sign in sign up
<<
Home , Gonadotropin, Gonadotropin receptor

Proc. Nati. Acad. Sci. USA Vol. 77, No. 8, pp. 4459-4463, August 1980 Biochemistry Direct inhibition of testicular function by -releasing : Mediation by specific gonadotropin-releasing hormone receptors in interstitial cells (testis/cyclic AMP/) RICHARD N. CLAYTON, MOHAN KATIKINENI, VIVIAN CHAN, MARIA L. DUFAU, AND KEVIN J. CATr Endocrinology and Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205 Communicated by Klaus Hofmann, April 21, 1980

ABSTRACT Agonist analogs of gonadotropin-releasing sought to analyze the nature and location of GnRH receptors hormone (GnRH) have been shown to exert antigonadal effects in the rat testis. in male and female animals. In hypophysectomized male rats treated with follicle-stimulating hormone, administration of a potent GnRH agonist caused depletion of MATERIALS AND METHODS and receptors and marked suppression of serum tes- Animals and . Male Sprague-Dawley rats 55 to tosterone levels. The possibility that such direct effects of GnRH 60 days old were used for all studies unless otherwise indicated. agonists on testicular function could be expressed through The direct effects of GnRH-A on testicular function were specific receptors located in the interstitial cells of the testis was evaluated in 100-g rats hypophysectomized 24 hr before hor- supported by the selective concentration of a 125I~labeled GnRH agonist by e testis in vivo. Specific receptors for the releasing mone treatment. hormone were demonstrated in testis particles and dispersed GnRH-A was kindly donated by J. Sandow (Hoechst, interstitial cells by direct binding analysis with the 125lIlabeled Frankfurt, West Germany). Other GnRH analogs were ob- GnRH agonist. The binding affinity (K. = 5 X 109 M-1) and tained through the Contraceptive Development Branch, Center specificity of the testicular GnRH binding sites were for Population Research (National Institute of Child Health and similar to those of and ovarian GnRH recep- tors. The presence of GnRH receptors in the testis indicates that Human Development). Purified human chorionic gonadotropin these sites mediate the direct inhibitory actions of GnRH ago- (hCG; CR 119,12,300 international units/mg by bioassay) was nists upon testicular endocrine function. provided by R. Canfield (Department of Medicine, Columbia University). Purified rat LH (rLH), LH reference preparation The antifertility effects of exogenous gonadotropin-releasing (RP-1, 0.03 X NIH LH SI), LH antiserum (S4), ovine LH (oLH; hormone (GnRH) agonists in male and female animals are NIH S18), ovine FSH (oFSH; LER 2056), and prolactin (NIH well-recognized and have recently been found to include a SlO) were obtained through the National Pituitary Agency direct inhibitory action at the gonadal level. In hypophysec- Hormone Distribution Program. Human tomized female rats, supraphysiological doses of these com- (hGH; HS 1216C) was obtained from A. Wilhelmi, (Depart- pounds antagonize the tropic actions of exogenous gonadotropic ment of Biochemistry, Emory University, Atlanta, GA). Crude hormones (1-3). In addition, GnRH agonists inhibit hor- hCG (Pregnyl, 2540 international units/mg) was purchased mone-stimulated steroidogenesis in isolated ovarian granulosa from Organon. (3) and luteal cells (4, 5) by acting through specific high-affinity Assay of GnRH LH, and Prolactin Receptors. [125I]Iodo- receptors in the respective cell membranes (4, 5). GnRH-A (l25I-GnRH-A; specific activity, 1000 ,gCi/,g; 1 Ci In the intact male rat, administration of GnRH agonist causes = 3.7 X 1010 becquerels) was used for GnRH binding marked impairment of testicular endocrine function and studies (4, 17). Briefly, 10 pM 125I-GnRH-A was incubated with (6-14). These effects are partly attributable the tissue preparation (400-500,gg of protein) and either in- to gonadotropic depletion (10, 11) and to the creasing concentrations of unlabeled hormone or a near-satu- ensuing reduction in production in response to en- rating concentration (1.5 nM) of labeled plus unlabeled peptide. dogenous gonadotropins (14). However, an additional inhibitory Nonspecific binding was determined in the presence of 0.2 ,M effect of GnRH agonists may arise from the direct antagonism GnRH-A. Incubations were for 80 min at 4VC with membrane of gonadotropic hormone action in the testis. Follicle-stimu- preparations or for the same time at 240C with interstitial cells. lating hormone (FSH)-stimulated testicular weight gain and Testicular GnRH receptors were characterized in a teased testis induction of luteinizing hormone (LH) receptors is inhibited particulate fraction prepared as described (18). Testicular LH, by GnRH agonists in hypophysectomized male rats (15, 16), FSH, and prolactin receptors were measured in binding assays implying the presence of specific binding sites for these with 125I-labeled hCG, hFSH, and hGH tracers as described (18, in the testis. 19). The above reports indicate that direct testicular actions of Preparation and Incubation of Interstitial Cells and pharmacological doses of GnRH agonists contribute to their Hormone Assays. An interstitial cell suspension was prepared antifertility effects, although the extent of this contribution to by collagenase of fresh testes (20). The dispersed cells the overall effect of GnRH remains to be determined. We ob- were resuspended in medium 199 containing 25 mM Hepes served direct actions of the GnRH agonist analog [D- buffer, 0.1% bovine serum albumin, and 0.1 mM isobutyl- Ser(tBu)6]des-Gly'0-GnRH N-ethylamide (designated methylxanthine for in vitro experiments and binding analyses. GnRH-A) upon testicular LH and prolactin receptors and Abbreviations: GnRH, gonadotropin-releasing hormone (gonadoli- The publication costs of this article were defrayed in part by page berin); FSH, follicle-stimulating hormone (follitropin); LH, luteinizing charge payment. This article must therefore be hereby marked "ad- hormone (lutropin); GnRH-A, GnRH agonist analog; hCG, human vertisement" in accordance with 18 U. S. C. §1734 solely to indicate chorionic gonadotropin; hGH, human growth hormone (somato- this fact. tropin). 4459 Downloaded by guest on September 27, 2021 4460 Biochemistry: Clayton et al. Proc. Natl. Acad. Sci. USA 77 (1980) Triplicate vials containing 0.5-1 X 106 cells per ml were incu- bated at 34"C for 3 hr with increasing concentrations of rLH or hCG in the presence or absence of GnRH-A (0.01-10 ,gg). and cyclic AMP contents of the incubation me- dium were determined by radioimmunoassay as described (21-23). Serum LH was measured by radioimmunoassay as described (24). Serum testosterone concentrations were deter- 2 ~~~~~0.15 mined by radioimmunoassay after methylene chloride ex- traction and LH-20 column chromatography (21, 25). 04 22.5 RESULTS '0 ~~~B/F 0 Localization and Characterization of Testicular GnRH 0.0750 Receptors. The specific uptake of 125I-GnRH-A by the 27,000 X g teased testis particles was linear between 100 and 1000 jig of protein per ml (B/T up to 0.08). Uptake of tracer hormone by interstitial cells was also linear, although a large number of cells (7 X 106/ml) was required to detect reasonable binding 0~~~~ (B/T = 0.06). When measured with a near-saturating ligand 0 ~~~20 40 concentration (1.5 nM), specific GnRH-A binding in particles Bound, pM from the whole testis homogenate was 4.6 fmol/mg of protein 3 6 (Fig. 1). In the teased testis preparation, which has lost most of Total analog, nM its seminiferous tubules, GnRH-A binding was increased by 3- FIG. 2. Saturation curve and Scatchard plot (Inset) of GnRH-A to 4-fold relative to the particles from unteased testes. This binding to 27,000 X g particles from teased testes ofadult male rats. enrichment parallels that of 125I-hCG and 125I-hGH binding, Data from one of three identical experiments are shown as the mean which was 6-fold and 2.5-fold higher than in the homoge- of triplicate determinations which agreed within 109%o of the mean. nate-derived particles, respectively. These findings indicate Protein concentration was 2.2 mg/ml; GnRH binding site concen- that GnRH receptors are located in the Leydig cells, because tration was 16.2 fmol/mg protein. Kd = 3 X 10-10 M; K. = 4 X 109 tubules and Sertoli cells are devoid of LH and prolactin re- M-1. ceptors (18). Also, no specific binding of GnRH-A was detected in 27,000 X g particles derived from homogenates of seminif- and the receptor concentration was 22 + 3.5 fmol/mg protein, erous tubules. No GnRH receptors and very few LH and pro- equivalent to 93 + 3 fmol per testis. Measurement of the re- lactin sites were detected in the 250 X g pellet (low-speed pellet) ceptor concentration in three separate preparations, with a discarded during preparation of the teased testis particles. The near-saturating GnRH-A concentration, gave a mean (+ SEM) particles from the teased frozen testes contained the same of 17.6 ± 2.1 fmol/mg for the total concentration of GnRH-A concentrations of GnRH (5 fmol/mg), LH (50 fmol/mg), and binding sites, in good agreement with the values obtained by prolactin (14.5 fmol/mg) receptors as the unteased testis ho- Scatchard analysis. mogenate, indicating the presence of a large non-Leydig-cell The hormonal specificity of testicular GnRH-receptors is component in this preparation. shown in Fig. 3. A number of small peptides and larger pituitary The properties of interstitial cell binding sites for GnRH were analyzed in the 27,000 X g particulate fraction from teased fresh testes, in which GnRH receptors were saturated by ligand 1 C SO concentrations between 3 and 6 nM (Fig. 2). The data from a representative binding-inhibition curve was best described by A a linear Scatchard plot (Fig. 2 Inset), indicating the presence 0.8 ' 91 of a single class of GnRH-A binding sites. The equilibrium as- sociation constant (Ka) was 5.1 + 0.6 (SEM) X 109 M-1 (n = 3)

0~~~~~~~0

~04

0.2

C 10-1 -10 10- 10-1 10-7 10-6 Hormone added, M FIG. 3. Specificity of binding to testicular GnRH receptors. 1251-GnRH-A (10 pM) was incubated with 27,000 X g particles from A B C D teased testes at 1.3 mg/ml. Initial specific binding (B/Bo) was 13% FIG. 1. Content of GnRH-A (o), LH (3), and prolactin (-) re- (3900 cpm). Each point is the mean oftriplicate determinations which ceptors in testis fractions. Total receptor concentrations were mea- agreed within 10% of the mean. 0, GnRH (natural sequence); &, sured with near-saturating amounts of ligand (GnRH-A and hCG). [D-Lys]6GnRH; 0, GnRH-A; *, [D-pGlul, D-Phe2, D-Trp3'6]GnRH; Values are the mean of triplicate determinations which agreed within *, hCG; *, oLH; Q, ovine prolactin; i, rFSH; 4, hGH; 0, thyrotropin 10%o ofthe mean. A, whole testis homogenate; B, low-speed pellet from releasing hormone; *, corticotropin; O. angiotensin I; *, angiotensin fresh teased testis; C, teased fresh testis; D, teased frozen testis. II; *, . Downloaded by guest on September 27, 2021 Biochemistry: Clayton et al. Proc. Natl. Acad. Sci. USA 77 (1980) 4461 hormones, including gonadotropins, prolactin, and growth hormone, did not interact with the GnRH receptor. 3. Natural-sequence GnRH completely inhibited GnRH-A binding at concentrations of 0.1 MM and above. Other analogs .4.f 2 of GnRH, with one to three amino acid substitutions, bound to the testicular GnRH receptor with high affinity (Table 1). For the peptides tested, the testis receptors appeared to exhibit X1046 - I binding specificity identical to that of pituitary and ovarian GnRH receptors. However, the concentration of binding sites in the testis particles was only 10% of that in pituitary and 50% 0 6 I I Ir of that in luteinized ovarian particles. In assays, GnRH-A did not inhibit 9 binding of l25I-hCG, 125I-hFSH, or 125I-hGH to testicular 0 or particles at concentrations up to 5 MM. toe Testicular Uptake of l25IGnRH-A in Vivo. The in vivo 0I 0 interaction between GnRH agonists and the testis was examined in adult rats injected subcutaneously with 125I-GnRH-A (-1 x-a ,MCi or 1 ng, dissolved in phosphate-buffered saline containing Go" 4) 0.1% bovine serum albumin). Nonspecific uptake of radioac- .0 tivity was determined in rats receiving the tracer together with C) 4 4 Mg of unlabeled hormone. GnRH-A uptake by the pituitary x was maximal 30 min after injection (Fig. 4), and returned to background levels at 4 hr. However, little 125I-GnRH-A ap- aJ peared in the testis until 1 hr, and the peak of uptake occurred at 2 hr, followed by a slow decline to background levels at 6 hr after injection. There was no significant specific uptake of 125I-GnRH-A by the , , spleen, adrenal, hypothal- 0 1 2 3 4 5 6 amus, cerebral cortex, or cerebellum. The slower testicular Mme, hr uptake may be attributable to local differences in tissue pene- FIG. 4. Specific in vivo uptake of '25I-GnRH-A by testes (Lower) tration rather than a difference between the pituitary and testis and pituitary (Upper) after subcutaneous administration. Each point receptor-GnRH binding interaction, because the affinity con- represents the mean (+ SEM) of values from three animals. stants of the two receptors were identical. This experiment >200 pmol/ml, with a median effective dose of 3 pM which confirms the biological relevance of the in vitro binding of was not attenuated by the presence of GnRH-A. GnRH-A to specific receptors in testis particles. Effect of GnRH-A on Testicular Gonadotropin Receptors Action of GnRH-A on Leydig Cells in Vitro. We recently in Vivo. The in vivo effects of GnRH-A on testis receptors were observed an inhibitory action of GnRH in isolated ovarian cells evaluated in hypophysectomized adult male rats, one group of that was manifested as a shift to the right in the which received 150Mg of oFSH daily in an attempt to maintain dose-response to hCG and epinephrine stimulation in the LH receptors. In both groups the effects of a single subcuta- presence of GnRH-A (4, 5). To determine whether such effects neous injection of 1 Mg of GnRH-A on testicular gonadotropin were produced acutely by GnRH-A on the testis in vitro, we receptors were compared with those of 100 Mg of oLH, hor- examined the effect of GnRH-A (1 MM) on the LH- and mone administration beginning 24 hr after hypophysectomy. hCG-stimulated steroidogenic response of collagenase-dispersed In the control hypophysectomized rats, there was a prompt interstitial cells. Neither basal nor hCG-stimulated testosterone decrease in testicular LH receptors, to about 40% of the values production was inhibited by GnRH-A at 5 MM during 3-hr in- in intact rats (Fig. 6). One and 2 days later, the LH receptor cubations (Fig. 5). In another experiment, GnRH-A (0.01-10 - Mg) and native GnRH (up to 10 AM) had no inhibitory effect 4 on testosterone production by submaximal concentrations of 0 LH (5-50 ng) (data not shown). There was a marked cyclic r. 200 - AMP response to hCG, from basal values of 1-2 pmol/ml to -.4. Table 1. Gonadal GnRH receptors: Binding capacity and 0 50 4- 0.4 affinities of GnRH and some related peptides 0 °0 6) Ka (109 M-1} 100 ,0a Testis 150 R0 40 Peptide interstitium Pituitary U) ._ GnRH (natural sequence) 0.5* 0.5 0.7 Ez 501C) [D-LyS6]GnRH 1.7 3.9 2.9 Co [D-Ser(tBu)6ldes-Gly10- 0a GnRH N-ethylamide 5.7 6.6 4.8 [D-pGlul, D-Phe2, -13 10-12 D-Trp3,61-GnRH 1.7 2.8 4.9 hcG, M Binding site capacity, FIG. 5. Effect of GnRH-A (1 ,gM) on hCG-stimulated testoster- fmol/mg protein 22 i 3.5 51 + 12 235 ± 17 one and cyclic AMP production by enzyme-dispersed testis interstitial n 3 3 13 cells (0.6 X 106 cells per ml) in vitro. Cells were incubated for 3 hr at 340C with increasing concentrations ofhCG (CR 119) in the presence * Mean of triplicate estimates from one assay with intra-assay vari- of0.1 mM isobutylmethylxanthine. Mean 4 SEM oftriplicate incu- ation <10%. bations is shown. -, Without GnRH-A; - - -, with GnRH-A. Downloaded by guest on September 27, 2021 4462 Biochemistry: Clayton et al. Proc. Natl. Acad. Sci. USA 77 (1980)

Day 0, Day 1 Day 2

1.01 010

0.5 ._.U, -6

20.25- Cl1 H- 24 r0

0 m FSH FSH

FIG. 6. Effect of a single subcutaneous injection of 1 Mg of GnRH-A (M) or 100 Mg of oLH (l) on testicular LH receptors of hypophysectomized rats. cs, Hypophysectomized controls. One group of animals received 150,Mg of oFSH once daily beginning 24 hr after hypophysectomy on day 0. Data are mean + SEM of values from groups of 5 rats. For day 0: M, intact; O3, hypophysectomized. FSH FSH FIG. 7. Testicular prolactin receptors in the same groups of rats content of the hypophysectomized controls was further de- depicted in Fig. 6. creased to 0.25 pmol per testis, an effect not prevented by FSH treatment. After administration of GnRH-A or oLH, LH re- DISCUSSION ceptors were decreased to even lower levels, and fell to the same GnRH agonists have been shown to exert marked antifertility extent in the FSH-treated group. The effect of hypophysectomy actions when administered to intact male rats (6-11). These on prolactin receptors in the testis was less dramatic, with only effects were initially attributed to gonadotropin-induced de- a slight and insignificant decrease at 24 hr after operation (Fig. pletion of LH receptors and desensitization of testicular ste- 7). At 1 and 2 days there was a significant decrease in hGH roidogenesis to subsequent hormonal stimulation (7, 11, 26). binding, to 69% and 61% of intact values, respectively, which However, in the hypophysectomized rat, GnRH agonists inhibit was accentuated by GnRH-A in both untreated and FSH- FSH-induced testis weight gain and LH receptors and decrease treated groups. In contrast, the effect of oLH treatment on the in vitro hCG-stimulated testosterone responses of testes from lactogenic receptors was transient: decreased hGH binding by treated animals (15, 16), demonstrating a direct testicular site 50% on day 1 and rapid recovery to the control values on day of GnRH action. Furthermore, the deleterious effect of chronic 2. The completeness of hypophysectomy in these experiments GnRH-A treatment on accessory sex organ and testis weights was evaluated by measurement of serum LH after the subcu- in intact rats could be largely prevented by concurrent ad- taneous administration of 100 ng of GnRH-A. One hour later, ministration of (8), suggesting that GnRH-induced seminiferous tubule is serum LH levels in these animals were the same (15-50 ng/ml) dysfunction secondary to testicular rather than the result of direct as unstimulated levels (14-20 ng/ml) (Table.2), indicating that action of GnRH on the tubules themselves. Our findings are no responsive pituitary tissue was present. Thus, the observed consistent with such a mechanism because testicular GnRH changes in LH and prolactin receptors cannot be attributed to receptors are present only in the interstitial cell fraction of the an increase in endogenous LH by the original dose of GnRH testis. used to evaluate receptor depletion. In this study, we have demonstrated that in Vitro binding of Two days after hypophysectomy (day 1), serum testosterone labeled analog to GnRH receptors in the testis is saturable, was decreased to 10% of the intact control value (28 vs. 278 specific, and of high affinity. In addition, in vivo uptake ng/100 ml). This decrease was reversed by LH alone or in of 125I-GnRH-A was confined to the testis and pituitary. combination with FSH but not by FSH alone on day 1 (Table Competition studies with GnRH and other agonist and antag- 2). GnRH-A alone or in combination with FSH had no effect onist analogs demonstrated that the recognition properties of on day 1. However, on day 2, GnRH-A completely prevented these sites are similar to those of GnRH receptors in the ovary the stimulation of serum testosterone levels by FSH. and pituitary. However, it remains possible that slight differ-

Table 2. Effect of GnRH-A on serum hormone levels in hypophysectomized male rats Serum LH, ng/ml Serum testosterone, ng/100 ml Dayl Day2 Dayl Day2 Intact rats 57 + 11 278 12 Hypox. alone 19 + 6 10± 2 28 2 45 ± 7 Hypox. + GnRH-A 35 + 4 18 + 5 33 4 18 ± 2 Hypox. + LH 40 + 5 12 ± 3 473 42 209 + 10 Hypox. + FSH 51 + 4 44 ± 6 56 7 152 + 38 Hypox. + FSH + GnRH-A 47 + 5 34± 2 50 9 16 + 3 Hypox. + FSH + LH 46 ± 2 37 ± 4 287 30 261 + 44 Days 1 and 2 refer to 2 and 3 days after hypophysectomy (hypox.), respectively. GnRH-A (1 Mg) or oLH (100,ug) was given subcutaneously once on day 0. The data shown are mean ± SEM for five rats per group. Downloaded by guest on September 27, 2021 Biochemistry: Clayton et al. Proc. Natl. Acad. Sci. USA 77 (1980) 4463

ences in the structural requirements of the gonadal receptor ticular function awaits elucidation. If they serve to mediate the might become apparent upon more detailed analysis. actions of a naturally occurring ligand, this would have to be In the ovary, gonadotropin-stimulated steroidogenesis by present in adequate concentration (about 0.1 nM) to activate isolated granulosa cells(3) or luteal cells (4,5) is attenuated by the receptors. Available evidence indicates that the circulating GnRH-A during short-term in vitro incubations. However, we concentration of hypothalamic GnRH is too low to exert a observed no diminution of the steroidogenic response of rat regulatory action upon the . Nevertheless, it is evident interstitial cells during incubation for 3 hr with LH or hCG in that the antifertility effects of exogenous GnRH agonists include vitro. Others, using natural-sequence GnRH as well as an a direct inhibitory action that is mediated by the GnRH re- agonist analog, also observed no inhibition of hCG-induced ceptors we have identified in the . steroidogenesis under similar conditions (27). However, cells prepared from the testes of hypophysectomized or intact ani- We are grateful to Mr. K. Schumacher for performing many of the mals pretreated with GnRH-A in vWvo exhibit dramatically hormone assays. reduced steroid responses to gonadotropins in vitro (14, 15). This effect is not the result of GnRH-A competition for gona- 1. Rippel, R. H. & Johnson, E. S. (1976) Proc. Soc. Exp. Biol. Med. dotropin receptors in the testis, and its occurrence in hypo- 152,432-436. animals indicates that it is not solely attributable 2. Ying, S-Y. & Guillemin, R. (1979) Nature (London) 280,593- physectomized 595. to the desensitizing actions of endogenous gonadotropins. 3. Hsueh, A. J. W. & Erickson, G. F. (1979) Science 204, 854- GnRH-A antagonizes gonadotropin- and epinephrine- 855. stimulated progesterone production by cells from fully lu- 4. Clayton, R. N., Harwood, J. P. & Catt, K. J. (1979) Nature teinized in vitro (4, 5) yet has no effect upon hCG- (London) 282,90-92. stimulated testosterone and cyclic AMP production by Leydig 5. Harwood, J. P., Clayton, R. N. & Catt, K. J. (1980) Endocrinology cells during short-term incubations under similar conditions. 107,407-413. This difference might be due to a slower response of Leydig 6. Pelletier, G., Cusan, L., Auclair, C., Kelly, P. A., Desy, L. & La- brie, F. (1978) Endocrinology 103,641-643. cells to the direct action of GnRH, whereas in luteal cells the 7. Labrie, F., Auclair, C., Cusan, L., Kelly, P. A., Pelletier, G. & natural process of regression may be accelerated by GnRH-A. Ferland, L. (1978) Int. J. Androl., Suppl. 2,303-316. It is noteworthy that a single injection of GnRH-A significantly 8. Rivier, C., Rivier, J., Vale & W. W. (1979) Endocrinology 105, reduces serum progesterone concentrations within 4 hr in rats 1191-1201. with fully luteinized ovaries (28). In contrast serum testosterone 9. Rivier, C. & Vale, W. (1979) Life Sci. 25, 1065-1074. levels in adult male rats treated daily with GnRH-A do not fall 10. Auclair, C., Kelly, P. A., Labrie, F., Coy, D. H. & Schally, A. V. (1977) Biochem. Blophys. Res. Commun. 76,855-862. until 1.5-4 days after treatment (9), even though testicular 11. Auclair, C., Kelly, P. A., Coy, D. H., Schally, A. V. & Labrie, F. gonadotropin receptors are markedly reduced within 10 hr after (1977) Endocrinology 101, 1890-1893. a single injection (13). Thus, the in vio data are consistent with 12. Huhtaniemi, I. & Martikainen, H. (1978) Mol. Cell. Endocrinol. the possibility that the Leydig cell is more resistant to the direct 11, 199-204. effects of GnRH-A than is the luteal cell. The mechanism 13. Catt, K. J., Baukal, A. J., Davies, T. F. & Dufau, M. L. (1979) through which the GnRH-receptor interaction leads to inhi- Endocrinology 104, 17-25. bition of progesterone production in the luteal cell includes a 14. Dufau, M. L., Cigorraga, S., Baukal, A. J., Sorrell, S., Bator, J. M., Neubauer, J. F. & Catt, K. J. (1979) Endocrinology 105, reduction in hormone-stimulated cyclic AMP formation (5). 1314-1321. However, in the Leydig cell, no direct antagonism by GnRH-A 15. Hsueh, A. J. W. & Erickson, G. F. (1979) Nature (London) 281, of hCG-stimulated cyclic AMP production could be demon- 66-67. strated in vitro. Nevertheless, the possibility that GnRH-A 16. Arimura, A., Serafini, P., Talbot, S. & Schally, A. V. (1979) Bio- causes inhibition of cyclic AMP and testosterone production chem. Blophys. Res. Commun. 90,687-693. after longer exposure, or in vivo, is not precluded by these 17. Clayton, R. N., Shakespear, R. A., Duncan, J. A. & Marshall, J. C. (1979) Endocrinology 105, 1369-1381. data. 18. Catt, K. J., Ketelslegers, J.-M. & Dufau, M. L. (1976) in Methods There is direct action of GnRH-A on Leydig cell function in in Receptor Research, ed. Blecher, M. (Decker, New York), Part iwo because GnRH-A prevented the increase in serum testos- 1, pp. 175-250. terone in FSH-treated hypophysectomized rats. Also, GnRH-A 19. Ketelslegers, J.-M. & Catt, K. J. (1978) Biochim. Biophys. Acta consistently decreased both LH and prolactin receptors in the 541,360-371. testes of hypophysectomized rats, the effect on LH receptors 20. Dufau, M. L., Mendelson, C. & Catt, K. J. (1974) J. Clin. Endo- being similar to that seen after oLH. However, the prolactin crinol. Metab. 39,610-613. 21. Mendelson, C., Dufau, M. L. & Catt, K. J. (1975) J. Biol. Chem. receptor loss after oLH, although initially of the same magni- 250, 8818-8824. tude as after GnRH, was transient, and receptors had recovered 22. Solano, A. R., Dufau, M. L. & Catt, K. J. (1979) Endocrinology by day 2. This discrepancy between the effects of GnRH and 105, 872-.81. LH on prolactin receptors has been previously observed in in- 23. Dufau, M. L., Tsuruhara, T., Homer, K. A., Podesta, E. & Catt, tact male rats (unpublished data) and in the luteinized ovary K. J. (1977) Proc. Natl. Acad. Sci. USA 74,3419-3423. (29) and suggests that GnRH and the gonadotropic hormones 24. Dufau, M. L., Catt, K. J., Tsuruhara, T. & Ryan, D. (1972) Clin. Chim. Acta 37, 109-116. operate through different mechanisms to regulate prolactin 25. Bartke, A., Steele, R. E., Musto, N. & Caldwell, B. V. (1973) En- receptors in the gonads. docrinology 92, 1223-1228. The present studies have shown that GnRH exerts direct 26. Cusan, L., Auclair, C., Belanger, A., Ferland, L., Kelly, P. A., inhibitory effects in the rat testis, including loss of prolactin Sequin, C. & Labrie, F. (1979) Endocrinology 104, 1369- receptors as well as decrease of LH receptors and testosterone 1376. secretion. These direct actions of GnRH agonists on gonado- 27. Badger, T. M., Beitins, I. Z., Ostrea, T., Crisafulli, J., Little, R. & Saidel, M. (1980) Endocrinology 106, 1149-1153. receptors may contribute to the reduction in 28. Harwood, J. P., Clayton, R. N., Chen, T. T., Knox, G. & Catt, K. testosterone secretion by testes of intact animals treated for J. (1980) Endocrinology 107,414-421. prolonged periods with these peptides (7-9, 11). The physio- 29. Davies, T. F., Katikineni, M., Chan, V., Harwood, J. P., Dufau, logical relevance of these sites in the regulation of normal tes- M. L. & Catt, K. J. (1980) Nature (London) 283,863-865. Downloaded by guest on September 27, 2021