$ To whom all correspondence should be addressed. Anita H. Payne,+ Kar-Lit Wong, and Margarita M. Vega

Transcription

1 THE JOURNAL OF B~OLOCICAL CHEMISTRY Vul No. 15. Issue of August 10. pp , 1980 Prmted cn (I S. A. Differential Effects of Single and Repeated Administrations of Gonadotropins on Luteinizing Hormone Receptors and Testosterone Synthesis in Two Populations of Leydig Cells* (Received for publication, March 20, 1980) Anita H. Payne,+ Kar-Lit Wong, and Margarita M. Vega From the Departments of Obstetrics and Gynecology and Biological Chemistq, The University of Michigan, Ann Arbor, Michigan hormone essential for maintenance of testicular steroid hormone production (I). LH mediates its effect on steroidogenesis by binding to high affinity membrane receptors inleydig cells. In recent years, numerous investigators have reported of Leydig cells after different modes of in vivo gonado- that administration of a single high dose of LH or hcg brings tropin treatments. Dispersed cells from decapsulated about a reduction in testicular LH receptors (1-7) accomparat testes were purified and separated into two distinct nied by a decrease in cyclic AMP (4, 6, 7) and testosterone populations of Leydig cells, I and II, by centrifugation production (1, 4, 6, 7) to subsequent stimulation by LH or in a 0 to 40% Metrizamide gradient. The number of LH hcg. Recent studies from our laboratory, however, have receptor sites per lo6 Leydig cells, as measured by I- demonstrated a dissociation between loss oflh receptor hcg binding, was similar in the two populations. Leynumber and steroidogenic response of the testis to LH followdig cell responsiveness was markedly greater in Leydig ing administration of LH to mature rats (1, 8). We observed cell population I1 than I. Twice daily treatment with 6 or 12.5 pg of LH for 6 days produced a marked increase that ody a single high dose of LH, 100 yg or greater, brought in responsiveness in population I, in spite of a 44 and about a decrease in testicular responsiveness, while twice daily 47% loss in receptor sites. The same treatment regimen injections of LH for 6 days or longer were accompanied by a produced only a slight increase in responsiveness in time-related increase in testicular testosterone synthesis in population 11, while LH receptor sites were reduced by responseto an in vivo stimulatory dose of LH. The LH- 34 and 61% in these cells. A single subcutaneous admin- induced loss in testicular LH receptors was similar in rats that istration of 150 pg of LH, a dose equivalent to the total received a single high dose of LH or the twice daily injections. dose of LH that was administered to rats on a twice These observations indicate that the mode of the in vivo LH daily regimen of 12.5 pg, had no effect on responsive- treatment will result in markedly different effects on testicular ness in population I, but decreased in uitm testosterone responsiveness to subsequent LH-hCG stimulation. Interpreproduction in response to hcg from 250 to 70 ng/106 tation of these results, however, is complicated by the possi- Leydig cells in population II. A 57 and 79% loss in LH bility that chronic pituitary hormone treatment may change receptors was observed in populations I and 11, respec- 1) the sensitivity of the Leydig cells to LH; 2) the secretion or tively, after a single administration of LH. The changes clearance rate of testosterone; or 3) total number of Leydig produced in Leydig cell responsiveness after different cells. To clarify these possibilities, changes in LH receptor treatments with gonadotropins were not a result of a concentration and Leydigcellresponsiveness to LH after change in sensitivity of the Leydig cells. different modes of in vivo hormone treatment need to be The results demonstrate that 1) LH has different assessed in the same cells. effects on each of the populations of Leydig cells; 2) the In light of our recent finding of two distinct populations of effect of LH on Leydig cell steroidogenesis is dependent Leydig cells (9), the present study investigates the different on the mode of administration of the hormone and does modes of in vivo gonadotropin treatment OR the number of not appear to relate to loss of LH receptor sites; and 3) LH receptor sites and on in vitro testosterone synthesis in Leydig cell steroidogenic desensitization does not occur response to hcg of Leydig cells in each of the populations. except with a single administration of a high, nonphysiological dose of LH or hcg, suggesting that LH-in- MATERIALS AND METHODS duced desensitization is not a likely mechanism by which Leydig cell steroid production is regulated in Preparation of Leydig Cells-Male Sprague-Dawley rats (55 to 60 days old) were obtained from Spartan Research Animals, Inc., vivo. Haslett, MI. Ovine LH (olh) was kindly provided by Dr. Darrell Ward of the University of Texas Systems Cancer Center, Houston, TX. Animals were injected subcutaneously twice daily with either 6 or The pituitary hormone LH has been shown to be the ody Luteinizing hormone (LH) receptor sites and in vitro testosterone production in response to human chorionic gonadotropin (hcg) stimulation (Leydig cell responsiveness) were studied in two distinct populations * This investigation was supported by National Institutes of Health Grant HD This work was presented in part at the Sixth International Congress of Endocrinology (1980), Melbourne, Australia, p The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom all correspondence should be addressed. The abbreviations used are: LH, luteinizing hormone; olh, ovine pg of olh for 6 days or given a single subcutaneous injection of I50 pg of olh (2.43 x NIH-LH-S1) or 100 IU of hcg. Control animals received equal volumes of isotonic saline (0.9% NaCl solution) on the same injection schedule. Animals were killed by decapitation either at 15 h after the last of the multiple injections or 3 days after a single injection. Leydig cells were obtained by treatment of decapsulated testes with collagenase as described by Dufau et al. (10). Dispersed luteinizing hormone; hcg, human chorionic gonadotropin; LHRH, luteinizing hormone-releasing hormone; BSA, bovine serum albumin.

2 cells from each treatment group were subjected to centrifugation at 3,300 x g for 5 min in a 0 to 40% Metrizamide (Nyegard and Co. supplied by Accurate Chemical and Scientific Corp.) gradient. Onemilliliter fractions were collected from the top of the gradient. Cells from Fractions 17 to 21 were combined as Leydig cell population I while cells from Fractions 25 to 28 were combined as Leydig cell population I1 (9). Cells comprising population I1 were washed and resuspended in 2.5 ml of Medium 199 (Grand Island Biological Co., Grand Island, NY), 0.1% BSA. Cells comprising population I were subjected to a second centrifugation in a 0 to 40% Metrizamide gradient to separate any contaminating cells from population I1 (9). Fractions 17 to 21 from the second gradient were combined, washed, and resuspended in 2.5 ml of Medium 199, 0.1% BSA. Total cells in each population were determined by counting aliquots in a Coulter Counter. The percentage of Leydig cells in each population was established by staining for 3b-hydroxysteroid dehydrogenase as previously described (9). Determinution of LH Receptor Sites by '2sZ-hCG-Aliquots (0.1 ml) of the resuspended cells were incubated for 90 min at 34 C under 95% 02, 5% COS with a saturating concentration (>250 p ~ of ) '"1- hcg (11,OOO IU/mg) prepared as previously described (9). Nonspecific binding was measured in tubes containing the same aliquots of cell suspension and Iz5I-hCG in the presence of 2.5 pg of unlabeled hcg. After incubation, 4 ml of cold Medium 199,0.1% BSA were added to each tube and cells were collected by Centrifugation. Cells were washed once more in 2 ml of Medium 199,0.1% BSA. Cell-bound Iz51- hcg was determined by an automatic spectrometer (Packard) with 50% counting efficiency for '*'I. LH receptor sites per lo6 Leydig cells are expressed as femtomoles of '2sI-hCG bound based on the measured specific activity of the Iz5I-hCG preparations. The specific activity of the radiolabeled hcg was determined by comparing the 50% value on self displacement curves using increasing concentrations of "'I-hCG with the 50% value on inhibition curves using unlabeled hormone (11). The active fraction of the 12sI-hCG was determined by measuring the percentage of labeled hormone capable of specifically binding to an excess of testicular LH receptors. Determination of Testosterone Production by Isolated Leydig Cells-Aliquots of resuspended cells obtained after Metrizamide gradient centrifugation were incubated in Medium 199, 0.1% BSA containing 0.1 mm 1-methyl-3-isobutyl xanthine and increasing concentrations of purified hcg (11,OOO IU/mg) in a total volume of 1 ml. Incubations were carried out for 3 h at 34 C under 95% 02, 5% COS. At the end of incubation, 2 ml of cold Medium 199, 0.1% BSA were added to each vial and the cells plus medium were transferred to glass tubes (12 X 75 mm) before placing in a boiling water bath for 5 min. The tubes were cooled and centrifuged for 20 min to remove denatured protein. Supernatants were frozen and saved for subsequent testosterone determination by radioimmunoassay as previously described (12, 13). Each incubation was carried out in duplicate. Statistical Analysis-Statistical variance or Student's t test. RESULTS Effects of Gonadotropin Treatment on Leydig Cells analysis was done by analysis of Number of LH Receptor Sites and Leydig Cell Responsiveness in Two Populations of Leydig Cells-As shown in Fig. 1, the two populations of Leydig cells, separated by Metrizamide gradient centrifugation, exhibited similar concentrations of LH receptor sites per lo6 Leydig cells but marked differ- ences in testosterone production in response to increasing concentrations of hcg. Testosterone production in Leydig cell population I1 increased from 15 to 250 ng/106 Leydig cells, but only to 80 ng/106 Leydig cells in population I. Maximum production of testosterone was observed at similar concentrations of hcg in each of the populations of Leydig cells. Effect of Daily LH Treatment on Leydig Cell Population I-Twice daily treatment with 6 or 12.5 pg of LH for 6 days resulted in a marked increase in the in vitro responsiveness to hcg in Leydig cell population I (Fig. 2). Maximum testosterone production increased from about 55 ng/106 Leydig cells in cells from control animals to 275 and 390 ng/106 Leydig cells in cells obtained from rats treated with 12 or 25 pg of LH/day, respectively. The loss in LH receptors/106 Leydig cells was 44 and 47% for rats treated with 12 or 25 pg of LH/day, respectively. Although higher maximum testosterone production FIG. 1. Number of LH receptor sites and testosterone synthesis in response to hcg in two populations of Leydig cells. For testosterone production, duplicate aliquots of Leydig cells from Fractions 17 to 21, population I, or from Fractions 25 to 28, population 11, obtained after Metrizamide gradient centrifugation, were incubated for 3 h at 34OC with increasing concentrations of hcg. Testosterone was measured in cells plus medium. Each point represents the mean k S.E. of 11 separate experiments. The number of LH receptor sites, expressed in femtomoles per IO6 Leydig cells, was measured by incubating separate duplicate aliquots of each population with a saturating concentration of "'I-hCG for 90 min at 34 C. Results are the mean of 11 separate experiments. FIG. 2. Testosterone production and LH receptor sites in population I Leydig cells after a 6-day treatment with LH. Rats were injected subcutaneously twice daily for 6 days with either saline (O), 6 pg (H), or 12.5 pg (A) of olh. Animals were killed on Day 7 and Leydig cells from Fractions 17 to 21 were incubated in duplicate as described in the legend to Fig. 1. Each point represents the mean f S.E. of data from two separate experiments. The number in parentheses represents the percentage of LH receptor sites per IO6 Leydig cells relative to control. was seen with rats that received 25 pg of LH/day compared to rats receiving 12 pg of LH/day, the difference in testosterone production per lo6 Leydig cells was not significantly different at any dose of hcg except at 30 PM. Leydig cells from LHtreated rats produced significantly more basal testosterone than Leydig cells from nontreated rats ( p < 0.01). Testosterone concentration measured in Leydig cells from treated and nontreated rats prior to incubation was not significantly different and was tl ng/106 Leydig cells. Therefore, the difference in testosterone production after incubation of Leydig cells from treated versus nontreated rats was not due to an accumulation of testosterone in Leydig cells as a result of the in viuo LH treatment. Effect of Daily LH Treatment on Leydig Cell Population 11-The effect of twice daily treatment with 6 or 12.5 pg of

3 7120 Gonadotropin Effects of Treatment LH for 6 days on Leydig cell population I1 is illustrated in Fig. 3. These in vivo treatments with LH did not induce the marked increase in testosterone production in response to hcg as was observed with Leydig cell population I. The amount of in vitro testosterone production at 10 and 30 PM hcg, however, was significantly greater (p < 0.01) in Leydig cells from treated rats compared to Leydig cells from nontreated control rats (Fig. 3). The amount of testosterone production did not differ in Leydig cells from rats treated with either 12 or 25 pg of LH/day. In contrast, the number of LH receptor sites per lo6 Leydig cells was decreased more in cells from rats treated with 25pgof LH/day compared to rats treated with 12 pg of LH/day (61 versus 34%). Effect of a Single Subcutaneous Injection of LH or hcg on Leydig Cell Population I-Testosterone production, in response to hcg, of Leydig cell population I after a single subcutaneous injection of 150 pg of LH is shown in Fig. 4A. This single dose of 150 pg of LH is equal to the total multiple dose that was administered to rats on a twice daily regimen of 12.5 pg for 6 days. Leydig cells from rats that received a single 400 ' 1 I hcg (DM) FIG. 3. Testosterone production and LH receptor sites in population U Leydig cells after a 6-day treatment with LH. Rats were injected subcutaneously twice daily for 6 days with either saline (O), 6 pg (H), or 12.5 pg (A) of ovine LH. Animals were killed on Day 7 and Leydig cells from Fractions 25 to 28 were incubated in duplicate as described in the legend to Fig. 1. Each point represents the mean k S.E. of data from two separate experiments. The number in parentheses represents the percentage of LH receptor sites per lo6 Leydig cells relative to control. T on Leydig Cells administration of hormone were isolated 72 h after the injection. This time interval was used since we have previously reported that the number of testicular LH receptor sites and in vivo testicular responsiveness to LH were at a minimum 3 days after a single subcutaneous administration of LH (1, 8). A single subcutaneous administration of 150 pg of LH had no significant effect on in vitro responsiveness of Leydig cell population I to hcg (Fig. 4A). Increasing the concentration of hcg to as high as 1,000 PM did not change maximum testosterone production (-65 ng/106 Leydig cells) of Leydig cells from LH-treated rats. The effect on Leydig cell responsiveness observed with a single administration of LH is in marked contrast to the effect observed with multiple injections (25 pg/day) in spite of a similar loss in LH receptor sites. The number of LH receptor sites was decreased to 43% of control values in Leydig cell population I 3 days after a single injection of150pgof LH compared to 53% observed with multiple injections. A single subcutaneous injection of 100 IU of hcg resulted in complete desensitization of Leydig cell population I (Fig. 4B). Leydig cells from these hcg-treated rats did not have the capacity to respond to hcg, at a concentration up to 300 PM, as measured by testosterone production. Leydig cells from these animals exhibited a 98% loss of LH receptor sites. Effect of a Single Subcutaneous Injection of LH or hcg on Leydig Cell Population 11-A single subcutaneous injection of 150 pg of LH markedly densensitized Leydig cell population I1 to in vitro stimulation by hcg (Fig. 5A). Maximum testosterone production in response to hcg decreased from 250 ng to approximately 70 ng/106 Leydig cells. Maximum testosterone production in these Leydig cells was not increased by increasing the concentration of hcg up to 1,000 PM. Therefore, a single subcutaneous injection of LH produces a markedly different effect on Leydig cell responsiveness to hcg compared to twice daily injections of LH for a 6-day period. The number of LH receptor sites was decreased to 21.3% of nontreated control rats. A single subcutaneous injection of100 IU of hcg completely desensitized Leydig cells from population I1 and resulted in a 93% loss in LH receptor sites (Fig. 5B). This type of administration of hcg resulted in the complete absence of an increase in testosterone production by Leydig cells of either population in response to in vitro hcg stimulation. Effect ofdifferent Modes of Treatment with Gonadotropins on Leydig Cell LH Receptor Sites-A comparison of different in vivo treatments with LH or hcg on loss of LH receptor sites in each population of Leydig cells is illustrated in Fig. 6. The two populations of Leydig cells differ in their sensitivity to LH, depending on the dose and mode of administration of hcg(pm1 FIG. 4. Testosterone production and LH receptor sites in population I Leydig cells after a single administration of LH (A) or hcg (B). Rats were injected with one subcutaneous injection of either saline (O), 150pg of LH, or 100 IU of hcg (A). Animals were killed 72 h after the injection. Aliquots of Leydig cells from Fractions 17 to 21 were incubated in duplicate as described in the legend to Fig. 1. Each point represents the mean f S.E. of data from two separate experiments. The number in parentheses represents the percentage of LH receptor sites per IO6 Leydig cells relative to control.

4 Effects of Gonadotropin Treatment on Leydig Cells '0" t hcg(pm1 " l o a FIG. 5. Testosterone production and LH receptor sites in population 11 Leydig cells after a single administration of LH (A) or hcg (B). Rats were injected with one subcutaneous injection of either saline (O), 150 pg of LH, or 100 IU of hcg (A). Animals were killed 72 h after the injection. Aliquots of Leydig cells from Fractions 25 to 28 were incubated in duplicate as described in the legend to Fig. 1. Each point represents the mean * S.E. of data from two separate experiments. The number in parentheses represents the percentage of LH receptor sites per lo6 Leydig cells relative to control.."i - LH 12pgbay 6 days hcg 100 tu Single POPULATION II FIG. 6. LH receptor sites in two populations of Leydig cells after single or multiple administrations of gonadotropins. Rats were injected with the indicated amount of hormone for the indicated duration. The number of LH receptor sites per Leydig cells from each population was determined in duplicate as described in the legend to Fig. 1. Each value represents the mean k S.E. of at least three separate experiments. the hormone. Population I Leydig cells did not exhibit a difference in loss of LH receptor sites as a result of doubling the daily dose of LH. A single injection of LH (150 pg) produced a slightly greater loss in LH receptor sites of population I Leydig cells than the same total dose of LH administered over a 6-day period. In contrast, Leydig cell population 11, with daily hormone injections, showed a dose-dependent loss in LH receptor sites. In addition, population I1 Leydig cells exhibited a greater sensitivity to a single administration of LH than to the same dose of LH administered for a 6-day period. Administration of a single high dose of hcg (100 IU) resulted in greater than 90% loss of LH receptor sites in each of the populations. DISCUSSION sulted in a loss of testicular LH receptors, only a single high dose, 100 pg or greater to intact rats, was accompanied by a decrease in testicular responsiveness to an in vivo stimulatory dose of LH (1, 8). Twice daily injections of 30 pg of LH/day for up to 10 days resulted in a similar loss of testicular LH receptors as a single high dose of LH, but brought about a time-related increase in in vivo testicular responsiveness. Similar observations were made in mature hypophysectomized rats (1, 8). Induced endogenous high concentrations of LH in male rats have led to apparent contradictory observations. De Kretser et al. (14) have reported that a 4-fold increase in serum concentrations of LH in adult male rats 1 month after the induction of cryptorchidism produced a loss of testicular LH receptors, but no change in basal production of testosterone. In vitro stimulation of decapsulated testes by hcg resulted in significantly greater production of testosterone by cryptorchid than by control testes. These observations by de Kretser et al. (14) are consistent with our previous in vivo studies (1,8) and with the demonstration in the present study that chronic exposure of the testis to LH brings about increase an in Leydig cell responsiveness in spite of a loss of LH receptors. Since testes of cryptorchid rats contain a greater concentration of Leydig cells than normal testes, it cannot be determined from the data of de Kretser et al. whether the increased responsiveness of the cryptorchid testis reflects an increased steroidogenic capacity of the Leydig cells or is the result of an increase in the number of Leydig cells. Our data demonstrate that chronic treatment with LH markedly increases the steroidogenic capacity of population I Leydig cells and suggests that our previous demonstration of increased in vivo testicular responsiveness in rats treated twice daily with LH reflects increased steroidogenic capacity of population I Leydig cells. This marked increase in steroidogenic capacity of Leydig cell population I is not accompanied by a decrease in sensitivity of Leydig cells from treated animals. Half-maximal testosterone production in Leydig cell populations I and I1 from treated or nontreated rats varied between 0.32 and 0.86 PM hcg. This observed difference in hcg concentration needed to produce half-maximal testosterone production was not related to var- The present study demonstrates that the effect of LH or hcg on Leydig cell steroidogenesis is dependent on the mode of administration of the hormone and does not appear to relate to loss of LH receptor sites. Previous reports on gonadiation due to treatments but to variation between experiments. In contrast to our observations and the observations of de Kretser et al. (14), Cusan et al. (15) reported that chronic administration of LHRH or of an LHRH agonist, [D-Ala,desotropin-induced loss of testicular LH receptors accompanied Gly-NHzl']LHRH ethylamide, to adult male rats produced by a decrease in in vitro testosterone production in response high serum concentrations of LH, 70% inhibition of testicular to further stimulation by LH or hcg were based on studies of LH receptors, and a signficant decrease in weight of the a single administration of hcg or a single dose of LH (2-7). androgen-dependent organs, the seminal vesicle and the pros- Studies from our laboratory in mature male rats (1, 8) indi- tate. The decrease in the androgen-dependent organs was cated that although all variations of treatment with LH re- interpreted to be a result of decreased testicular testosterone

5 7122 Effects of Gonadotropin Treatment secretion, due to desensitization of testicular steroidogenesis caused by elevated serum LH concentrations. The observations by Cusan et al. (15) with LHRH or LHRH agonists can be explained by the recent findings of Hsueh and collaborators that LHRH and LHRH analogs exert a direct inhibition on testicular LH receptors and steroidogenesis in immature hypophysectomized (16) and in adult hypophysectomized rats (17). Cigorraga et al. (18) have reported that a single subcutaneous administration of hcg to male rats produced a 65 to 85% loss of Leydig cell LH receptors 3 days after gonadotropin administration. Incubation of these Leydig cells resulted in a marked reduction in testosterone production in response to hcg. The reduction in testosterone production of these Leydig cells could be overcome by a 20-fold increase in the concentration of hcg. Thus, subcutaneous administration of hcg did not result in a decrease of maximum testosterone production, but in a marked decrease in the sensitivity of Leydig cells to gonadotropin stimulation. In our study, a single subcutaneous administration of 150 pg of LH resulted in a 79% decrease of Leydig cell population I1 LH receptors and a 72% decrease in testosterone production. This decrease in testosterone production could not be overcome by increasing the hcg concentration 300-fold (1,000 PM). This single dose of LH decreased population I LH receptors by 57% but did not have a significant effect on testosterone production in response to hcg. It is possible that the Leydig cells studied by Cigorraga et al. (18) were a mixture of Leydig cell populations I and 11. The use of a 0 to 80% Metrizamide gradient for purification of Leydig cells as utilized by Cigorraga et al. did not separate Leydig cells into two distinct populations.2 The absence of in vitro responsiveness of Leydig cells of both populations 3 days after a single subcutaneous injection of 100 IU of hcg is most likely the result of the extensive loss in LH receptors, i.e. >90% (Fig. 6) (18). The reason for the different effect on Leydig cell steroidogenesis produced by a single injection compared to multiple injections of LH is not apparent. It is possible that the twice daily injections of LH have a trophic effect on steroidogenic enzymes. We previously reported that twice daily treatment with LH of adult hypophysectomized rats resulted in a marked increase in 3P-hydroxysteroid dehydrogenase activity in isolated interstitial tissue (19). We also demonstrated that chronic LH treatment to intact adult rats increased testicular aromatase activity (20). An indication that chronic LH treatment has a trophic action on steroidogenic enzymes is presented in this report. Leydig cells from rats that received the 6-day treatment with LH produced significantly more basal testosterone during in vitro incubation than Leydig cells from nontreated control rats (Fig. 2). This increase in in vitro production of basal testosterone by Leydig cell population I was not observed in Leydig cells from rats that received a single injection of 150 pg of LH. The desensitization of Leydig cell steroidogenesis after a single high administration of LH or hcg could be due to inhibition of endogenous secretion of A. H. Payne, K.-L. Wong, and M. M. Vega, unpublished observations. on Leydig Cells LH by the pituitary resulting in a decrease of steroidogenic enzyme activity or a result of a specific block in steroidogenesis as reported by Cigorraga et al. (18). Future studies on specific enzyme activities in each of the populations of Leydig cells after different in vivo treatments with gonadotropins should answer these questions. In conclusion, the present study clearly demonstrates that Leydig cell steroidogenic desensitization does not occur except with a single administration of a high, nonphysiological dose of LH or hcg. Since the testis is normally exposed to only small pulsatile LH discharges and not to massive LH surges, LH-induced Leydig cell steroidogenic desensitization as described by several authors (2-7) is not a likely mechanism by whichleydigcell steroid production is regulated in vivo. Furthermore, the demonstration that LH has different effects on the two populations of Leydig cells needs to be taken into consideration in future studies on hormonal regulation of Leydig cell function. Acknowledgments-The superb assistance of Mary Dockrill is most gratefully acknowledged. We thank Dr. Darrell N. Ward for generously supplying the ovine LH. REFERENCES 1. Zipf, W. B., Payne, A. H., and Kelch, R. P. (1978) Biochim. Biophys. Acta 540, Sharpe, R. M. (1976) Nature 264, Hsueh, A. J. W., Dufau, M. L., and Catt, K. J. (1976) Biochem. Bwphys. Res. Commun. 72, Hsueh, A. J. W., Dufau, M. L., and Catt, K. J. (1977) Proc. Nutl. Acad. Sci. U. S. A. 74, Purvis, K., Torjesen, P. A., Haug, E., and Hansson, V. (1977) Mol. Cell. Endocrinol. 8, Tsuruhara, T., Dufau, M. L., Cigorraga, S., and Catt, K. J. (1977) J. Biol. Chem. 252, Saez, J. M., Haour, F., and Cathiard, A. M. (1978) Biochem. Bwphys. Res. Commun. 81, Payne, A. H., and Zipf, W. B. (1978) Znt. J. Androl. 1, Suppl. 2, Payne, A. H., Downing, J. R., and Wong, K.-L. (1980) Endocrinology 106, Dufau, M. L., Mendelson, C. R., and Catt, K. J. (1974) J. Clin. Endocrinol. Metab. 39, Rao, M. C., Richards, J. S., Midgley, A. R., Jr., and Reichert, L. E., Jr. (1977) Endocrinology 101, Payne, A. H., Kelch, R. P., Murono, E. P., and Kerlan, J. J. (1977) J. Endocrinol. 72, Hauger, R. L., Chen, Y.-D. I., Kelch, R. P., and Payne, A. H. (1977) J. Endocrinol. 74, de Kretser, D. M., Sharpe, R. M., and Swanson, I. A. (1979) Endocrinology 105, Cusan, L., Auclair, C., Belanger, A., Ferland, L., Kelly, P. A., Seguin, C., and Labrie, F. (1979) Endocrinology 104, Hsueh, A. J. W., and Erickson, G. F. (1979) Nature 281, Bambino, T. H., Schreiber, J. R., and Hsueh, A. J. W. (1980) Endocrinology, in press 18. Cigorraga, S. B., Dufau, M. L., and Catt, K. J. (1978) J. Bid. Chem Shaw, M. J., Georgopoulos, L. E., and Payne, A. H. (1979) Endocrinology 104, Valladares, L. E., and Payne, A. H. (1979) Endocrinology 105,