7, P.; d, significnt difference etween d 3 nd d 6 7, P.. d c existing knowledge y suggesting tht the dominnt site of E feedck is, in fct, t the hypothlmus. One of the chllenges to studying endogenous gondotropin feedck mechnisms in humns is developing n experimentl model cple of fithfully recreting norml sex steroid milieu. A mjor strength of this study is tht we were successful in this regrd. KC ws effective in reducing T nd E to cstrte levels. The T dd-ck regimen we used restored T concentrtions to BL vlues in oth norml nd GnRH-deficient men. In keeping with the previous demonstrtion tht KC inhiits romtse ctivity (17, 1), we sw no significnt increse in E levels fter T dd-ck. Similrly, the dose of trnsderml estrogen we used restored E levels to BL vlues in the IHH men; E levels in the norml men fter E ddck, lthough slightly higher thn BL, were still within the physiologicl rnge t 3 pg/ml. In contrst, other groups hve filed to chieve dequte E suppression using KC regimen similr to ours except for omission of the loding dose (3). Similrly, previous studies y our group () nd others (3) used estrogen replcement regimens tht resulted in suprphysiologicl E levels. In previous studies, we showed tht the increse in LH in response to selective E suppression ws significntly lower thn tht seen fter suppression of oth T nd E, suggesting tht T hs oth direct effects on LH medited through the ndrogen receptor s well s indirect effects medited y romtiztion (9). However, in tht study, we were unle to determine whether it ws the hypothlmic or pituitry effects of T tht required romtiztion. The demonstrtion y Bgtell nd collegues () tht the nonromtizle ndrogen dihydrotestosterone cuses no suppression of LH levels in GnRH-deficient men undergoing GnRH dministrtion suggested tht romtiztion is needed for T s negtive feedck t the pituitry. Bsed on the demonstrtion tht ddition of n romtse inhiitor completely locks the inhiition of LH secretion y T in men, Schnorr et l. (3) suggested tht T s restrint of LH secretion is lrgely conditionl on its in vivo romtiztion t oth the hypothlmic nd pituitry level. The present study demonstrtes tht T cn normlize GnRH pulse frequency in helthy men when E levels remin suppressed, therey indicting tht T s feedck t the hypothlmus is direct ndrogen effect. In contrst, the filure of T dd-ck to suppress either men LH levels or LH mplitude in GnRH-deficient men mintined t constnt GnRH pulse frequency indictes tht T s negtive feedck t the pituitry requires romtiztion s previously suggested (). These dt confirm previous work from our group, which using n romtse inhiitor to cuse selective suppression of Downloded from y guest on 6 August 1

6 J Clin Endocrinol Met, Mrch, 93(3):7 791 jcem.endojournls.org 79 A Helthy B C Helthy # GnRH pulses/1h # GnRH pulses/1 h LH Amp (IU/L) LH Amp (IU/L) No ddck T ddck E ddck c n = 9 n = 6 n = d n = n = E in norml nd GnRH-deficient men, showed tht E hs oth hypothlmic nd pituitry sites of negtive feedck in the mle (). Using humn ltion nd replcement model, the present study confirms hypothlmic site of ction of E y demonstrting decrese in GnRH pulse frequency in norml men in response to selective physiologicl E dd-ck, wheres the suppression of men LH levels nd LH pulse mplitude in GnRH-deficient men provide evidence for pituitry site of negtive feedck. The filure of severl previous studies to show ny impct of exogenous estrogen dministrtion on LH pulse frequency in norml men ( 6) is likely due to the suoptiml -min smpling prdigm for LH tht ws employed resulting in erroneous estimtions. An n = 9 n = 6 n = 6 D n = n = 1 1 BL D3- D6-7 BL D3- D6-7 BL D3- D6-7 T E FIG.. GnRH pulse frequency nd LH pulse mplitude in helthy mle volunteers (A nd C) nd IHH men therpy mintined t constnt h frequency (B nd D) during medicl cstrtion for 7 d with either no sex steroid dd-ck or replcement with T or E from d 7., Significnt difference from BL, P.;, significnt difference from BL, P.; c, significnt difference etween d 3 nd d 6 7, P.; d, significnt difference etween d 3 nd d 6 7, P.. d 1 1 c dditionl strength of the present study is the use of FAS secretion rther thn LH to nlyze chnges in GnRH pulse frequency. We previously demonstrted the superiority of this mrker of GnRH secretion over LH t fst GnRH pulse frequencies (). Hd we relied on LH s surrogte of GnRH pulstility in response to medicl cstrtion, we would hve picked up % fewer pulses on d3ofsexsteroid ltion nd filed to show suppressive effect of E pulse frequency s result. Hd this study een confined to helthy men, the demonstrtion tht E reduces men LH levels y lowering LH pulse frequency without ny pprent chnges in LH pulse mplitude could hve led one to conclude tht E feedck occurs exclusively t the hypothlmus. However, previous work from our group hs shown tht there is n inverse reltionship etween GnRH pulse frequency nd LH mplitude (7). Therefore, if the dominnt effect of E is to slow pulse frequency, n dditionl suppressive effect of E t the pituitry level might e msked y the tendency for LH mplitude to increse t slower frequencies. An lterntive possiility is tht E suppresses the mplitude of GnRH-induced LH pulses to such degree tht some LH pulses re no longer detectle, leding to the erroneous conclusion tht LH pulse frequency is ctully reduced. In the present study, the inclusion of GnRH-deficient men mintined on fixed dose nd frequency of exogenous GnRH llowed pituitry site of ction to e identified sed on the suppression of oth men LH levels nd LH mplitude fter E dd-ck. Our conclusion from the present study tht the hypothlmus is the dominnt site of E feedck on LH in the mle is supported y the following oservtions. First, selective suppression of E levels in helthy men cuses mrked increse in GnRH pulse frequency, despite concomitnt increse in endogenous T levels tht would normlly serve to restrin the GnRH pulse genertor (). Second, dministrtion of ntiestrogens to norml men lso cuses n increse in LH pulse frequency ( 3). The demonstrtion in this study tht circulting E cn modify GnRH secretion in the presence of cstrte T levels (the mjor sustrte for centrl romtse ctivity) provides indirect evidence tht estrogen effects t the hypothlmus re not dependent on centrl romtiztion. Two recent studies lso support this hypothesis. The first tkes dvntge of n experiment of nture, nmely men with congenitl romtse Downloded from y guest on 6 August 1

7 79 Pitteloud et l. Differentil Regultion of LH y Sex Steroids in Men J Clin Endocrinol Met, Mrch, 93(3):7 791 deficiency. In this unique humn model of oth peripherl nd centrl estrogen deficiency, normliztion of E levels with trnsderml estrogen reduces LH pulse frequency, LH pulse mplitude, nd GnRH-stimulted LH secretion (3). These dt provide evidence not only for dul sites of E negtive feedck ut lso highlight the importnce of circulting, s opposed to loclly generted, estrogen in mediting this effect. One could rgue tht this genetic model hs the limittion tht congenitl estrogen deficiency my hve interfered with mturtion of the hypothlmic-pituitry gondl xis. However, using clever experimentl prdigm, Rven et l. (33) exmined the importnce of circulting vs. centrlly generted estrogens in helthy dult mles y first suppressing romtse ctivity with letrozole nd then restoring physiologicl E levels with trnsderml estrogen. Dt from this experimentl model confirm tht restoring physiologicl concentrtions of circulting E cn normlize gondotropin secretion in men receiving n romtse inhiitor. These dt therefore rgue ginst proposed models of gondotropin feedck, which emphsize greter role for in situ estrogen formtion within the rin thn for circulting E concentrtions in regulting LH secretion (3). This humn investigtive model employing sex steroid ltion nd selective physiologicl sex steroid dd-ck in helthy nd GnRH-deficient men provides novel insights into the study of LH regultion in men. These dt suggest model of sex steroid feedck wherey 1) T nd E hve independent effects on LH secretion, ) inhiition of LH y T requires romtiztion for its pituitry ut not its hypothlmic effects, nd 3) E hs dul sites of feedck, ut its predominnt effect is t the hypothlmus. Acknowledgments Address ll correspondence nd requests for reprints to: Frnces Hyes, MB, FRCPI, Reproductive Endocrine Unit, Msschusetts Generl Hospitl, Fruit Street, Boston, Msschusetts 1. E-mil: Hyes.Frnces@MGH.Hrvrd.edu. This work ws supported y Ntionl Institutes of Helth Grnts R1 HD17-1, DK7-, nd M1-RR-66 nd y the Ntionl Center for Reserch Resources, Generl Clinicl Reserch Centers Progrm. Disclosure Summry: The uthors hve nothing to disclose. References 1. Finkelstein JS, Whitcom R, O De LS, Longcope C, Schoenfeld DA, Crowley Jr WF 1991 Sex steroid control of gondotropin secretion in the humn mle. I. Effects of testosterone dministrtion in norml nd GnRH deficient men. J Clin Endocrinol Met 73:69 6. Finkelstein JS, O De LS, Whitcom R, Crowley Jr WF 1991 Sex steroid control of gondotropin secretion in the humn mle. II. Effects of estrdiol dministrtion in norml nd GnRH deficient men. 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