LH Receptor Content Research Articles

Inhibition of Spermatogenesis and Steroidogenesis During Long‐Term Treatment with hCG in the Rat

The effects of chronic treatment with hCG (100 I.U. s.c. every second day) on testicular morphology, LH receptor levels and concentration of steroid intermediates of the δ4 and δ5 biosynthetic pathways were studied in adult rats for periods extending from one to 12 weeks. Treatment with hCG causes a decrease in testis weight, a maximal inhibitory effect being observed after two and four weeks of treatment. At these time intervals, the loss of testis weight is accompanied by degenerative changes in most seminiferous tubules and hypertrophy of Leydig cells. Administration of hCG for one week leads also to an almost complete loss of LH binding sites and to a marked stimulation of the levels of testicular steroids of the A and pathways, as well as to an increase in weights of secondary reproductive organs. The initial increment of testicular steroid levels is followed after two weeks of hCG administration by an apparent decrease of 17, 20‐desmolase activity suggested by a reduction in the levels of androst‐5‐ene‐3β, 17β‐diol, androstenedione, testosterone, and 5α‐dihydrotestosterone and an increase in the concentrations of pregnenolone, 17‐OH‐pregnenolone, progesterone and 17‐OH‐progesterone. Plasma and pituitary LH levels are maximally reduced at one and four weeks of treatment, respectively, while plasma and pituitary FSH levels are only slightly reduced after four weeks of hCG administration. The effects of hCG on all of the above‐mentioned parameters, except for testicular morphology and testis weight, are completely reversible at the eight and 12 week intervals. This transiency in the effects of hCG is accompanied by a gradual increase in plasma levels of hCG antibodies. The present data show that chronic treatment of adult rats with hCG induces a marked degeneration of the seminiferous tubules and an inhibition of spermatogenesis that accompanies the well‐known loss of testicular LH receptors and inhibition of the steroidogenic pathway.

Observations on the effects of prolactin on LH-receptors and steroidogenesis in corpus luteum and testis of the hypophysectomized rat.

Abstract. In this study the effects of hypophysectomy and autotransplantation of the pituitary gland on the concentration of hCG-binding sites (LH-receptors) and steroidogenesis in the corpus luteum and the testis of the rat were investigated. It was found that during pseudopregnancy both hCG-binding to homogenates of isolated corpora lutea and the progesterone levels in blood increase until day 7 and subsequently decrease until day 13. Hypophysectomy on day 5 led to a decrease of the number of LH-receptors and the serum progesterone level. By contrast hypophysectomy followed by autotransplantation of the pituitary gland increased the LH-receptor concentration and progesterone synthesis in spite of non-detectable LH-levels. Progesterone implants in hypophysectomized rats did not influence the number of LH-receptors. Hypophysectomy on day 0 without pituitary gland transplantation did not prevent the formation of some luteal LH-receptors measured on day 5 although progesterone was not secreted. A similar effect of prolactin secreted by pituitary autografts on LH-receptors was seen in the testis. The rapid decline of the number of binding sites normally observed after hypophysectomy was prevented by the presence of two pituitary autografts. Testosterone and LH-levels were non-detectable in the operated male rats. These data show that progesterone secretion by the corpora lutea is always associated with the presence of LH-receptors, regardless whether serum LH-levels are detectable or not. Moreover it appears that prolactin maintains or even increases the amount of LH-receptors in hypophysectomized male and female rats.

Prolactin and luteinizing hormone receptors in marsupial corpora lutea: relationship to control of luteal function.

Prolactin and LH receptor concentrations in tammar wallaby corpora lutea (CL) have been examined and related to the control of luteal function in this and other marsupial species. During embryonic diapause, quiescent CL contained high concentrations of prolactin receptors. This was consistent with an earlier suggestion that prolactin may act directly on the CL to maintain its quiescent state. However, despite an apparent seasonal change in the mechanism by which the CL is maintained in quiescence, the number of luteal prolactin receptors remained constant throughout the year. Reactivation of quiescent CL led to an approximate halving of prolactin receptors on a per cell basis, but pregnancy had no effect. None of the wallaby CL showed any significant degree of LH binding, although wallaby follicles and testicular tissue did show binding. This lack of LH binding was consistent with previous findings that quiescent wallaby CL reactivate in response to hypophysectomy and are refractory to LH when cultured in vitro. Parturition, which occurs approximately 2 days before ovulation in this species, had no effect on numbers of either prolactin or LH receptors despite the occurrence of a peak of plasma prolactin at this time. The red kangaroo, which also exhibits embryonic diapause, showed a similar pattern of luteal receptor numbers with high prolactin and negligible LH binding, whereas the brush-tailed possum, which does not exhibit embryonic diapause, gave a pattern more like that seen in Eutheria with many more LH than prolactin receptors.

Low LH (hCG) receptor concentration in ovarian follicles in endometriosis.

The concentration of LH (hCG) receptors in the granulosa cells of ovarian follicles was assayed in 20 patients with histologically proven endometriosis and in 6 control patients. The levels of receptors in 15 patients with ovarian endometriosis (0.48 +/- 0.46 fmol/mg protein) and in 5 patients with endometriosis without ovarian involvement (0.51 +/- 0.52 fmol/mg protein) were lower (p less than 0.01 and p less than 0.05, respectively) than in control patients (1.32 less than 0.61 fmol/mg protein). The severity of the disease was not reflected in the results. The low receptor concentration in endometriosis suggests a failure in the mechanism mediating LH action in the follicle.

Delayed puberty induced by chronic suppression of prolactin release in the female rat.

To study the effect of PRL deficiency on the onset of puberty, PRL release was chronically inhibited by treating immature female rats with the dopaminergic receptor agonist, bromoergocriptine (CB-154). The resulting alterations in the time of puberty onset, and in other associated parameters, such as serum levels of pituitary hormones, ovarian responsiveness to gonadotropins and ovarian hCG receptor content were then evaluated. CB-154 was provided in the drinking water from day 22 onward at the concentration of 20 and 100 micrograms/ml. The treatment resulted in almost complete suppression of serum PRL levels throughout the entire period studied (day 22 to first diestrus). In contrast, serum GH, FSH, and LH levels were not depressed. Likewise, pulsatile release of FSH was not affected and only a subtle alteration in pulsatile LH release was apparent. The onset of puberty, as determined by the age at vaginal opening, and at first diestrus after the first estrus and by the presence of corpora lutea at sacrifice (first diestrus), was markedly delayed in the hypoprolactinemic (HPO) rats. This inhibitory effect of CB-154 was completely prevented by concomitant administration of PRL. Ovarian weight was significantly decreased in HPO rats at the three ages studied (27, 32, and 36 days of age). By day 36, 50% of the control animals had already ovulated, as compared with only 9% of the HPO rats. Microscopic examination of ovaries from HPO rats revealed a retarded follicular development. In vitro ovarian progesterone response to hCG studied at day 32 and 36 of age was reduced in the HPO rats. Uterine growth was also depressed in HPO rats, the ovaries of which, when incubated in vitro, failed to show the prepubertal increase in estrogen response to hCG seen in control rats between day 32 ad 36. Aromatase activity, as measured by the in vitro release of estradiol from ovaries incubated in the presence of an excess of androgen substrate, was depressed in HPO rats. hCG receptor content in the ovaries from HPO rats (counts per min [125]hCG bound per micrograms DNA) was also lower than that of control animals at day 32 and 34 but not at day 36. However, at this later time the hCG receptor content per milligram of ovary was still significantly reduced in HPO rats. The results support the view that PRL plays an important role in the process of ovarian development that leads to the onset of puberty in the female rat and that this effect is, at least in part, exerted through a positive influence of PRL on ovarian LH receptor content.

Changes in ovarian luteinizing hormone and follicle-stimulating hormone receptor content and in gonadotropin-induced ornithine decarboxylase activity during prepubertal and pubertal development of the female rat.

Ovarian hCG and FSH receptor content was measured at different postnatal ages (days 4-32), at 4-day intervals, and expressed as counts per min 125I-labeled hormone bound/micrograms DNA. Specific FSH binding was minimal at day 4 and increased to a maximum at day 28, with the greatest rate of increase between day 4 and 16. hCG receptor content increased 6-fold between day 4 and 32, but contrasting with FSH, its greatest rate of increase occurred between day 16 and 28. The effectiveness of in vivo treatment with LH in inducing ovarian ornithine decarboxylase (ODC) activity, measured in 20,000 X g supernatants by the in vitro formation of 14CO2 from labeled ornithine, increased significantly during prepubertal development (days 21-33), paralleling the changes in hCG receptors. Ovarian hCG and FSH receptor content was also measured during the time of puberty. Specific hCG binding increased from anestrus (juvenile 32-day-old animals) to 1300 h of the first proestrus. The levels declined significantly by 1600 h of first proestrus and reached minimal values by the morning of estrus. Specific FSH binding also increased from anestrus to first proestrus, but to a much lesser degree, the levels showing only minor fluctuations during the rest of the cycle. hCG receptors measured in granulosa cells during puberty increased to an even greater extent than in the whole ovary; binding exhibited an 8-fold increase from anestrus to 1300 h of first proestrus and then declined to much lower values on estrus and the first diestrus. The capacity of LH to induce ovarian ODC activity increased markedly between anestrus and first proestrus, declining thereafter, and again paralleled the changes in LH receptor content. Evaluation of the kinetics of binding of hCG and FSH with their respective receptors in ovaries of 28-day-old rats revealed that the binding of hCG reached equilibrium within 4-8 h, whereas the binding of FSH attained equilibrium between 12-24 h. In both cases, however, the labeled hormones were clearly dissociable from their respective receptors by addition of an excess of unlabeled hormone. Moreover, although the ovarian receptor content of both hCG and FSH increased during prepubertal development, the equilibrium association constant of either hormone with its receptor remained unchanged. The results suggest that the infantile increase in FSH receptors may be induced by the high serum FSH levels present at that age and that, as shown by others in hypophysectomized rats, the subsequent increase in hCG receptors is, at least in part, an FSH-dependent phenomenon. The parallel changes in hCG receptors and LH-induced ODC activity during pre- and peripubertal development strongly suggest that an increase in LH binding capacity plays a fundamental role in the process of ovarian growth. It is also suggested that the changes in the steroidogenic ovarian response to hCG previously observed in peripubertal rats are, at least in part, determined by changes in ovarian LH receptor content.

Direct inhibitory effect of glucocorticoids upon testicular luteinizing hormone receptor and steroidogenesis in vivo and in vitro.

The direct effects of glucocorticoids on testicular LH receptor content and steroidogenesis were studied in vivo and in vitro. Immature hypophysectomized rats were treated with varying doses of dexamethasone, corticosterone, or a synthetic progestin, 17,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020). Some animals were also treated concomitantly with FSH to prevent the hypophysectomy-induced decrease in testis functions. At the end of 5 days of treatment, testicular LH/hCG receptor content was measured by [125I]hCG binding assay while steroidogenic responsiveness was measured by in vitro incubation of testes. Dexamethasone decreased testicular LH receptor in control and FSH-treated hypophysectomized rats in doses as low as 10 microgram/day, whereas corticosterone (10 microgram/day) decreased testicular LH receptor in the FSH-treated rats but had no effect in rats not treated with FSH. In contrast, R5020 had no effect on testicular LH receptor content. In vivo treatment of hypophysectomized rats with FSH increased both basal and hCG-stimulated production of androstanediol in vitro. In contrast, concomitant treatment with dexamethasone, but not R5020, decreased both basal and hCG-stimulated testicular androstanediol production. The direct effect of glucocorticoids on testicular steroidogenic potentials was also studied in primary culture of testicular cells obtained from adult hypophysectomized rats. Treatment of cultured testicular cells wtih hCG increased testosterone production. The addition of various natural and synthetic glucocorticoids, but not R5020, to hCG-treated cells decreased testosterone production in a dose- and time-related manner (triamcinolone greater than or equal to dexamethasone greater than cortisol greater than or equal to corticosterone). A 40% decrease in testosterone production was apparent at 6 h after addition of 10(-7) M dexamethasone to hCG-treated cells. These results demonstrate the direct inhibitory effect of glucocorticoids on testicular LH receptor content and steroidogenesis, suggesting the adrenal glucocorticoids may regulate testis functions.

Follicle-stimulating hormone induction of luteinizing hormone receptor in cultured rat granulosa cells: an examination of the need for steroids in the induction process.

The induction of LH receptors by FSH in cultured rat granulosa cells and the effects of ovarian steroids on this process were examined. Granulosa cells were isolated from the ovaries of untreated immature rats (25 days old) and cultured with highly purified FSH (Sairam; 250 ng/ml). After culture (48-96 h in chemically defined media), both the binding of [125I]hCG and the responsiveness (cAMP and progesterone production) to an acute LH stimulus (100 ng/ml; NIH B8) were measured. The appearance of LH/hCG-binding sites and LH responsiveness indicates the presence of functional LH receptors. The induction of LH receptors by FSH requires a lag period of 24-48 h. After 48 h, the concentration of LH receptors in cultured granulosa cells continues to increase with time in culture with FSH; the continuous presence of FSH is required to maintain the induction process. If granulosa cells are cultured without hormone for 24-48 h before FSH is added, no induction of LH receptors occurs. However, if 17 beta-estradiol (5 X 10(-7) M) is added during this initial period, then the cells are responsive to the later addition of FSH. This maintenance of FSH responsiveness is not observed when dihydrotestosterone or progesterone is substituted for 17 beta-estradiol in the initial culture period. The FSH-dependent induction of LH receptors in cultured rat granulosa cells can be blocked if an inhibitor of steroidogenesis, such as aminoglutethimide phosphate (AGP; 1 mM), is added along with FSH at the initiation of the culture. The inclusion of progesterone, dihydrotestosterone, or 17 beta-estradiol (but not 20 alpha-dihydroprogesterone) in the culture together with FSH and AGP will overcome the inhibition by AGP and restore the induction of LH receptors. The results suggest that steroids produced by the developing follicle can modulate the FSH-dependent induction of LH receptors, and this may play a role in the development of follicular responsiveness to LH.

Hyperprolactinemia-induced precocious puberty: studies on the mechanism(s) by which prolactin enhances ovarian progesterone responsiveness to gonadotropins in prepubertal rats.

Hyperprolactinemia induced in immature female rats by treatment with sulpiride, a dopaminergic receptor blocker, increased the in vitro release of ovarian progesterone (P) in response to different doses of both highly purified hCG and human FSH. The increased P response to gonadotropins was also observed in ovaries of animals injected with ovine PRL or in rats in which the hyperprolactinemic condition was induced by pimozide, a more typical dopaminergic receptor blocker. In addition, the pimozide treatment advanced the time of puberty in a manner similar to that previously observed with sulpiride. In other experiments in which only the ovarian response to hCG, but not that to FSH, was evaluated, it was found that the in vivo treatment of hypophysectomized immature rats with sulpiride did not modify the almost undetectable serum PRL levels of these animals and failed to increase the in vitro ovarian P response to hCG. By contrast, sc injection of PRL to hypophysectomized rats clearly enhanced the in vitro release of P in response to the gonadotropin. Adrenalectomy of otherwise intact rats significantly decreased the in vitro ovarian P response to hCG and blunted the increase in the P response induced by hyperprolactinemia. These effects, however, were almost completely reversed by concomitant corticosterone replacement therapy. The ovarian content of hCG receptor in normal rats was found to increase during juvenile development (days 22-31). Hyperprolactinemic animals showed a greater ovarian hCG receptor content than age-matched 31-day-old controls. In contrast, the FSH receptor contents were similar in both groups. The increase in hCG receptor content induced by hyperprolactinemia was even more clearly manifested in isolated granulosa cells, but, as in the case of the whole ovaries, the FSH receptor content in these cells remained essentially the same in hyperprolactinemic and control rats. The results indicate that the enhanced ovarian P response to hCG induced by PRL in prepubertal rats is, at least in part, mediated by an increase in the LH receptor content of the granulosa cells of the developing follicle. It also appears that the sensitizing effect of PRL on the prepubertal ovarian P response to gonadotropins is modulated by the adrenal gland through an effect exerted by corticosterone. The mechanisms by which PRL enhances the ovarian P response to FSH do not appear to involve changes in FSH receptors. However, the occurrence of enlarged uteri in hyperprolactinemic rats suggest that the PRL-induced increase in the P response to FSH may be related to the presence of more mature, estrogen-secreting follicles resulting from the hyperprolactinemic condition.

Activation of growth hormone short loop negative feedback delays puberty in the female rat.

Implantation of GH in the median eminence of the hypothalamus of 23-day-old female rats tonically inhibited serum GH levels throughout prepubertal development (days 23-36) and depressed GH diurnal pulsatile release. Puberty was significantly delayed in GH-deficient animals, a delay associated with a blunted in vitro ovarian steroidal responsiveness to gonadotropins (particularly estradiol) and a marked decrease in prepubertal uterine weight, as evaluated 4, 7, and 13 days after GH implantation. The prepubertal body weight increase was also depressed. Neither ovarian weight nor serum levels of LH, FSH, PRL, or TSH were consistently altered by the GH implant. In addition, evaluation of pulsatile PRL release in 33-day-old rats revealed no difference between control and GH-deficient animals. Ovarian LH receptor content was lower in GH-implanted rats than in controls, suggesting that a decrease in LH receptors may be one of the mechanisms by which a chronic decrease in serum gH depressed the prepubertal ovarian estradiol and, to a lesser extent, the progesterone response to gonadotropins. A direct ovarian site of action for GH was indicated by the results of experiments in which GH was administered to immature hypophysectomized estrogen-treated rats. The in vitro ovarian progesterone response of the GH-treated animals to both hCG and human FSH was distinctly increased by prior in vivo GH treatment. This effect of GH was not reproduced either by the in vivo administration of LH at a dose calculated by RIA to be contaminating the GH preparation or by FSH at a dose that induced a marked increase in aromatase activity in the same ovaries. GH treatment of hypophysectomized rats failed to affect either aromatase activity or hCG-induced estradiol release, indicating that GH does not directly facilitate the production of estradiol by the ovary. The fact that intact rats with GH implants did not actually show a decrease in the ovarian estradiol response to hCG but, rather, failed to show an increased response at the same time as control animals strongly suggests that ovarian maturation was delayed in GH-deficient rats. It is suggested that, in addition to PRL, GH may also play a role in the process of prepubertal reproductive development by enhancing the steroidal response of the ovary to gonadotropins.

Failure of short-term hyperprolactinaemia to increase the number of gonadotrophin receptors in the ram testis.

Hyperprolactinaemia was induced during March in 2-year-old rams which had regressed testes. Five animals were intravenously infused with prolactin for 3 days at a rate that raised concentrations of prolactin in plasma to levels equivalent to those generally found in summer. Five untreated animals acted as controls. The treatment did not change plasma levels of LH and FSH and had no effect on testicular content of LH and FSH receptors. These results indicate that prolactin is not directly involved in the acute regulation of gonadotrophin receptors in the ram testis.

LH(hCG) receptor in benign and malignant tumors of human ovary.

LH(hCG) receptors were identified with [125I]hCG in 38 benign and 26 malignant ovarian tumors of the human ovary. Eighteen per cent of all the benign and 27% of all the malignant tumors were LH(hCG) receptor-positive. Four of 12 benign serous tumors and 3 of 17 benign mucinous tumors displayed definitive binding of [125I]hCG. Two Brenner tumors failed to bind [125I]hCG. Six of 21 malignant epithelial tumors displayed definitive binding of [125I]hCG. Only one out of 4 malignant granulosa cell tumors bound [125I]hCG, while the other sex cord stromal tumors as well as one dysgerminoma failed to bind [125I]hCG. LH(hCG) receptor content in the benign and malignant receptor-positive tumors was low compared with the normal ovarian tissue. The presence of LH(hCG) receptors in certain benign and malignant ovarian tumors may be a sign of the gonadotropic control of these tumors. The possible applications of these findings for the diagnosis and treatment of human ovarian tumors is discussed.

Direct Inhibitory Effect of Gonadotropin-Releasing Hormone upon Luteal Luteinizing Hormone Receptor and Steroidogenesis in Hypophysectomized Rats*

The effects of gonadotropin-releasing hormone (GnRH) and its potent agonist [des-Gly10, D-Leu6-N alpha Me) Leu7, Pro9,NHEt-GnRH (GnRH-A)] on ovarian luteal functions maintained by PRL were studied in vivo and in vitro. Hypophysectomized, diethylstilbestrol-treated female rats were primed with FSH for 2 days, followed by an ovulating dose of LH or hCG. Two days later, ovarian luteal functions were maintained by daily injections of 250 microgram PRL for 3 days. PRL treatment increased the serum progesterone level from 13.0 +/- 0.5 to 298 +/- 24 ng/ml and increased the ovarian hCG-binding capacity from 5.8 +/- 1.3 to 584 +/- 86 ng bound hCG/ovary. In contrast, concomitant treatment with GnRH or GnRH-A resulted in dose-dependent decreases in the PRL-induced increase of serum progesterone and ovarian LH/hCG receptor content. GnRH at 100 microgram/day caused a 60% decrease in serum progesterone and an 80% decrease in ovarian LH receptor content, whereas GnRH-A was effective at a 1-microgram dose level. Neither GnRH nor GnRH-A affected the binding affinity (Kd) of ovarian LH receptor. The direct inhibitory effects of GnRH and GnRH-A upon granulosa-luteal cell function were also tested in vitro. FSH treatment for 2 days induced functional LH and PRL receptors in cultured PRL, increased (by approximately 3-fold) progesterone production by these granulosa-luteal cells, whereas concomitant treatment with GnRH-A inhibited progesterone production in a dose-dependent manner. Thus, these studes demonstrated that GnRH and GnRH-A exert direct inhibition on ovarian luteal functions by decreasing LH receptor and progesterone production in vivo as well as inhibiting progesterone production by cultured granulosa-luteal cells in vitro.

Gonadotropin-releasing hormone and its agonist inhibit testicular luteinizing hormone receptor and steroidogenesis in immature and adult hypophysectomized rats.

The direct effect of gonadotropin-releasing hormone (GnRH) and its agonist on testicular LH receptor and steroidogenesis was studied in hypophysectomized immature and adult rats. Hypophysectomized rats were treated daily with varying doses of GnRH or [des-Gly10,D-Leu6(N alpha Me)Leu7, Pro9-NHEt]GnRH(a potent agonist). Some animals were also treated concomitantly with FSH, PRL, GH and/or LH to prevent the hypophysectomy-induced loss of testicular LH receptor and steroidogenic capacity. At the end of 5 days of treatment, testicular LH/hCG receptor concentration was measured by a [125I]-hCG-binding assay and steroidogenic responsiveness was determinded by in vitro incubations. GnRH and the GnRH agonist reduced testicular LH receptor in control and FSH-treated hypophysectomized immature rats. As little as 0.5 microgram agonist/day induced a greater than 40% decrease in the LH receptor content, whereas GnRH was less potent, with 50 micrograms/day inducing about a 50% decrease. The inhibitory effect of GnRH was shown to be the result of decreases in the concentration of LH receptor rather than changes in the receptor affinity (Kd = 1.1 X 10(-10)M). GnRH did not interfere with the [125I]hCG receptor assay. Treatment with PRL, GH, and FSH, alone or in various combinations, increased the testicular LH receptor content. The stimulatory effect of these pituitary hormones was depressed by concomitant treatment with the GnRH agonist. Similar inhibitory effects of GnRH and the agonist on testicular LH receptor were demonstrated in adult hypophysectomized rats. In vitro studies demonstrated that treatment with the GnRH agonist in vivo inhibited both basal and hCG-stimulated androgen production in FSH-primed immature hypophysectomized rats. Associated with decreases in androgens (testosterone and androstenedione) and reduced androgens (dihydrotestosterone, androstanediol, and androsterone), there was marked suppression of 17 alpha-hydroxylated precursors and C-21 steroid intermediates in animals treated with the GnRH agonist, thus suggesting that the inhibitory effect of the GnRH agonist was associated with possible defects in 17 alpha-hydroxylase and side-chain cleavage enzymes. Likewise, treatment with the GnRH agonist inhibited in vitro testicular steroidogenic responses in adult hypopysectomized rats. These results demonstrate the extrapituitary inhibitory effect of GnRH on testicular LH receptor content and Leydig cell steroidogenesis in immature and adult hypophysectomized rats.

Evidence that changes in tonic luteinizing hormone secretion determine the growth of preovulatory follicles in the rat.

Physiological concentrations of progesterone (20-100 ng/ml), maintained by the insertion of implants into 30-day-old rats, delayed first ovulation, and withdrawal of progesterone on day 47 of age synchronized first ovulation in rats. Inhibition of ovulation involved negative feedback regulation of tonic LH and FSH secretion, blockage of gonadotropin surges, and suppression of preovulatory, but not antral, follicular growth. Removal of implants resulted in a rapid decline in serum progestrone from 100 to 5 ng/ml within 0-12 h. Between 0-36 h there were progressive increases in serum concentrations of LH and FSH, enhanced accumulation of estradiol by individual follicles incubated in vitro with or without exogenous substrate, and marked progressive increases in the content of LH (but not FSH) receptors in both thecal and granulosa cells. These events were followed by gonadotropin surges at 48 h (1800 h on day 49), ovulation, and morphological and biochemical signs of luteinization, including decreases in follicular gonadotropin receptor content and estradiol accumulation, evident by 60 h. With the exception of changes in basal LH, this sequence of events is remarkably similar in time and pattern to that after the decline of progesterone on diestrous day 2 and ovulation on proestrus of a 5-day cycle. Although a direct effect of progesterone on ovarian follicular cell function cannot be excluded, the data suggest that subtle but sustained increases in LH (and possibly FSH) are required for the enhanced follicular accumulation of estradiol and LH-binding activity occurring between diestrus and proestrus of the rat estrous cycle. Thus, perhaps some of the mystery surrounding the endocrine events between diestrus and proestrus can be ascribed to changes in serum LH that have been too small and/or variable for current nonserial sampling methods and RIAs to detect reliably.

Further Studies on the Inhibitory Effect of [D‐A1a6, des‐Gly‐NH210]LHRH Ethylamide on Spermatogenesis and Steroidogenesis in the Rat: Reversibility and Effect of Androgen Administration

Following the recent observation that chronic treatment with [D‐Ala6, des‐Gly‐NH210]LHRH ethylamide, a potent LHRH agonist, leads to an almost complete degeneration of seminiferous tubules in the rat, the time‐course of recovery of spermatogenesis after cessation of treatment was investigated. Adult rats injected with the LHRH agonist (100 ng, every second day) for four weeks were studied four, eight, and 16 weeks later. Although progressive improvement was noted from four to 16 weeks after cessation of treatment, about 25% of tubules still appeared completely degenerated at the end of the recuperation period. One month following cessation of treatment, testicular LH and prolactin receptor concentrations, testicular testosterone (T) and dihydrotestosterone (DHT) levels, and ventral prostate and seminal vesicle weights had returned to normal values. Testis weight, however, which was decreased by 40% one month after treatment with the LHRH analog, increased more slowly toward normal and was still at 85% of control value four months after treatment. Plasma LH and FSH levels, which were increased approximately 100% after treatment with the LHRH analogue, returned to within normal values between one and two months following cessation of treatment, respectively. Administration of androgens (T or DHT) concomitantly with the LHRH agonist only partially prevented the deleterious effects of the peptide on spermatogenesis. On the other hand, administration of the LHRH agonist had no effect on testicular morphology in hypophysectomized rats. These data show a rapid return to normal of the hypophyseal testicular elements responsible for androgen formation after desensitization by LHRH agonist treatment in adult rats. These results suggest that the inhibitory effect of chronic LHRH agonist administration on spermatogenesis is at least partially reversible, and that a decrease in androgen production is probably not the only factor involved in the inhibition of spermatogenesis by LHRH agonist treatment.

Regulation of Primate Testicular Luteinizing Hormone Receptors and Steroidogenesis

The testicular luteinizing hormone (LH) receptors of the rhesus monkey and human have many features in common, including high equilibrium association constant, marked species specificity, and relatively low binding capacity. We have, therefore, used rhesus monkeys as models for human LH-receptor regulation in vivo during gonadotropin treatment. In four adult male monkeys, treated with 10,000 IU human chorionic gonadotropin (hCG), serum and testicular steroidogenic responses were monitored at 24-h intervals during the following 4 d, and LH-receptor concentrations were measured by (125)I-hCG binding to 15,000-g particles prepared from testis biopsy specimens. In treated animals, serum hCG was maximal on day 1 at 322+/-16 ng/ml and declined to 24.4+/-2.3 ng/ml by day 4. Serum testosterone was increased threefold during the subsequent 4 d (from 6.5+/-2.0 to 18.6+/-4.4 ng/ml) but serum progesterone remained unchanged. In contrast, serum 17alpha-hydroxyprogesterone increased 12-fold to 5.5+/-0.5 ng/ml at day 1 and was increased fourfold during the subsequent 3 d. The LH-receptor binding capacity of the primate testis was reduced by 18.3+/-6.0% on day 1, 51.7+/-7.4% on day 2, and 45.3+/-2.4% on day 4. Occupancy of the LH receptors by endogenously bound hCG was significant on day 1 but was negligible by day 4. These data demonstrate that gonadotropin-induced LH-receptor depletion occurs in the rhesus testis and indicate that primate gonadotropin receptors are susceptible to the regulatory processes recently described in the rat. In addition, the simultaneous and disproportionate accumulation of 17alpha-hydroxyprogesterone indicates that 17,20-desmolase was rate-limiting under these conditions in the primate testis Leydig cell.

Mechanism of the Postcoital Contraceptive Effect of Luteinizing Hormone-Releasing Hormone: Ovarian Luteinizing Hormone Receptor Interactions*

LHRH, via inducing inordinately high LH levels, can disrupt pregnancy in the rat. Based on studies indicating that LH can reduce gonadal content of its own receptor and thereby modify LH-dependent steroidogenesis, the effect of contragestational LHRH treatment on ovarian LH receptor dynamics was investigated. Pregnant rats were administered sc 200 ng LHRH/day during days 1–7 (pre- and periimplantation) or days 7–12 (postimplantation) of pregnancy. Vehicle-injected animals served as controls. At 1-day intervals, beginning the day after the first injection and continuing until the day after the final injection, animals were sacrificed for determination of pregnancy state, ovarian LH binding, and, in the postimplantational group, serum hormone levels. LHRH treatment resulted in a marked decrease in the number of normal implantation sites as observed by day 6 in the preand periimplantational groups and by day 11 in the postimplantational group. Ovarian LH binding in both LHRH treatment groups was significantly decreased within 24 h after a single injection and continued to be depressed throughout the course of treatment. The degree of LH binding to isolated luteal homogenates from postimplantational animals autopsied on day 9 after 2 days of LHRH or vehicle treatment suggests that the differences seen using ovarian homogenates reflect changes in luteal LH binding. Scatchard analyses indicate that the decreased binding is due to a lower LH receptor concentration (LHRH, 0.2–0.7 × 1015 M/mg wet wt; controls, 3.9–4.4 × 10-15 M/mg wet wt) rather than a change in receptor equilibrium constants (LHRH, Kd = 4.2–6.8 × 10-10 M; controls, Kd = 8.3- 10.0 × 1O-10 M). In both LHRH groups, the progressive failure of pregnancy and depression of LH binding were associated with decreased serum progesterone levels. The results from these experiments suggest that the mechanism by which LHRH terminates pregnancy involves a hypersecretion of LH which produces a decrease in ovarian LH receptor content (downregulation) resulting in a disruption of ovarian steroidogenesis, thereby removing gestational support. (Endocrinology 105: 139,1979)

Ovarian Follicular Development during the Rat Estrous Cycle: Gonadotropin Receptors and Follicular Responsiveness1

The LH and FSH receptor content in thecal and granulosa cells of large antral follicles was measured by in vitro binding assays and in vivo autoradiography during the rat estrous cycle. Specific binding of l’25I]-hCG to granulosa cells increased from <1000 cpm/llg DNA on estrus, metestrus and diestrus to 6000 CPM/Mg DNA on proestrus. Specific binding of l’2511-hFSH to granulosa cells was consistently between 2000 and 3000 cpm/ g DNA throughout the cycle. Specific hCG binding to thecal cells also increased between diestrus and proestrus, while specific hFSH binding to thecal cells was essentially nondetectable. The increase in LH receptors in large antral follicles from diestrus to proestrus could be correlated with an increased ability of these follicles to produce cyclic AMP and estradiol during in vitro incubations with increasing concentrations (0.06-60 pg/mI) of 0LH. Incubation of antral follicles collected on metestrus, diestrus and proestrus with and without testosterone revealed a progressive increase in both follicular aromatase activity and endogenous androgen production. It is concluded that in the presence of low and unchanging LH and FSH concentrations from metestrus to proestrus large antral follicles become more responsive to LH. This responsiveness is associated with an increase in LH receptors both in thecal and granulosa cells and increased production of estradiol. Although the hormonal stimulus for the increase in granulosa cell LH receptor on proestrus is not known, it is suggested that increased follicular estradiol might play a pivotal role.

Prolactin, Growth Hormone, Luteinizing Hormone Receptors, and Seasonal Changes in Testicular Activity in the Golden Hamster*

In adult male hamsters, 2 months of exposure to a short photoperiod (5 h of light:19 h of darkness) caused testicular regression and a precipitous decline in plasma PRL, in agreement with earlier reports from other laboratories. Depressed release of PRL cannot be explained by a reduction in testicular steroidogenesis, because castration of males kept in a long photoperiod did not reduce PRL levels and administration of testosterone to males kept in a short photoperiod failed to reverse the decline in plasma PRL concentration. Treatment of such "regressed" animals with PRL, GH, or ectopic pituitary transplants stimulated growth of the testes and the accessory reproductive glands, increased the concentration of LH receptors in the testes, and elevated plasma testosterone levels. A single injection of 250 microgram PRL was sufficient to increase testicular LH binding, and chronic treatment with pituitary grafts completely reversed testicular regression. The effectiveness of exogenous PRL in stimulating testicular growth and LH receptors was significantly influenced by the timing of the injection. In some experiments, gonadotropin levels appeared elevated in animals injected with PRL, but these differences were not statistically significant. In hamsters with gonadal regression induced by exposure to a short photoperiod, daily administration of 20 microgram H and/or 150 microgram FSH had no apparent effect on testicular function. However, treatment with large doses of hCG and/or PMS gonadotropin resulted in significant stimulation of testicular growth and steroidogenesis. Chronic treatment of males maintained in a long photoperiod (14 h of light:10 h of darkness) with an inhibitor of PRL release, 2-Br-alpha-ergocryptine, resulted in a decreased weight of the testes and seminal vesicles. Administration of this inhibitor for a longer period (2 months) produced a significant increase in body weight but had little effect on testicular function. These results indicate that changes in the release of PRL (and possibly also GH) may plan an important role in mediating the effects of the photoperiod on testicular function in the golden hamster.