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Opioid peptides inhibit LH secretion and the opiate antagonist naloxone provokes increases in plasma LH levels by release of endogenous GnRH from the hypothalamus. To explore the effect of endogenously released GnRH on the mobilization of bioactive LH pools, the bioactive LH response to a single iv bolus dose of 20 mg naloxone has been evaluated and compared to the immunoactive pattern of the hormone in eight young normal male volunteers. Blood samples were withdrawn at 15, 30, 45, 60, 90, 120 min after naloxone injection and LH levels were measured by RIA and rat interstitial cell testosterone (RICT) bioassay. A significant increase in both bio and immuno active LH was observed in all subjects after 15–30 min (p < 0.05 to p < 0.001), reaching maximal levels at 30–60 min for both forms of the hormone. The time course of the bioactive LH response magnified the immunoactive LH pattern, and the maximum fold increases were 1.4 and 1.3 fold (62.4 ± 5.5 SE and 25.0 ± 3.7 SE mlU/ml) from basal bio and immuno LH levels of 25.9 ± 4.3 SE and 11.1 ± 2.0 SE mlU/ml respectively. An early single peak response of bio and immunoactive LH was observed in six subjects while a biphasic pattern was observed in two subjects with a clearly defined and prominent early pool followed by a second pool of higher magnitude. Both bio and iummunoactive LH levels began to decline at 45–60 min, but in most subjects remained significantly elevated by about 30% above the basal values at 120 min. The finding of a significant early bioactive LH increase gives further support to the clinical use of naloxone as a test to investigate the hypothalamic function in man.

The effects of estrogen upon testicular gonadotropin receptors and Leydig cell responses were studied. Immature hypophysectomized rats were treated for 7 days with 50 μg FSH, with or without diethylstilbestrol (DES). Daily injections of FSH induced a marked increase in testis weight; this weight increase was inhibited by concomitant DES treatment. FSH also induced an increase in testicular LH/hCG receptor content; the increase in [125I]iodo-hCG binding was not affected by daily injections of low doses (0.01 and 0.2 μg) of DES but was prevented by treatment with high doses (5 and 20 μg) of DES. In contrast, no significant change in testicular [125I]iodo-FSH-binding capacity was detected in any treatment group. Functional responses of the Leydig cells of hypophysectomized rats were studied by incubation of decapsulated testes with or without a saturating concentration of hCG. Daily treatment of FSH caused a marked increase in both basal and stimulated levels of testosterone production. In contrast, with or ...

The in vitro bioassay for LH/hCG employing isolated rat testes has been significantly improved by utilization of collagenase-dispersed interstitial cells in the presence of phosphodiesterase inhibitors. The sensitivity of the assay system has been extended to 20μIU (2 picograms) of hCG, and the index of precision of the method (λ) is less than 0.04. The high sensitivity of the interstitial cell assay permits bioassay of circulating gonadotropins in human plasma.

The development of an in vitro bioassay of high specificity, sensitivity and precision for the measurement of low circulating concentrations of biologically active glycoprotein hormones has offered exciting new insights into the in vivo secretion and metabolic clearance of luteinizing hormone (LH) in various pathophysiological states. Moreover, the most recent combined application of the rat interstitial cell testosterone (RICT) bioassay and a novel multiple-parameter deonvolution model has allowed investigators to dissect plasma concentration profiles of bioactive LH into defined secretory bursts, which have numerically explicit amplitudes, locations in time, and durations, and are acted upon by determinable subject- and study-specific endogenous metabolic clearance rates. Here, we have: (i) reviewed the ability of the endogenous GnRH pulse signal to regulate the in vivo secretion of biologically active LH molecules as assessed in the RICT and by deconvolution mechanics; (ii) demonstrated that low-dose exogenous GnRH pulses effectively mimic spontaneous bioactive LH pulsatility; (iii) investigated the role of endogenous androgen and estrogen in modulating bioactive gonadotropin secretion in men and women; and (iv) described significant alterations in endogenous LH bioactivity in puberty and healthy aging.

Ovarian and Leydig cell LH/hCG receptors purified to homogeneity were identified as a single protein of M, 80,000 and 90,000 respectively. The homogeneity of this protein was confirmed by microsequencing of the first 18 amino acids of the ovarian receptor. The unblocked N-terminal peptide consisted of NH 2 -R-E-L-S-G-S-R-X-P-E-P-X-D-X-A-P-D-G. These receptors are N-linked sialoglycoproteins which accounts for the size difference between testicular and ovarian receptors and may participate in the interaction with gonadotropin. Crosslinking of pure receptor with hCG with 125 I label in either subunit indicated significant interaction of α-hCG with the receptor, while β-hCG seems involved mostly through association and conformational influence on the α-subunit. Comparison of M r , derived from SDS with those from FPLC suggested that the native LH receptor are dimers of identical subunits. Autoradiographs of blotted receptors demonstrated that both monomeric and dimeric forms can bind hCG. Receptors from both tissues can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase and phosphopeptide maps were identical. Receptor occupancy by agonist leads to a conformational change which facilitates its phosphorylation during initial binding and reduces the rate of phosphorylation after more prolonged exposure to gonadotropin. Aggregation or dimerization of the hCG/LH receptors could promote clustering and or crosslinking of receptors in the membrane favouring the initial transduction steps in the action of these hormones.

Discrepancies between adenosine 3′:5′-cyclic monophosphate (cAMP) and steroid production have been frequently observed in isolated target cells stimulated by low concentrations of trophic hormone. This dissociation is particularly marked in the interstitial cells of the testis, where testosterone production is elicited by gonadotropin concentrations in the picomolar range. Because of these observations, and a disparity between steroidogenesis and protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activation in Leydig cells, the role of cAMP as a mediator of the acute steroidogenic response has been questioned. This problem has been further analyzed by assay of free and occupied cAMP-binding sites of the regulatory subunit of protein kinase in basal and hormone-stimulated cells. Free sites were measured by a [ 3 H]-cAMP-binding assay, and occupied sites were measured by radioimmunoassay of endogenous cAMP eluted from receptor protein. After stimulation of purified Leydig cells with 0.1-10 pM human chorionic gonadotropin, a dose-dependent decrease in available [ 3 H]cAMP-binding sites was observed, with no change in binding affinity. The reduction in cAMP-binding sites was equivalent to the increase in occupancy of cAMP receptors by endogenous nucleotide formed during gonadotropin action. Fractional occupancy of cAMP receptors rose progressively from basal values of 0.2-0.40 to full saturation as intracellular cAMP rose 10- to 30-fold during hormone stimulation. The testosterone dose-response curve was coincident with the initial part of the cAMP-receptor occupancy curve. These changes in endogenous cAMP binding to the regulatory subunit were accompanied by a significant increase in protein kinase activity in gonadotropin-stimulated Leydig cells. These observations provide direct evidence for the role of cAMP and protein kinase during hormonal activation of steroidogenesis in the Leydig cell by low concentrations of gonadotropin.

The metabolism of pregnenolone-7α-3H has been studied in vitro in an aldosterone producing tumor and in the adrenal tissue adjacent to the tumor. The predominant products in the incubation of the tumor tissue were progesterone, and lesser amounts of deoxycorticosterone and aldosterone, with no cortisol. The adjacent adrenal tissue converted pregnenolone to androstenedione and cortisol, with no aldosterone. Although 17-oxygenated steroids were produced by both tissues, the tumor failed to convert 17-hydroxyprogesterone to 11-deoxycortisol or cortisol. Androstenedione was formed in the tumor incubation but was not converted to an 11-hydroxylated product. These findings suggest that the biosynthetic pathways in the aldosterone secreting tumor were directed both by the presence of 18-hydroxylase system and by the relative absence of alternate pathways leading to cortisol or androgen synthesis.

The gonadal receptor for luteinizing hormone LH and chorionic gonadotropin was extracted in soluble form from a particulate binding fraction of the interstitial cells of the rat testis by treatment with the nonionic detergent Triton X-100. During binding studies with the soluble receptor and 125I-labeled human chorionic gonadotropin (hCG), receptor-bound and free forms of the hormone were separated by a double precipitation procedure with polyethylene glycol. The soluble gonadotropin receptors retained hormonal specificity and high affinity for LH and hCG, and showed rapid and reversible gonadotropin binding during incubation with 10-11 m 125I-hCG. The initial rate of binding of hCG by soluble receptors was higher at 34° than at 24° or 4°, but degradation of receptors occurred more rapidly at the higher temperature with corresponding loss of binding activity. The equilibrium association constant of the soluble hormone-receptor complex at 24° (0.5 to 1 x 1010 m-1) was detectably lower than that of the particulate receptors for hCG (2.4 x 1010 m-1). The optimum pH for gonadotropin binding was 7.4, and no effects of buffer composition, ionic strength, or calcium concentration upon binding were demonstrable. Exposure of particulate and soluble receptors to trypsin caused loss of gonadotropin binding activity, indicating the protein nature of an essential component of the receptor site. In addition, a significant role of phospholipid in the structural and functional properties of the receptor was suggested by the reduced binding activity observed after treatment of particulate and soluble receptors with phospholipase A, and by the aggregation which occurred after exposure of the soluble receptors to phospholipase C. Gel filtration and density gradient centrifugation of the free receptors, and the receptor-hormone complex formed by equilibration of the soluble receptors with 125I-hCG, were performed in solutions containing 0.1% Triton. The soluble receptor and receptor-hormone complex showed adsorption to Sepharose 6B during gel filtration, and were quantitatively bound by blue dextran. For these reasons, blue dextran could not be used as a front marker during gel filtration studies, and 0.01% bovine serum albumin was included in buffers employed for chromatography on Sepharose 6B. The distribution coefficient (Kav) of the receptorhormone complex on Sephadex G-200 was 0.09, and that of free hCG was 0.33. On columns of Sepharose 6B, the Kav of the free receptors and the receptor-hormone complex was 0.32, and that of free hCG was 0.56. By reference to the behavior of standard proteins during filtration on Sepharose 6B, the hydrodynamic radius of the receptor was calculated to be 64 A. Sucrose density gradient centrifugation showed that the sedimentation constant of the free receptor was 6.5 S, and that of the hormone-receptor complex was 7.5 S. Dialysis of the complex to remove Triton X-100 caused conversion to an 8.8 S form, but no aggregation occurred. The density of the 7.5 S hormone-receptor complex in cesium chloride gradients was 1.289. From these values, the molecular weights of the 6.5 S (free) and 7.5 S (combined) forms of the receptor were calculated to be 194,000 and 224,000, respectively, and the axial ratios (prolate) of the two forms were 12 and 10.2, respectively. The properties of the gonadotropin receptor extracted by Triton X-100 were consistent with those of a highly asymmetric molecule, predominantly of protein nature, with a minor but functionally important phospholipid component. The retention of high specificity and affinity by the solubilized gonadotropin receptors indicates that the receptor macromolecules possess relatively high conformational stability, and provides an approach to the structural analysis of the hormone binding site.

Aldosterone and corticosterone were assayed by double isotope derivative dilution in serial samples of adrenal vein blood and i n the entire gland after collection, in morphine-pentobarbital treated male rats. Each animal served as its own control, and the secretion rate was compared to the adrenal pool of each steroid to obtain the adrenal turnover rate. Continuous angiotensin infusion (3.6 μg/min) caused highly significant increases in the secretion of aldosterone, but no significant changes in corticosterone secretion. The fractional adrenal turnover rate of the glands showed a decreased turnover for aldosterone and no change for corticosterone. Angiotensin in pharmacologic dosage can selectively stimulate aldosterone secretion in the rat. (Endocrinology 83: 180, 1968)

The effects of removal of the terminal sialic acid and galactose residues of hCG on the immunological and biological properties of the hormone were studied in order to further clarify the role of the carbohydrate moieties in the action of glycoprotein hormones on their target tissues. hCG treated sequentially with neuraminidase and β–galactosidase was evaluated for biological in vivoactivity, for binding activity by radioimmunoassay and radioligand—receptor assay, and for in vitro biological activity by assay of testosterone production by the isolated rat testis. Metabolism of the asialo— and asialo—agalacto—hCG preparations labeled with 125I was compared with that of the intact hormone after intravenous injection. The asialo—agalacto—hCG preparation possessed biological activity in vivo of 2.3 IU/mg, comparable to that of the desialylated hormone (0.7 IU/mg) and considerably below that of the stored intact hormone (7400 IU/mg). By contrast, the ability of the modified hormones to compete with intact hCG ...

Aldosterone and corticosterone were assayed by double isotope derivative dilution in serial samples of adrenal vein blood and in the entire adrenal gland after collection, in groups of 4–6 intact, anesthetized male rats. Each animal served as its own control, and secretion rate was compared to the adrenal pool of each steroid to obtain the adrenal turnover rate. Chronic treatment with angiotensin-II-amide, 0.25 mg sc daily in oil for 14 days, caused highly significant increases in adrenal content and secretion of bothaldosterone and corticosterone. The fractional adrenal turnover of aldosterone decreased, whereas that of corticosterone increased during stimulation. Acute infusions of angiotensin produced similar results and the aldosterone response preceded that of corticosterone. These pharmacologic effects may indicate a role of angiotensin or a related substance in the secretion of aldosterone by the intact rat. (Endocrinology 82: 29, 1968)

Plasma testosterone levels and testis morphology were studied in 4 immature rhesus monkeys treated with human menopausal gonadotropin (hMG) in graded daily doses of 5–40 IU for 50–70 days. Testosterone values were measured twice weekly, and testis biopsies were taken before treatment, at each increase in dose, and at the end of treatment. Before treatment, testosterone levels were low and variable (range: 30–250 ng/100 ml) in all 4 animals, and biopsies of testis from 3 monkeys showed immature tubules containing only Sertoli cells and type A spermatogonia; relatively advanced spermatogenesis was found in the fourth animal. During administration of hMG in doses at or above 10 IU/ day, plasma testosterone rose to adult levels (600– 1600 ng/100 ml) and type B spermatogonia followed by spermatocytes appeared in biopsies from the three juvenile animals. After about 6 weeks of treatment, plasma testosterone in all 4 monkeys declined to pretreatment levels despite continued administration of hMG. The presence of...

Ten adult female rhesus monkeys were mated 11 days after the onset of menstruation. Blood samples were taken on the first day of mating and subsequently at 10-day intervals throughout gestation as well as one week postpartum. Plasma levels of 17β-estradiol and progesterone were determined by radioimmunoassay and competitive protein-binding assay, respectively. Continuous daily urine collections were made in early (days 7–35) and late (ISO days until 7 days post-partum) pregnancy for quantitation of chorionic gonadotropin and estrone excretion. Estradiol concentrations in plasma were elevated 3 times during the fertile cycle and pregnancy: (1) preovulatory, (2) at 20 days in pregnancy, and (3) during a plateau that was maintained from day 100 until term. The 20-day estradiol peak was associated with the time of highest chorionic gonadotropin activity. During the initial 40 days of gestation, peak levels of plasma progesterone were associated with nadirs in estradiol and vice versa. However, both progestero...

Cell monolayers were cultured from dissected rat seminiferous tubules in Eagle’s minimum essential medium for 7 days. The culture media were then replaced by fresh individual media containing pregnenolone-7α-3H, 17α-hydroxyprogesterone-7α-3H, dehydroepiandrosterone- 7α-3H and Δ5-androstenediol-7α-3H. After 48 hr incubation, the labeled steroid products were purified and identified by column, paper and thin layer chromatography, with derivative formation and crystallization to constant specific activity. The cell cultures showed minor conversion of pregnenolone to progesterone, 5α-pregnane-3,20-dione and dehydroepiandrosterone, and of 17α-hydroxyprogesterone to testosterone and androstenedione. By contrast, substantial conversions of dehydroepiandrosterone to testosterone and androstenedione, and Δ5-androstenediol to testosterone were observed. These findings show that cell cultures derived from seminiferous tubules possess the capacity for testosterone formation from proximate precursors and for reductive...

Radioimmunoassay of testosterone in plasma extracts purified by thin layer chromatography has been applied to the development of a highly sensitive and specific method for measurement of plasma testosterone. Antibodies to testosterone coupled to bovine gamma globulin via the 3 position were raised in sheep, and an antiserum of high association constant ( K = 4.5 × 10 9 M −1 ) was employed for assay. Isolation of plasma testosterone prior to assay was performed by thin layer chromatography, and extraction losses were corrected for recovery of tritiated testosterone indicator. For assay, antiserum (1:2500) and tritiated testosterone (40 000 dpm) were incubated at 4° for 16 h, followed by separation of bound and free tracer with 500 μg dextrancharcoal. The sensitivity of the assay was 10 pg, between-assay precision was ± 12% and testosterone recovery was 95–100%. Plasma testosterone levels in individual samples were similar to those obtained by competitive binding assay, and the levels observed in males (709 ± 143; 450–1100 ng/100 ml) and females 54 ± 22; 25–80 ng /100 ml ) were in agreement with those reported by other methods. The combination of radioimmunoassay and thin layer chromatography provides a sensitive and reliable method for the specific determination of testosterone in small plasma samples.

All glycoprotein hormone subunit preparations possess low biological activity when subjected to conventional bioassays. To determine whether the subunits of hCG and LH possess intrinsic biological activity, a variety of hCG and bovine or ovine LH subunits of known biological activity in vivo were assayed in vitro for target cell binding and steroidogenic activity in the rat testis. Binding-inhibition activity in the rat testis homogenate-125I-hCG radioligand-receptor assay system, and steroidogenic activity in the isolated rat testis, were found in all cases to be of similar magnitude to the biological activity of subunits measured in vivo by conventional bioassays. Because enhanced binding-inhibition or steroidogenic properties of isolated subunits were not demonstrable during the appropriate in vitro studies, it was concluded that the individual subunits do not possess preferential binding or activation functions in the dissociated state. In addition, the close agreement between the results of bioassay ...

The binding of 125I-Labelled human luteinizing hormone (LH) and chorionic gonadotropin (HCG) to the interstitial cell fraction of the rat testis has been applied to the development of a radioligand-receptor assay system for gonadotropins.

Measurement of testosterone production by the rat testis in vitro provides an extremely sensitive response system for the biological activities of luteinizing hormone (LH) and human chorionic gonadotrophin (HCG) activity in vitro. The sensitivity of the response to gonadotrophins depends upon the structural integrity of the isolated testis, as physical disruption by teasing apart the testis tubules results in substantial loss of the steroidogenic response to trophic hormones. The intact testis responds to as little as 0.2 ng HCG or 1 ng ovine LH, and shows maximal stimulation with 2 ng HCG or 10 ng ovine LH. Testosterone production in vitro was also markedly enhanced by Sepharose-coupled ovine LH and by dibutyryl cyclic AMP, consistent with the view that LH acts upon cell-membrane receptors to stimulate steroidogenesis in the testis via activation of adenyl cyclase. Desialylated gonadotrophins retained considerably greater biological activity in vitro than in vivo, indicating that the sialic acid residues are not essential for interaction of gonadotropins with their tissue receptor sites.

Agonist-induced changes in expression and mRNA levels of luteinizing hormone (LH) receptors were compared during stimulation of ovarian follicular maturation and luteinization by gonadotropic hormones. Three major species of LH receptor mRNA, 5.8, 2.6 and 2.3 kb, were present throughout differentiation and changed similarly, the 5.8 kb species being consistently more abundant than the smaller forms. The increased expression of plasma-membrane LH receptors in preovulatory follicles and luteinized ovaries and their homologous down-regulation during follicular and luteal desensitization were closely correlated with the steady-state receptor mRNA levels. The reappearance of LH receptors following desensitization during the luteal stage was preceded by an increase in mRNA levels. These studies have demonstrated that the expression of LH receptors during follicular maturation, ovulation and desensitization is related to the prevailing levels of receptor mRNA in the ovary.

Specific gonadotropin receptors of the rat testis were purified 15,000-fold after detergent extraction by a single affinity chromatography step on agarose coupled to human chorionic gonadotropin. Receptors were eluted in the free form at pH 3.2 and did not aggregate in the absence of detergent. The purified receptors displayed about 50% of the maximum theoretical binding activity, and retained the high binding affinity and hormonal specificity of the original gonadotropin receptors of the cell membrane.

Receptor sites for lactogenic hormones such as prolactin (PRL), human growth hormone (HGH), and placental lactogens, are widely distributed in mammalian tissues, including mammary glands1, steroid-secreting cells of the adrenal, testis, and ovary, and target cells of steroid hormone action such as liver, prostrate, and kidney2,3. Although the biological functions of lactogen binding sites remain uncertain, a relationship between prolactin and lipoprotein metabolism is implied by the occurrence of prolactin receptors in steroidogenic cells of the gonads and adrenal, and by the ability of prolactin to increase esterified cholesterol in the testis4. Recently, loss of testicular prolactin receptors has been observed following elevation of circulating luteinising hormone (LH) concentrations by the gonadotropin releasing hormone (GnRH) and its agonist analogues5,6. The hormone dependence of lactogen receptor sites in steroid-secreting cells was further analysed in rat testis, ovary, and adrenal glands after treatment with the respective trophic hormones, gonadotropin and ACTH. In each of these tissues, rapid and transient loss of lactogen receptors was observed after trophic hormone stimulation. These findings indicate that increased turnover of lactogen receptors is an important component of the target-cell response, and suggest that prolactin receptors might be involved in the transport of lipoprotein precursors for steroid biosynthesis.

GONADOTROPHIN receptor sites with high specificity and affinity for luteinizing hormone (LH) and human chorionic gonadotrophin (HCG) have been demonstrated in particulate and soluble interstitial cell fractions of rat testis1–4. Such hormone binding sites have been used in radioligand receptor assays for LH and HCG1,2 and in studies on the structural determinants of gonadotrophin binding to target tissue5–7. Other parameters of gonadotrophin action have been examined during incubation of intact rat testes with HCG by radioimmunoassay of the cyclic AMP and testosterone produced during trophic hormone stimulation in vitro8–11. Decapsulated intact rat testes are highly sensitive to gonadotrophic stimulation in vitro, responding to HCG concentrations as low as 0.1 ng ml−1 (10−12 M) with synthesis and release of testosterone and to higher concentrations with synthesis and release of cyclic AMP10. Release of cyclic AMP during incubation with HCG has been shown to reflect cyclic AMP synthesis by simultaneous measurement of 14C-adenine incorporation and radioimmunoassay of cyclic AMP9,10.

PEPTIDE hormone receptors have been studied in homogenates and membrane preparations from a variety of target tissues and in some cases have been shown to be associated with adenylate cyclase activity in such particulate fractions1-7. They have a moderately high stability to physical fractionation procedures; more detailed analysis of their properties has been impeded by the complexity and insoluble nature of the membrane-bound receptor sites, so that various techniques were used to obtain soluble forms of receptor for structure-function studies. Receptors for adrenocorticotrophic hormone were successfully dispersed into minute membrane fragments by sonication and pressure disruption of adrenal tumour particles in the presence of phosphatidylethanolamine3. Treatment with detergents such as ‘Lubrol’ and ‘Triton X-100’ has been used to prepare soluble forms of the cardiac β adrenergic receptor8 and the insulin receptors of liver and adipose tissue9.

Recent studies in several endocrine target cells have revealed moderate to marked dissociations between steroidogenesis and cAMP formation during hormone action (1–3). In the corpus luteum, low concentrations of LH or hCG have been found to evoke full steroidogenic responses with little or no detectable change in cAMP formation (4). However, a rise in protein kinase activity has been observed in corpus luteum slices incubated with gonadotropin concentrations which produce a dose-related stimulation of progesterone synthesis, albeit in the absence of a discernible rise in cAMP (5). Such findings suggest that an increase in cAMP synthesis or turnover is stimulated by even the lowest doses of hCG that evoke a steroid response, and that measurement of this change should be possible under. appropriate experimental conditions. A similar dissociation between steroidogenesis and cAMP during hormone action in the testis and adrenal has been recently resolved by optimization of incubation conditions and detailed analysis of cAMP levels in hormone-stimulated Leydig cells and and adrenal fasciculata cells (6,7). To evaluate the role of cAMP during hormone action in ovarian luteal tissue, we have analyzed the relationships between cAMP production and binding to protein kinase receptors, and stimulation of progesterone synthesis in isolated luteal cells.

We have investigated whether cholera toxin (CT)- or pertussis toxin (IAP)-sensitive G proteins are involved in ovine (o) PRL-stimulated mitogenesis in the lactogen-dependent rat Nb2 node lymphoma cell line. Addition of IAP to medium caused a biphasic effect on oPRL-stimulated cell number. Low doses (10(-3) ng/ml) enhanced (mean +/- SEM, 15 +/- 3%) whereas higher doses (greater than or equal to 10 ng/ml) inhibited (24 +/- 3%) mitogenesis stimulated by a submaximal dose of oPRL (0.1 ng/ml) compared to control values. The cAMP analog 8-bromo-cAMP also had a biphasic effect on cell division stimulated by submaximal doses of PRL. Low doses (10(-5) M) enhanced whereas higher doses (10(-3) M) inhibited Nb2 cell growth in response to PRL. Incubation with CT only inhibited oPRL-stimulated mitogenesis in a dose-dependent manner. Maximal inhibition (63 +/- 7%) occurred at a concentration of 10 ng/ml or more. Phorbol myristate acetate (PMA) enhanced mitogenesis stimulated by PRL alone and in the presence of either stimulatory or inhibitory doses of IAP, but PMA did not block IAP inhibition. In contrast, PMA had no effect on cells incubated with CT; the inhibition of PRL-stimulated cell division by CT remained unchanged. Lactogenic receptor-binding sites per cell and affinity were not significantly affected by PMA, IAP, or CT, suggesting a postreceptor mechanism of action. In summary, these data demonstrate that cAMP modifies PRL-stimulated Nb2 cell mitogenesis. The differences between IAP and CT (i.e. biphasic effect, degree of inhibition, and differential effect of PMA) suggest that these agents could also modulate PRL actions in the Nb2 cell through different mechanisms, including a cAMP-independent pathway.

Significant gonadotropin-like bioactivity, as measured by rat interstitial cell testosterone assay, is present in the serum and placenta of pregnant rats. The patterns of activity for this putative rat chorionic gonadotropin (rCG) in serum and placenta are distinct from those observed for rat placental lactogen (rPL) radioreceptor activity. Placental and serum rCG (and rPL) activities are substantially increased, as are placental weights, following removal of the maternal pituitary on day 12 of pregnancy. These findings strengthen the evidence for the existence of a rat CG and suggest a novel role for the maternal pituitary in regulating placental function during normal gestation.

Solubilization and characterization of particulate high-affinity gonadotropin receptors derived from the interstitial cell fraction of the rat testis [l-3] , and homogenates of the pseudopregnant rat ovary [4,5], has been performed by extraction with the non-ionic detergents Triton X-l 00, Lubrol PX and Lubrol WX. Binding studies with 1 25 I-labeled hCG have shown that such soluble preparations contain a single class of gonadotropin binding sites, with Ka = 0.6 X 10” M-’ and binding capacity of 0.02 0.13 X 1 O-l2 mol/mg protein [l-4] . The mean affinity constant of the solubilized receptors is significantly lower than that observed in the original particulate receptors (Ka = 2-4 X 10” M-‘) from which the Tritonextracted fraction was prepared. During incubation at 4°C and 34”C, the free or uncharged soluble receptors were found to be relatively unstable, probably due to enzymatic degradation of the gonadotropin binding site. By contrast the formed hormone-receptor complex was much less susceptible to degradation after solubilization, suggesting that the binding sites were stabilized by association with the trophic hormone [2,3,6]. More recently, serial measurement of the binding capacity of solubiked free receptors during storage have revealed the presence of a small proportion of high-affinity receptors with association constant similar to that of the parent preparation. The demonstration and possible significance of these high-affinity sites are described in the present report.

Hypophysectomy of prepubescent (3-week-old) rats prevented the pubertal development of testicular, but not pulmonary, angiotensin-converting enzyme (EC 3.4.15.1). Additionally, hypophysectomy resulted in a loss of testicular converting enzyme activity in 10-week-old rats that had achieved puberty and had developed enzyme activity. Hormone regimens consisting of FSH/LH (7.5 U/rat X day), hCG (10 U/rat X day), or testosterone (1 mg/rat X day) were employed to ascertain their ability to maintain activity in hypophysectomized rats. All three of the above hormone regimens, if initiated on the first day after hypophysectomy of 10-week-old rats, were capable of maintaining testicular converting enzyme activity. Centrifugal elutriation of dispersed testicular cells indicated that the majority of enzyme activity in mature rats was associated with the germinal cells, a result consistent with the data accumulated from the hormonal studies. Lastly, [3H]captopril bound specifically to cellular fractions enriched in germinal cells. The above studies suggest that the pituitary gland is required for the development and maintenance of testicular angiotensin-converting enzyme in the rat by stimulating steroidogenesis in the testes. Furthermore, the sensitivity of converting enzyme activity to androgen coupled with the centrifugal elutriation and [3H] captopril binding studies strongly support the notion that testicular converting enzyme is associated with germinal cells.

Activation and regulation of Leydig cell function is exerted primarily by LH, which is secreted in pulses of high biological activity and interacts with membrane receptors. Other hormones and factors secreted by the Leydig cell or from the tubular compartment can influence Leydig cell differentiation and acute or chronic actions of LH on steroidogenesis. Conversely, hormones produced in the Leydig cell could modulate tubular function (e.g. beta-endorphin, oxcytocin). The LH receptor has been purified to homogeneity in sufficient quantities to allow its peptide sequence to be determined and its gene structure to be elucidated as well as functional reconstitution studies to be performed. The LH receptor subunit of Mr 90,000 can be phosphorylated by cAMP-dependent protein kinase. The native receptor appears to exist in the membrane as a dimer of identical subunits associated by noncovalent interactions. It is likely that receptor dimerization and further aggregation are necessary for signal transduction to occur, and receptor phosphorylation by one or more kinases may be involved in regulating gonadotropin action. Stimulation of the androgen pathway occurs mainly through a cAMP-mediated mechanism. The stimulatory event can be negatively influenced by the action of certain peptide hormones through the guanyl nucleotide inhibitory subunit of adenylate cyclase. Such an inhibitory action of angiotensin has further emphasized the importance of the cAMP pathway in the Leydig cell. The hormone also appears to facilitate androgen production by a cAMP-independent mechanism located at the plasma membrane or intracellular sites. A Ca2+ sensitive kinase system is present in the Leydig cell membranes. The presence of nM amounts of Ca2+ induces membrane phosphorylation of a protein Mr 45,000. Adenylate cyclase activation also is affected by Ca2+. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH-induced actions in the activated Leydig cell membrane. In the adult rat testis, the ability of Leydig cells to respond to sustained gonadotropic stimulation with increased androgen production is limited by the development of a refractory state associated with loss of LH receptors and steroidogenic enzymes. Gonadotropin-induced steroidogenic lesions in adult rat testes include a late steroidogenic lesion at the site of conversion of progesterone to androgen and an early lesion before pregnenolone formation that leads to a decreased in vitro pregnenolone and testosterone response to hCG.

Publisher Summary This chapter discusses the gonadotropin receptors and regulation of steroidogenesis in the testis and ovary. The regulation of testicular and ovarian function by gonadotropic hormones is mediated by specific, high-affinity receptors that are located in the plasma membrane of the respective target cells. A common feature of receptors for gonadotropins and other protein hormones is their extremely high affinity for the respective ligands, with association constants of about 1010 M−1. The initial demonstration of gonadotropin receptors in the rat testis and ovary by isotopic tracer methods was performed by in vivo localization of radioiodinated lutenizing hormone (LH) or human chorionic gonadotropin (hCG) upon the LH receptor sites. The morphological evidence for the existence of gonadotropin receptor sites in the gonads has been complemented by a considerable body of in vitro data demonstrating that specific hormone binding sites are present in slices, cell suspensions, and homogenates of the testis and ovary. The location of LH receptors in the plasma membrane of the Leydig cell of the testis and the luteinized cells of the corpus luteum is also elaborated in the chapter.

Regulation of peptide hormone receptors and target cell responses by increased concentrations of the homologous hormone has been demonstrated in a number of endocrine-dependent tissues (1–6). This process, described variously as desensitization, tachyphylaxis, refractoriness, tolerance, and down-regulation, is particularly marked in the testis and ovary following exposure to elevated gonadotropin levels (5–12). The finding that insulin (1) and growth hormone (2) are able to regulate their own receptor sites in target cells was followed by the demonstration of an inverse relationship between ligand concentration and receptor number for several other hormones, including catecholamines (3), thyrotropin releasing hormone (4) and gonadotropins (5–12). Although such negative regulation of receptor sites is now recognized to be a wide-spread property of peptide hormones, it should be noted that certain hormones, such as prolactin (13) and angiotensin (14) exert positive regulation upon their receptor sites, with an increase in binding capacity of the target cells in response to elevated hormone concentration.

Cultured rat anterior pituitary cells were continuously perfused with medium 199 and exposed to short (5 min), intermediate (30 min), or long (6 h) pulses of a maximally effective concentration of gonadotropin-releasing hormone (GnRH). Assay of the effluent by radioimmunoassay and interstitial-cell bioassay revealed a biphasic response to GnRH and indicated that 3 pools of luteinizing hormone (LH) are present in the gonadotroph. A rapidly releasable peak of bioactive LH comprising about 2% of the total cellular LH was mobilized within 1 min of GnRH addition, lasted for 3–4 min and was independent of the duration of stimulation. The second, larger pool of bioactive LH varied from 15 to 50% of the total LH as the duration of exposure to GnRH was increased from 5 min to 6 h. A third LH pool comprising up to 50% of the total LH could be mobilized by 50 mM potassium but not by continuous GnRH treatment, due to refractoriness of the cells to prolonged stimulation by the decapeptide. In contrast, repeated pulses of GnRH evoked a series of biphasic LH peaks with profiles similar to that observed during a single response to GnRH, indicating that continuous exposure to GnRH is necessary for desensitization. Release of LH from the perfused cells was calcium-dependent and the bio-immuno ratio of the first and second pools of LH was similar. The in vitro secretion profile of cultured rat cells is comparable with the early and late phases of LH release observed in GnRH-infused man, but occurs much more rapidly and demonstrates heterogeneity of the LH release process at the level of the gonadotroph. The superfusion technique provides a powerful tool to further investigate the bioactivity of GnRH and its analogs for use in fertility control.

To characterize the physiological pattern(s) of bioactive LH secretion in normal women, serial blood samples were withdrawn at frequent intervals in each of six women at three different stages of the menstrual cycle. Plasma LH concentrations were quantitated in each sample by both rat interstitial cell testosterone ( RICT ) bioassay and immunoassay (RIA). When the resulting RICT and RIA LH profiles were systematically compared, we found that mean (and integrated) plasma concentrations of bioactive LH were approximately 2-fold higher than immunoactive LH levels at all stages of the menstrual cycle. In addition, unequivocal prominent pulsations of bioactive LH could be demonstrated in these women throughout the normal menstrual cycle. For all stages of the menstrual cycle, bioactive to immunoactive LH ratios within LH pulses were significantly (P less than 0.01) increased over these ratios in the interpulse periods. The frequency of bioactive LH pulses increased dramatically from 0.44 +/- 0.24 (+/- SD) pulses/h in the early follicular phase to 1.21 +/- 0.07 pulses/h in the late follicular phase (P less than 0.003), and then declined to only 0.25 pulses/h in the luteal phase (P less than 0.001). Notably, significant discordance existed between bioactive and immunoactive LH pulses, with 30% of immunoactive and 14% of bioactive LH pulses occurring alone. In conclusion, using the RICT , we demonstrated that biologically active LH is secreted in discrete episodic pulsations in normal women. Estimates of the bioactive to immunoactive LH ratio indicate that these pulses of LH are preferentially enriched in biologically active compared to immunoactive hormone. The properties of bioactive LH pulses are under physiological control, since the amplitude and frequency of bioactive LH pulses vary significantly in relation to phases of the menstrual cycle. Since significant discordance exists between immunoactive and bioactive LH pulsations in normal women, we suggest that estimates of the circulating concentrations of biologically active LH (rather than immunoactive LH alone) are necessary to characterize fully physiological patterns of LH secretion during the menstrual cycle.

It is well established that beta-endorphin has a regulatory influence on the reproductive function at the level of the hypothalamic-pituitary axis. However, recent immunohistochemical evidence demonstrated that beta-endorphin is also present in the Leydig cells of fetal, neonatal and adult mice and hamsters. In addition, beta-endorphin synthesis was localized in the Leydig cells of adult rats, leading to the hypothesis of a direct function of the peptide in the reproductive organs. Our interest was to investigate the role of beta-endorphin at testicular level. We have demonstrated the presence of high-affinity opioid binding sites (Kd in the nanomolar range) in tubular homogenates and Sertoli cells in culture of adult (50 days) and immature (18 days post-natal) rat testes. Also, chronic beta-endorphin treatment of the Sertoli cells significantly inhibited basal and FSH-stimulated androgen-binding protein production, this effect being prevented by the universal opiate antagonist naloxone. No opiate binding was observed on Leydig cell cultures. Furthermore, we have demonstrated that acute or chronic beta-endorphin treatment does not affect testosterone production by Leydig cells in vitro, consistent with the absence of receptors on these cells. On the other hand, fetal Leydig cells (21 days fetal life) in culture produced considerable amounts of beta-endorphin. Also, fetal Leydig cells represented a preferred in vitro system to study beta-endorphin release since in adult cell culture a marked degradation of the peptide was detected (greater than 50%). beta-endorphin accumulation for 3 and 5 days was markedly increased by inhibitors of steroid biosynthesis (1.5-fold); a significant reduction by GnRH at both days (by 50-30%) was observed, while by dexamethasone the reduction was only noted after 5 days of treatment (by 50%). Acute stimulation (3 h) of control cells with hCG enhanced by 10-12-fold the beta-endorphin secretion. The hormone stimulation of beta-endorphin production was not mediated by testosterone. On the contrary, inhibition of Leydig cells steroid biosynthesis markedly increased basal and hCG-stimulated beta-endorphin production (150-200%), suggesting autocrine negative modulation of Leydig cell beta-endorphin by androgen and/or its metabolites. In contrast, dexamethasone reduced basal and hCG-stimulated beta-endorphin production (by 50%). Altogether these findings indicate that beta-endorphin produced within the Leydig cells may behave as a paracrine inhibitor of the Sertoli cell function and demonstrate that the peptide production is under direct control by gonadotropins and is modulated by steroids.

Purified testicular and ovarian luteinizing hormone/human chorionic gonadotropin (hCG) receptors are phosphorylated at serine and threonine residues by the catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). Occupancy of the receptors by hCG significantly increased the rate but not the extent of phosphorylation. However, prolonged preincubation of receptors with hCG reduced the subsequent rate of receptor phosphorylation. Identical phosphopeptide maps were obtained for the phosphorylated ovarian and testicular receptors. The phosphorylated receptor, like the native receptor, bound to wheat germ lectin and hCG-Sepharose and migrated as a single band of Mr 90,000 (testis) and Mr 85,000 (ovary) on NaDodSO4/PAGE. Neuraminidase treatment of receptors caused reductions of molecular weight to 82,000 (testis) and 77,000 (ovary), and further treatment with O-Glycanase had minimal effect on molecular size. However, deglycosylation with N-Glycosidase and endoglycosidase F produced a single labeled polypeptide of Mr 59,000 for both gonadal receptors. Treatment of native receptors with neuraminidase caused no apparent change in binding of gonadotropin to blotted receptors, whereas deglycosylated receptors showed a major reduction in hormone binding. These results indicate that luteinizing hormone/hCG receptors are sialoglycoproteins with predominantly N-linked glycosyl residues that account for the size difference between testicular and ovarian receptors and that may participate in the interaction with gonadotropin. Receptor occupancy by agonist leads to a conformational change that facilitates its phosphorylation during initial binding and reduces the rate of phosphorylation after more prolonged exposure to hCG.

The interaction of [125I]hCG with the primate LH receptor was investigated in particulate fractions and cell suspensions from human and rhesus testicular tissue. Binding to testis homogenates was saturable, dose and time dependent, and displaced by low concentrations of unlabeled human LH and hCG. Both the human and rhesus LH receptors were of high affinity (Ka = 2-3 X 1010 M-1) and low capacity (23-146 fmol/g), and both failed to cross-react with heterologous LH at concentrations up to 1 μg/ml. In contrast, the rat testis LH receptor interacted with several heterologous LH preparations in nanogram concentrations. The species specificity of the primate LH receptor was also demonstrated by recombination studies with isolated subunits of porcine LH and hCG. The presence of the β-subunit of hCG, irrespective of the recombined α-subunit, conferred the ability to inhibit binding of [125I]hCG by the primate testis homogenates. The porcine β-subunit, when recombined with the α-subunit of hCG, failed to ...

Rat interstitial cells were fractionated by centrifugal elutriation to facilitate the purification of Leydig cells for analysis of mechanisms of gonadotropin action in vitro. By this procedure, 10(9) collagenase-dispersed interstitial cells from adult rat testes were separated into 12 fractions in about 1 h. Fractions 1-7 (sedimentation velocities, 1.9-12 mm/h.g) comprised 80-85% of the total cells applied, including erythrocytes, lymphocytes, germinal cells, macrophages, endothelial cells, damaged Leydig cells, and contained less than 4% intact Leydig cells. Fractions 8-12 (sedimentation velocities, greater than 12 mm/h.g) comprised 15-20% of the original cells and contained 90-95% intact Leydig cells. Despite their different sedimentation velocities, the Leydig cell-rich fractions were similar in their LH receptor content (mean +/- SD, 36,115 +/- 4,815 sites/cell) and showed similar 5-fold increases above the original cell preparation in testosterone and cAMP responses to hCG. The pooled Leydig cell-rich fractions (8-12) were further resolved on 16-24% Metrizamide gradients into 5 bands. Bands II-V (density range, 1.075-1.110 g/ml) contained pure Leydig cells, and band I (1.048 g/ml) contained pachytene spermatocytes, the contaminating cell type present in the Leydig cell-rich fractions obtained by elutriation. Each of the 4 Leydig cell-rich bands showed similar morphology and functional activity. Essentially similar results were observed using 14-32% Metrizamide gradients. Leydig cells desensitized in vivo by hCG treatment and isolated by elutriation were also resolved by Metrizamide gradients into 4 bands, but showed a redistribution in the gradient, due to the shift of about 50% of the cells originally present in the heaviest bands to lower density fractions. However, in spite of their changes in density, the Leydig cell bands still showed similar degrees of receptor down-regulation and impairment of the steroid responses to hCG in vitro. This study has demonstrated that centrifugal elutriation is a rapid and effective method for obtaining large quantities of purified (greater than 90%) and active Leydig cells. Further resolution of the Leydig cell-rich fractions in Metrizamide gradients has allowed complete Leydig cell purification, which is not achieved by density gradient centrifugation alone. Since less responsive or inactive Leydig cells displayed various degrees of structural damage, such cells should not be considered as a population of physiological significance.(ABSTRACT TRUNCATED AT 400 WORDS)

Aromatase activity during gonadotropin action in vivo and in vitro was examined in purified Leydig cells to further define the early effects of LH and for elucidation of the enzymatic processes involved in the development of late lesions of androgen biosynthetic pathway. Aromatase was measured by the tritiated water release method using [1 beta-3H]testosterone as substrate. The enzyme activity was proportional to the amount of cells (0.1-1.0 X 10(6] incubated, increased with the incubation time (2-60 min), was inhibited by androstatriendione (ED50, 5.0 microM), and showed a Km for testosterone of 1.69 microM. Aromatase activity was stimulated (10-20%; P less than 0.05) 1 h after treatment of rats with a single sc dose of 5 micrograms hCG. This activation preceded the late steroidogenic lesion at the site of 17 alpha-hydroxylase and 17,20-desmolase activity by 2-5 h. A RIA of improved sensitivity (0.5 pg) was developed to detect the very low cellular and secretory levels of estradiol. The testicular contents of testosterone and estradiol showed small increases (P less than 0.05) within 40 and 60 min after hCG treatment, respectively. Testicular testosterone levels reached a peak by 1 h after injection and preceded the peak level of estradiol formation by 2 h. After in vitro treatment of cultured Leydig cells with 100 ng hCG, the aromatase activity was significantly increased within 30 min (P less than 0.05) and then returned to control levels for up to 16 h of culture. A similar temporal pattern for enzyme activation was observed after treatment of cultures with 8-bromo-cAMP or forskolin (23-27% above control; P less than 0.05), while cholera toxin stimulated aromatase activity at 2 h. Net testosterone accumulation in the incubation medium increased 30 min after the hCG treatment and reached a plateau by 4 h. A small but significant increase in estradiol levels (P less than 0.05) was also observed at 30 min, remaining constant until 120 min, which was followed by a sharp rise parallel to that of testosterone. These results suggest that the estradiol-mediated desensitization of the Leydig cell observed after hCG administration is consistent with an early cAMP-dependent activation of aromatase and a further rise in estradiol formation due to increased substrate availability.

In order to define the early lesion (before pregnenolone formation) of the androgen biosynthetic pathway induced by human CG (hCG) or LH in the Leydig cell, we initially have optimized the use of aminoglutethimide to obtain maximal and sustained inhibition of steroidogenesis in vivo and in vitro. Aminoglutethimide inhibited Leydig cell steroidogenesis in vitro at a dose of 100 micrograms/ml. The minimal serum concentration of aminoglutethimide necessary for maximal inhibition of testosterone in vivo was also 100 micrograms/ml (1 h after the ip injection of 20 mg aminoglutethimide). However, testosterone levels were normal 12 h later, coincident with a marked fall in the serum aminoglutethimide levels. The t 1/2 of the circulating aminoglutethimide was 5 +/- 0.7 h on the first day of treatment but was reduced to 3.0 +/- 0.4 and 2.25 +/- 0.35 h at 2 and 3 days of treatment. At the dose eliciting maximal and sustained steroid inhibition (60 mg/day) aminoglutethimide was able to prevent the estradiol-dependent late steroidogenic lesion (after pregnenolone formation) induced by 1 microgram hCG, with no effect on the early lesion (before pregnenolone formation) caused by 10 micrograms hCG. The aminoglutethimide-induced in vivo accumulation of cholesterol in the inner mitochondrial membrane (by 50%) was associated with an increase in the production of testosterone and pregnenolone by the Leydig cell when subsequently incubated in vitro. Similar increases in the steroidogenic capacity were observed after initial exposure of Leydig cells to aminoglutethimide in vitro, even after acid wash to remove the surface-bound endogenous LH. The steroidogenic cholesterol was also increased in desensitized Leydig cells (by 50-70%); however, the conversion of cholesterol to pregnenolone was substantially blocked in animals with the early lesion. Our findings define the requirement of increasing high levels of aminoglutethimide to inhibit cholesterol metabolism and provide a dose schedule suitable for studies on cholesterol availability and inhibition of steroidogenesis in the rat. These results support our proposal that the early lesion observed in desensitized Leydig cells is due to inhibition of the side-chain cleavage activity rather than to a decrease in the amount of metabolically available cholesterol.

Measurements of immunoactive and bioactive serum LH in the rat were performed after optimization of both RIA and rat interstitial cell-testosterone (RICT) assay to measure the low circulating LH levels of intact male rats. The effective sensitivity of the RIA was increased 3- to 5-fold to 6.2 or 4.7 ng RP1/ml by extraction of 1--1.5 ml serum, respectively. Serum LH levels of adult male rats were 48.5 +/- 16.3 ng RP-1/ml (n = 50), became undetectable after hypophysectomy, and were suppressed 50% after estrogen treatment. For RICT assay of serum rat LH, sensitivity was increased 5-fold by reduction of the incubation volume to 0.35 ml containing 2--4 x 10(6) interstitial cells/tube, with a detection limit of 3 ng RP-1 or 0.1 ng pure LH. The within-assay coefficient of variation for measurement of a pool of control male rat serum (126 +/- 19l6 ng RP-1) WAS +/- 12.5% and the between-assay variation was +/- 15%. Bioactive serum LH levels in adult male rats were 124.8 +/- 32.3 ng RP-1/ml or 2.74 +/- 0.71 ng rat LH/ml, with biopotency to immunopotency (B:I) ratios of 2.8 +/- 0.2 and 1.1 +/- 0.1, respectively, that were unchanged during elevations of serum LH by LHRH administration. Pituitary LH content was 726 +/- 183 micrograms RP-1 or 16.4 micrograms rat LH/gland, with B:I ratios of 2.3 +/- 0.4 and 1.0 +/- 0.2, respectively. The difference between B:I ratios in assays employing pure rat LH and assays using the RP-1 preparation was attributable to the presence of alpha-subunit and biologically inactive material in the RP-1 standard. Precise measurements of immunoactive and bioactive rat LH in male rat plasma can not be performed by these modified RICT and RIA procedures. The sensitive RICT assay can also be applied to the measurement of low levels of LH in the serum of primates and other species.

We have isolated and characterized a P-45017 alpha cDNA fragment from a rat testis library. The partial length rat P-45017 alpha cDNA (1Kb) has high overall nucleotide and deduced amino acid similarity with human and bovine P-45017 alpha cDNA's and contains the conserved tridecapeptide and heme regions, the termination codon and polyadenylation site. Using this rat testis cDNA probe we measured P-45017 alpha mRNA levels of rat Leydig cells from animals treated with hCG. Temporal studies with a low hCG dose showed an early increase in mRNA levels returning to control values at later times, while a higher desensitizing dose caused a marked reduction in the mRNA (24 h) and a small recovery at 48 h. Fetal rat Leydig cells maintained in the presence of LH treated with estradiol showed a 70% decrease in P-45017 alpha mRNA levels and testosterone production followed closely the changes in P-45017 alpha mRNA. These studies suggest that gonadotropin stimulation and desensitization of P-45017 alpha dependent enzymes in the adult rat testis as well as estradiol induced desensitization in fetal Leydig cells are related to levels P-45017 alpha mRNA.

Plasma chorionic gonadotropin levels were measured by three different assay methods during early pregnancy in four patients following induction of ovulation with Pergonal and hCG. Radioligand-receptor assay of unextracted samples was subject to non-specific interference by plasma proteins, causing an apparent elevation of gonadotropin levels during the first few days after fertilization. By contrast, the gonadotropin values measured by a highly sensitive LH/hCG bioassay were consistent with those obtained with a sensitive and specific radioimmunoassay for hCG, and showed that the first significant rise in plasma hCG occurred 9 to 13 days after ovulation. These results indicate that hCG does not appear in the maternal circulation until after the initiation of implantation of the blastocyst.A detailed study of the changes in plasma human chorionic gonadotropin (HCG) levels during early pregnancy was performed. Plasma HCG values, as determined by radioligand-receptor assay, were compared with those of radioimmunoassay and with a sensitive bioassay capable of measuring plasma levels of luteinizing hormone and HCG. Subjects were 4 women with secondary amenorrhea of hypothalamic origin. They were treated with human menopausal gonadotropin (Pergonal) followed by 10,000 IU of HCG to induce ovulation. Blood samples were collected at the time of HCG injection and at intervals of 1-2 days thereafter. Details of techniques used are given. Plasma proteins caused a nonspecific interference 1st significant rise in plasma HCG occurs 9-12 days after ovulation. Results indicate that HCG does not appear in the maternal circulation until after the implantation of the blastocyst.

We have characterized opioid binding sites in the Sertoli cells of adult and 18-day-old rat testes. Maximal specific etorphine binding was attained after 30 min at 4 C. The binding was reversible, with association and dissociation rate constants of 0.98 X 10(5) M-1 min-1 and 0.33 min-1, respectively. Scatchard analyses and saturation curves revealed a single class of high-affinity, low-capacity binding sites. No opioid binding was observed in Leydig cell cultures. Exposure to opioids for 3 days caused a significant increase in [3H]etorphine specifically bound to the Sertoli cells that was completely prevented by naloxone, demonstrating opioid up-regulation of its own receptor. Chronic opioid treatment of the cultures significantly inhibited androgen-binding protein production, and this effect was prevented by naloxone. Since the circulating concentrations of endorphins (10(-12) M) are lower than the Kd of testis opiate receptors, it is conceivable that opioids of Leydig cell origin act on the specific high-affinity receptors of the Sertoli cells, and may play a role in modulating their function.

Administration of human chorionic gonadotropin (hCG) to male rats was followed by dose-related changes in luteinizing hormone (LH) receptors in the testis. After treatment with a low dose of hCG (10international units), the number of LH receptors increased slightly over the first 24 hr, then fell to about 30% of the control value. These changes occurred with occupancy of only 8% of the available receptors, and were initially accompanied by increased basal testosterone production in vitro with no change in basal 3'5'-cyclic AMP production. During stimulation with hCG in vitro, such testes showed a transient decrease in cyclic AMP response on the first day after gonadotropin treatment and no change in testosterone response. By contrast, a 20-fold higher dose of hCG caused more rapid and complete loss of LH receptors, with major and transient occupancy of receptors at 24 hr and marked elevations of basal cyclic AMP and testosterone production in vitro. The initial occupancy of receptors was accompanied by a rapid fall in the cyclic AMP response to hCG in vitro, and was followed by marked receptor loss and inhibition of the cyclic AMP response for up to 5 days. The testosterone response to hCG in vitro was completely inhibited for about 3 days, then rose to the control level at 5 days, when only a small proportion of the original receptor sites and cyclic AMP response had begun to return. Such complete recovery of the steroidogenic response when only a fraction of the receptor population had returned was consistent with the presence of receptor reserve or "spare" receptors in the testis. These studies have demonstrated that negative regulation of LH receptors by exogenous gonadotropin is accompanied by consequent changes in cyclic AMP and testosterone responses to hCG in vitro. Hormone induced desensitization of interstitial cell responses was initially related to occupancy of LH receptors and later to a protracted loss of receptor sites.