Effects of follicle-stimulating hormone and testosterone on receptors of follicle-stimulating hormone in the testis of the immature Japanese quail.

Binding of radioactive rat follicle-stimulating hormone (FSH) to a particulate fraction of testicular homogenate of Japanese quail cockerels increased progressively with age when the cockerels were reared under long-day photoperiods from the day of hatch. The binding per unit weight of tissue (density of binding) showed a rapid increase from Day 23 to 29. It showed a decrease during the period between Days 29 and 36 when active spermatogenesis took place. The binding per testes (total binding) increased during the period from Day 23 to 36, Both density of binding and total binding remained low in cockerels reared under short-day photoperiods. Injections of testosterone to short-day cockerels at 1.0 mg/day for 3 days increased the density of binding 1.3-fold, but did not increase the testicular weight significantly. Injections of NIH-FSH-S12 at 16 μg/day for 3 days did not increase the density of binding significantly but increased the testicular weight 1.7-fold. The total binding was increased 1.8-fold by this treatment. Combined administration of FSH and testosterone increased the testicular weight 2.3-fold, the density of binding 1.7-fold, and the total binding 3.9-fold. The Scatchard plot analysis of the binding suggested that the hormonal treatment increased the number of binding sites but did not influence the affinity of binding. Histological examination of the testis indicated that the increase in testicular weight by hormonal treatment was mainly due to hypertrophy of the Sertoli cells. These results suggest that FSH induces, by acting synergistically with testosterone, its own receptors in the testis and consequently increases the sensitivity of the testis to FSH. This illustrates a kind of self-potentiating action of FSH. This self-potentiation mechanism, in addition to the synergism between FSH and testosterone, most likely enables the extremely rapid increase in testicular weight in photoperiodically stimulated birds.     

Somatomedin-C enhances induction of luteinizing hormone receptors by follicle-stimulating hormone in cultured rat granulosa cells

Recent studies have documented the ability of somatomedin-C (Sm-C) to synergize with FSH in the induction of rat granulosa cell progesterone biosynthesis. This direct cytodifferentiative action of Sm-C proved distinct from its established mitogenic property. To determine whether Sm-C partakes in the differentiation of granulosa cell functions other than progesterone biosynthesis, the role of Sm-C in the acquisition of LH receptors was investigated in vitro. Granulosa cells were obtained from immature, hypophysectomized diethylstilbestrol-treated rats and cultured under serum-free conditions for up to 72 h. Specific granulosa cell LH/hCG binding under basal conditions was relatively low and was not significantly affected by treatment with highly purified Sm-C (50 ng/ml). In contrast, treatment with FSH (oFSH; NIH FSH S14; 20 ng/ml) produced a 3.8-fold increase in LH/hCG binding to 3167 +/- 660 (+/- SE) cpm/10(6) cells. However, concurrent treatment with Sm-C (50 ng/ml) resulted in a 6.1-fold enhancement of the FSH effect. The ability of Sm-C to synergize with FSH in the induction of LH receptors was dose and time dependent, with an apparent median effective dose (ED50) of 6.2 +/- 0.6 (+/-SE) ng/ml, and a minimal time requirement of 24 h or less. Examination of the binding parameters of the LH/hCG receptor revealed that the Sm-C-mediated increase in LH/hCG binding was due to enhanced binding capacity, but not affinity (Kd = 4.4 X 10(-11) M). Decreasing dilutions (1:128,000-1:8,000) of a monoclonal antibody raised against Sm-C (sm 1.2), produced progressive and virtually complete immunoneutralization of the synergistic interaction between Sm-C and FSH, suggesting specificity of action. The ability of Sm-C to enhance LH receptor induction was associated with increased hCG-stimulated progesterone biosynthesis, suggesting that the newly acquired receptors are functional in nature. Our present findings indicate that nanomolar concentrations of Sm-C are capable of enhancing FSH-stimulated LH/hCG-binding capacity, but not affinity, in a time- and dose-dependent fashion. These observations suggest that the direct cytodifferentiative effect of Sm-C is not limited to the induction of progesterone biosynthesis, but other key granulosa cell functions may also be subject to modulation by this regulatory peptide.     

Evidence for the role of phospholipids in follicle-stimulating hormone binding to membrane-bound and soluble receptors from calf testis

The role of phospholipids in the interaction of FSH with receptors in the calf testis was explored by studying the effects of phospholipase-C (PL-C) on the binding of radioiodinated human FSH ([125I]iodo-hFSH) to three previously characterized but different preparations of FSH receptor: a membrane fraction derived from calf testis homogenate, a buffer-soluble receptor present in the cytosol of the calf testis homogenate, and a detergent-soluble receptor prepared from the membrane fraction by extraction with Triton X-100. Prior incubation with PL-C markedly reduced specific [125I]iodo-hFSH binding to the membrane-bound and buffer-soluble receptors. This loss in binding was associated with hydrolysis of phospholipids, was prevented by the addition of o-phenanthroline, an inhibitor of PL-C, but not by the addition of a protease inhibitor, and could not be reproduced by the addition of the products of PL-C hydrolysis. Enzyme treatment did not dissociate [125I]iodo-hFSH already bound to the buffer-soluble receptor and dissociated only 20% of [125I]iodo-hFSH bound to membrane receptor. PL-C treatment did not reduce [125I]iodo-hFSH binding to detergent-solubilized receptor, nor did it hydrolyze constituent phospholipids. The apparent resistance of the detergent-solubilized receptor to PL-C treatment was studied by incubating membranes with or without PL-C before detergent solubilization. Triton X-100 itself (2%) inhibited phospholipid hydrolysis by PL-C, but this could be overcome by reducing the detergent concentration 10-fold by dilution. Despite a marked decrease in specific [125I]iodo-hFSH binding to PL-C-treated membranes compared to that of untreated controls, the total amount of [125I]iodo-hFSH binding to Triton X-100 extracts of each membrane preparations was not significantly different. Addition of Triton X-100 to the buffer-soluble receptor restored 40% of the hormone binding that had initially been lost concomitant with enzymic hydrolysis of membrane phospholipids. It appears that constituent phospholipids play an important role in the interaction of FSH with membrane-bound or solubilized receptor and that Triton X-100 is able to substitute, although imperfectly, for phospholipids in that regard, probably because of its related amphipathic properties.     

Estrogens enhance the adenosine 3',5'-monophosphate-mediated induction of follicle-stimulating hormone and luteinizing hormone receptors in rat granulosa cells

The effects of estrogens on cAMP-induced FSH and LH receptor expression were studied in granulosa cells isolated from immature diethylstilbestrol-implanted rats. Although estradiol alone had negligible effects on granulosa cell maturation, estradiol concentrations from 10(-11)-10(-8) M progressively enhanced cAMP production and gonadotropin receptor formation in choleragen-stimulated cells. During 48 h of culture, estradiol augmented cAMP levels by 2-fold, LH receptors by 4- to 6-fold, and FSH receptors by 20-40%. Estradiol also enhanced the extent of LH and FSH receptor formation by other cAMP-inducing ligands, including FSH, prostaglandin E2, and forskolin. The stimulatory action of 8-bromo-cAMP on gonadotropin receptors was also increased by estradiol, indicating that part of the estrogenic effect was exerted on cAMP-activated processes. Scatchard analyses indicated that estradiol increased the number of choleragen-induced FSH receptors from 2,600 to 3,200/cell and of LH receptors from 13,000 to 86,000/cell with no changes in receptor binding affinity. Choleragen-stimulated cAMP accumulation was enhanced by estradiol during the later stages of culture (after 30 h), while increased LH receptors were detected by 30 h and FSH receptors by 43 h. The stimulatory effects of estradiol were not due to increased cellular proliferation and were also exerted by other estrogens, including estrone and diethylstilbestrol. Androgens, including testosterone and androstenedione, also amplified choleragen action. This effect was largely through conversion to estrogens, since dihydrotestosterone, a nonaromatizable androgen, did not markedly enhance LH receptor formation by choleragen. In contrast, progestins and pregnenelone had no facilitative effect on choleragen-induced responses. Although cortisol and dexamethasone increased choleragen-induced cAMP accumulation, only cortisol elevated LH receptors, and dexamethasone inhibited FSH receptor formation. These results demonstrate that estrogens enhance both ligand-induced cAMP production and cAMP-activated responses during granulosa cell differentiation. In particular, estrogens exert a major effect on the levels of gonadotropin receptors expressed in response to FSH and other cAMP-inducing ligands.     

Pituitary and plasma levels of luteinizing hormone and follicle-stimulating hormone in male and female rabbit fetuses

To study the ontogenesis of fetal pituitary gonadotrophin synthesis and release, LH and FSH were measured by radioimmunoassay in fetal rabbit pituitary glands and blood of both sexes from day 18 of gestation until birth. Results on levels of testicular and plasma testosterone were also included. Immunoreactive LH was first detected on day 19 in the pituitary gland and on day 20 in the plasma of fetuses of both sexes. Pituitary FSH was first measurable in both male and female fetuses at 24 days of gestation. Levels of FSH could not be detected in the blood of male fetuses at any time during gestation. In females, FSH could be measured in the circulation from day 27 of gestation until birth. These results show that (1) the ontogeny of pituitary LH and the onset of testosterone secretion are closely correlated and take place between 18 and 20 days of gestation just before the beginning of differentiation of the male genital tract, and (2) the highest concentrations of pituitary LH and FSH are observed, in both sexes, in late gestation after days 24-25.     

Pituitary and gonadal response to exogenous LH-releasing hormone in the male domestic cat

To determine the influence of exogenous LH-releasing hormone (LHRH) on serum LH and testosterone, ten adult male domestic cats received three treatments on a rotating schedule at 10-day intervals as follows: (I) 0.1 ml saline i.m. (control); (II) 10 micrograms LHRH i.m., single injection; (III) 10 micrograms LHRH i.m., two injections given at a 2-h interval. Serial blood samples collected over a 360-min interval were analysed by radio-immunoassay for LH and testosterone. Although baseline serum LH values in saline-treated animals (treatment I) varied markedly among individual cats (2.2-29.2 micrograms/l), there was no evidence of pulsatile LH release or alterations in testosterone over time within individual males. In treatment II, the single injection of LHRH induced a rapid rise in mean serum LH within 30 min in all cats (mean peak, 88.2 +/- 9.8 micrograms/l), which returned to baseline by 120 min after LHRH. Mean testosterone increased within 30 min in this group (from 6.03 +/- 2.18 to 18.55 +/- 3.36 nmol/l), peaked at the 60-min collection (19.76 +/- 2.77 nmol/l) and returned to baseline by the 150-min sample. After treatment III, serum LH peaked at 131.6 +/- 13.6 micrograms/l within 30 min of the initial LHRH injection. A second injection of LHRH produced another LH surge within 30 min, but in all cats this second response was of a lower magnitude (mean peak, 69.0 +/- 14.5 micrograms/l) and shorter duration (P less than 0.05). The second LHRH injection sustained peripheral testosterone levels for approximately 1 additional h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferative and steroidogenic effects of follicle-stimulating hormone during chick embryo gonadal development

The aim of this study was to evaluate the in vitro effect of human follicle-stimulating hormone (hFSH) on cellular proliferation and steroid hormone secretion in the left ovary, the right ovary, and the testis of the chick embryo. Gonads from 8- to 18-day-old chick embryo were cultured in a defined medium during 60 h under basal and hFSH-stimulated conditions (0.5 IU/ml of culture medium). At the end of the culture, the incorporation of ?(3)Hthymidine and the total number of cells were measured to estimate gonadal cell proliferation. The secretion of 17beta-estradiol and testosterone in the culture medium was radioimmunologically assayed in order to evaluate the steroidogenic function of the cultured gonadal cells. The response to hFSH stimulation was observed in the left ovary, the right ovary, and the testis from the 8-day-old chick embryo. In the left ovary, cellular proliferation was not augmented by hFSH in the 8-, 10-, and 13-day-old chick embryo; meanwhile, the proliferative stimulus of hFSH was observed in the 15- and 18-day-old embryos. In the same ovary, 17beta-estradiol and testosterone secretion were stimulated after hFSH treatment at all evaluated stages (8-18 days of chick embryo development). In the right ovary, an increment in proliferation and steroidogenesis was induced by hFSH in the 8-, 10-, and 13-day-old chick embryo. Afterward, the right gonad did not respond to hFSH. Testis cells displayed hFSH response as an increment in cell proliferation at all embryonic ages (8-18 days of chick embryo development). There was a transient lack of response to hFSH in testosterone secretion at 10 and 13 days of development. The in vitro effect of hFSH on cell proliferation and steroid hormone secretion changed in the ovary and the testis according to the age of the embryo. These changes could be attributed to the growth of the left ovary and the testis and the regression of the right ovary. Probably, paracrine factors modulated the gonadotropin effect on the target cells during embryonic development of chick embryo gonads.     

Properties of follicle-stimulating hormone receptors and changes during annual breeding cycle in the testis of short-tailed bandicoot rat, Nesokia indica

Some properties and seasonal changes of follicle-stimulating hormone (FSH) receptors in the testis of short-tailed bandicoot rat, Nesokia indica, were studied. The binding of FSH was highly specific for mammalian FSHs and located primarily in the testis. The Scatchard plot analyses of the binding of FSH to the testicular preparations of N. indica showed straight lines similar to those of albino Wistar rats, suggesting the presence of a single class of FSH-binding sites. The mean dissociation constants (Kds) for FSH receptors of N. indica were 1.416 (0.964-2.667, 95% confidence interval) nM in December, 1986; 1.348 (0.885-2.849) nM in April, 1987; and 3.039 (1.678-16.127) nM in August to October, 1987. No significant differences were found among the three groups, but they were all significantly greater than those in Wistar rats. The FSH binding per unit weight of tissue during non-breeding phase (June, 1987) was lower than those during other phases (December, 1986; April and August-October, 1987). Calculated numbers of FSH-binding sites showed no statistically significant differences among the latter three phases. In June preparations the number of binding sites was not determined due to their extremely low binding capacity.     

Circulating bioactive follicle-stimulating hormone and less acidic follicle-stimulating hormone isoforms increase during experimental induction of puberty in the female lamb.

The pubertal process with its multifaceted neuroendocrine control provides an excellent model for the study of the regulation of FSH heterogeneity. We tested the hypothesis that during the pubertal transition in the female lamb 1) an increase in both pituitary and circulating bioactive FSH concentrations occur and 2) that the increase in bioactivity is associated with a change in the distribution pattern of both pituitary and circulating FSH isoforms. Pituitary and serum immunoreactive (I), and bioactive (B, Sertoli cell bioassay) FSH concentrations were measured in six prepubertal lambs (18 +/- 1 weeks, 29.9 +/- 2.8 kg body weight; mean +/- SE) and compared to those of six others (24.2 +/- 2.2 weeks of age, 41.4 +/- 2.5 kg body weight) during the pubertal transition period. Puberty was synchronized by pulsatile iv administration of GnRH (2 ng/kg every 2 h for 24 h and then at hourly intervals for the next 12 h) in a manner mimicking the I-LH pulse patterns observed during the natural transition to adulthood. Blood samples were collected at 12-min intervals for 4 h from both groups of lambs; for the pubertal group this included the final 32-36 h of GnRH administration. At the end of the study, a 25 ml volume of peripheral blood was collected from both prepubertal and pubertal females for the determination of serum FSH distribution patterns; the lambs were then euthanised, and pituitaries were removed for determination of pituitary hormone content and FSH isoform distribution patterns. In addition, the distribution pattern of I-FSH isoforms in the pituitary and serum from both groups of lambs were compared. The pubertal stages of all lambs were verified by measuring the size of follicles, the circulating concentrations of estradiol (E2) and inhibin, and the I-LH pulse patterns. Prepubertal lambs had low frequency I-LH pulses, small (2-3 mm) size ovarian follicles and low circulating concentrations of E2 (4.1 +/- 0.4 pg/ml) and inhibin (38.0 +/- 2.9 U/ml WHO). By contrast, all the pubertal lambs had hourly I-LH pulse frequency (induced with exogenous GnRH), a large (5-6 mm) follicle (in one lamb a 4-mm follicle), follicular phase levels of E2 (7.1 +/- 0.8 pg/ml), and higher concentrations of inhibin (53.2 +/- 3.1 U/ml).(ABSTRACT TRUNCATED AT 400 WORDS)     

Pituitary glycoprotein hormone receptors in non-endocrine organs.

Although glycoprotein hormones are usually regarded as pituitary-endocrine signals, their receptors can be found in non-endocrine tissues. High expression of selected receptors in the pituitary-endocrine axis is key to mammalian endocrine regulation. We hypothesize that peripheral receptor distribution during development and in secondary organs reflects older but still-applicable functions, with their concentration in the pituitary a more recent evolutionary advancement. We extrapolate additional functions of these receptors by analogy of homologous receptors in older phyla, with emphasis on the bony fishes (teleosts). Studies of the multiple roles of the glycoprotein hormone receptors are likely to uncover novel endocrine functions and axes, and highlight the potential of these receptors as novel therapeutic targets.     

Pituitary and gonadal desensitization after continuous luteinizing hormone-releasing hormone infusion in normal females

Three normal females were studied during the early follicular phase of the menstrual cycle employing a continuous infusion of LHRH (1.4 micrograms/min) for 72 h. Blood samples were taken every 4 h. LH concentrations climbed from 9.3 mIU/ml basally to peak values of 120 mIU/ml at 12 h, then fell to a plateau between 23-31 mIU/ml from 36-72 h. FSH levels rose from 5.4 to 36 mIU/ml at 4 h of infusion and then returned to baseline. 17 beta-Estradiol increased from 48 to 184 pg/ml at 32 h, but subsequently fell toward basal concentrations. 17-Hydroxyprogesterone increased from 0.5 to 1.23 ng/ml at 12 h and remained elevated for the remainder of the infusion period. Serum progesterone levels remained constant. Three females with premature ovarian failure were studied, and the pattern of gonadotropin release was similar to that of normal subjects. Studies demonstrate that continuous LHRH infusion in normal females causes pituitary and gonadal desensitization. The desensitization of the pituitary is independent of gonadal activity.     

Pituitary and gonadal response to LH releasing hormone administration in the female and male cheetah

Luteinizing hormone releasing hormone (LHRH, 50 micrograms) or saline was administered (i.m.) to adult female and male cheetahs under anaesthesia to evaluate pituitary and gonadal response. Serum LH levels did not fluctuate over a 120-min sampling period in saline-treated animals. Serum LH concentrations were raised (P less than 0.05) in both female and male cheetahs after LHRH injection, the temporal response being similar to previously reported results in unanaesthetized, domestic carnivores. The magnitude of the LHRH-induced LH response was sex-dependent. Over a 120-min post-injection period both saline control and LHRH-induced LH levels were about twofold greater in males than females. Although LHRH had no acute influence on ovarian oestradiol-17 beta production in the female, serum testosterone levels were raised (P less than 0.05) in male cheetahs by 60 min after treatment. This study (1) provides introductory endocrine information on the cheetah, an endangered species, and (2) indicates that exogenous LHRH is effective in acutely altering pituitary (female) and pituitary/gonadal (male) function in an anaesthetized, non-domestic felid.     

Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone

The endocrine system displays highly complex interactions among its components. Excesses or deficiencies of hormone production in one gland may alter the production of hormones by others. Several physiological functions are affected by a balance among hormones acting either together or in sequence. For example, FSH secretion has been demonstrated to be affected by hypothalamic influences upon the anterior pituitary through a specific releasing factor, the decapeptide LRF. This decapeptide stimulates the release of both LH and FSH by the pituitary, and these gonadotropins cause the production of steroids by the testes and the ovaries. Gonadal steroids in the blood act directly upon the anterior pituitary to regulate the output of gonadotropins as originally proposed by Moore and Price in 1932 (3), or act indirectly upon the hypothalamus to adjust the output of pituitary hormones in accordance with the needs of the reproductive system. However, such a simple negative feedback of steroids on the hypothalamic-hypophysial axis cannot account for the differential secretion of FSH observed during the estrus cycle. Therefore, the concept that a gonadal protein, inhibin, specifically regulates FSH secretion was proposed. This concept has now been validated by the isolation and characterization of two forms of inhibin that exert their effects on the pituitary to suppress FSH secretion both in vitro and probably in vivo. Furthermore, the production of inhibin is stimulated by FSH, thus establishing a reciprocal relationship between the release of FSH and inhibin. Since hormones in the body are controlled through interlocking complexes of factors, a variety of secondary factors, in one way or another, may also exert influence on the regulation of FSH secretion. As an example, TGF beta, a protein growth factor found in all tissues, promotes the basal secretion of FSH by the pituitary and enhances FSH-mediated estrogen production by the granulosa cells. It is therefore not surprising that two forms of a novel protein, activin and activin A, isolated from the same FF from which inhibins were isolated, show bioactivities similar to those of TGF beta. These activins are formed as dimers of the two beta-subunits of inhibin, probably as a result of the rearrangement of the gene products. This novel observation that different arrangements of gene products can result in opposite biological activities may thus reflect a wholly different level of control of FSH secretion. If such a phenomenon occurs in other biosystems, it would represent an important form of homeostatic mechanism for controlling biologically active substances.(ABSTRACT TRUNCATED AT 400 WORDS)     

Auto-regulation of thyroid hormone receptors in the goldfish ovary and testis

Thyroid hormones, acting via their cognate thyroid receptors (TRs) act as mediators and modulators of several physiological processes and homeostasis. A clear role for the TRs in reproduction has not yet been established although several lines of recent evidence suggest that they are involved in the regulation of reproduction. To further study the role of TRs in control of reproduction, we investigated homologous regulation of TR subtypes in the gonads of goldfish, in vivo and in vitro. It was found that tri-iodothyronine (T₃) down-regulates the traditional TRs (TRα-1 and TRβ) and up-regulates a dominant-negative form, TRα-t. This indicates a ‘feedback’ mechanism whereby an acute treatment with T₃ down regulates further T₃ mediated response. The results provide novel information on auto-regulation of TRs in the goldfish ovary and testis, and support the hypothesis that thyroid hormones are involved in the control of reproduction.     

The relative role of gonadal sex steroids and gonadotropin-releasing hormone pulse frequency in the regulation of follicle-stimulating hormone secretion in men

OBJECTIVE: Our objective was to determine the importance of testosterone (T), estradiol (E(2)), and GnRH pulse frequency to FSH regulation in men. DESIGN: This was a prospective study with four arms. SETTING: The study was performed at the General Clinical Research Center. PATIENTS OR OTHER PARTICIPANTS: There were 20 normal (NL) men and 15 men with idiopathic hypogonadotropic hypogonadism (IHH) who completed the study. Intervention: Medical castration and inhibition of aromatase were achieved using ketoconazole x 7 d with: 1) no sex steroid addback, 2) T addback starting on d 4, and 3) E(2) addback starting on d 4. IHH men in these arms received GnRH every 120 min. In a further six IHH men receiving ketoconazole with no addback, GnRH frequency was increased to 35 min for d 4-7. Blood was drawn every 10 min x 12 h at baseline, overnight on d 3-4 and 6-7. MAIN OUTCOME MEASURES: Mean FSH was calculated from the pool of each frequent sampling study. RESULTS: In NL men FSH levels increased from 5.1 +/- 0.7 to 8.7 +/- 1.3 and 9.7 +/- 1.5 IU/liter (P < 0.0001). T caused no suppression of FSH. E(2) reduced FSH from 12.4 +/- 1.8 to 9.3 +/- 1.3 IU/liter (P < 0.05). In IHH men on GnRH every 120 min, FSH levels went from 6.0 +/- 1.6 to 9.0 +/- 3.0 and 11.9 +/- 4.3 (P = 0.07). T caused no suppression of FSH. E(2) decreased FSH such that levels on d 6-7 were similar to baseline. Increasing GnRH frequency to 35 min had no impact on FSH. CONCLUSIONS: The sex steroid component of FSH negative feedback in men is mediated by E(2). Increasing GnRH frequency to castrate levels has no impact on FSH in the absence of sex steroids. When inhibin B levels are NL, sex steroids exert a modest effect on FSH.