Basal Follicle-stimulating Hormone Secretion Research Articles

Spike protein of SARS-CoV-2 suppresses gonadotrophin secretion from bovine anterior pituitaries.

Coronavirus disease (COVID-19), the ongoing global pandemic, is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Recent evidence shows that the virus utilizes angiotensin-converting enzyme 2 (ACE2) as a spike protein receptor for entry into target host cells. The bovine ACE2 contains key residues for binding to the spike protein receptor-binding domain. This study evaluated the hypothesis that bovine gonadotroph expresses ACE2, and spike protein suppresses luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from cultured bovine anterior pituitary (AP) cells. ACE2 mRNA expression and ACE2 protein expression were detected in the bovine AP cells using reverse transcription PCR and western blot analysis. Immunofluorescence microscopy analysis with the anti-ACE2 antibody revealed the co-localization of ACE2 and gonadotropin-releasing hormone (GnRH) receptor on the gonadotroph plasma membrane. Approximately 90% of GnRH receptor-positive cells expressed ACE2, and approximately 46% of ACE2-positive cells expressed the GnRH receptor. We cultured bovine AP cells for 3.5 days and treated them with increasing concentrations (0, 0.07, 0.7, or 7 pM) of recombinant spike protein having both S1 and S2 regions. The spike protein (0.07–7 pM) suppressed both basal and GnRH-induced LH secretion (P < 0.05). Spike protein (0.7–7 pM) suppressed GnRH-induced (P < 0.05), but not basal FSH secretion. In contrast, pre-treatment with ERK 1/2/5 inhibitor (U0126) partially restored the GnRH-induced LH and FSH secretion from the spike protein suppression. Collectively, the results indicate that gonadotrophs express ACE2, a receptor for coronavirus 2 spike protein, which in turn suppresses LH and FSH secretion from AP cells.

Basal and GnRH-induced secretion of FSH and LH in anestrous versus ovariectomized bitches

The basal and gonadotropin releasing hormone (GnRH)-induced plasma concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were studied in four anestrous and four ovariectomized (OVX) bitches. Blood samples were obtained via jugular venipuncture 40 min before and 0, 10, 20, 30, 60, 90, and 120 min after the i.v. administration of synthetic GnRH in a dose of 10 μg/kg body weight. The basal plasma FSH and LH concentrations were significantly higher in the OVX bitches than in the anestrous bitches. In the anestrous bitches, the plasma FSH concentration was significantly higher than the pretreatment level at 10, 20, and 30 min, whereas the plasma LH concentration was significantly elevated at 10 and 20 min. The maximal GnRH-induced plasma FSH concentration in the anestrous bitches did not surpass the lowest plasma FSH concentration in the OVX bitches, whereas the GnRH-induced plasma LH concentrations in the anestrous bitches overlapped with the basal plasma LH concentrations in the OVX bitches. In the OVX bitches, GnRH administration did not induce a significant change in the plasma FSH concentration, whereas the plasma LH concentration increased significantly at 10 and 20 min. In conclusion, the results of the present study indicate that in anestrous bitches GnRH challenge results in increased plasma levels of both FSH and LH, whereas in the OVX bitches, in which the basal plasma FSH and LH concentrations are higher, only a rise in the plasma LH concentration is present after GnRH stimulation. The results also suggest that a test to measure plasma concentration of FSH in single samples appears to have potential in verification of neuter status in bitches.

Effects of Ghrelin upon Gonadotropin-Releasing Hormone and Gonadotropin Secretion in Adult Female Rats: In vivo and in vitro Studies

A reproductive facet of ghrelin, a stomach-derived orexigenic peptide involved in energy homeostasis, has been recently suggested, and predominantly inhibitory effects of ghrelin upon luteinizing hormone (LH) secretion have been demonstrated in rat models. Yet, the modulatory actions of ghrelin on the gonadotropic axis remain scarcely evaluated. We report herein a detailed analysis of the effects of ghrelin upon LH and follicle-stimulating hormone (FSH) secretion in the female rat, using a combination of in vivo and in vitro approaches. Intracerebroventricular administration of ghrelin (3 nmol/rat) evoked a significant inhibition of LH secretion in cyclic female rats throughout the estrous cycle (proestrus afternoon, estrus, metestrus), as well as in ovariectomized females. In good agreement, gonadotropin-releasing hormone (GnRH) secretion by hypothalamic fragments from ovariectomized females was significantly inhibited by ghrelin. In contrast, ghrelin dose-dependently stimulated basal LH and FSH secretion by pituitary tissue in vitro; a phenomenon that was proven dependent on the phase of estrous cycle, as it was neither detected at estrus nor observed after ovariectomy. Conversely, GnRH-stimulated LH secretion in vitro was persistently inhibited by ghrelin regardless of the stage of the cycle, whereas stimulated FSH secretion was only inhibited by ghrelin at estrus. In addition, cyclic fluctuations in mRNA levels of growth hormone secretagogue receptor (GHS-R)1a, i.e. the functional ghrelin receptor, were observed in the pituitary, with low values at estrus and metestrus. GHS-R1a mRNA levels, however, remained unchanged after ovariectomy. In summary, our data illustrate a complex mode of action of ghrelin upon the gonadotropic axis, with predominant inhibitory effects at central (hypothalamic) levels and upon GnRH-induced gonadotropin secretion, but direct stimulatory actions on basal LH and FSH secretion. Overall, our results further document the reproductive role of ghrelin, which might be relevant for the integrated control of energy balance and reproduction.

Biological Role of Pituitary Estrogen Receptors ERα and ERβ on Progesterone Receptor Expression and Action and on Gonadotropin and Prolactin Secretion in the Rat

Estrogen (E) is a key regulator of the synthesis and secretion of pituitary reproductive hormones [luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL)]. Until recently, it was thought that all biological actions of E at the pituitary were manifested through a single E receptor (R). The pituitary, like many other reproductive tissues, expresses two isoforms of ER, α and β, both activated by E. The relative contribution of α and β forms in E regulatory actions is largely unknown. To this end, 2-week-old ovariectomized (OVX) rats were injected over 3 days with 25 µg estradiol benzoate (EB), 1.5 mg of propylpyrazole triol (PPT), a selective ERα agonist, 1.5 mg of the selective ERβ agonist diarylpropionitrile (DPN) or a combination of PPT and DPN. Controls were injected with 0.2 ml oil. At 10:00 h on the day after treatment, trunk blood was collected to determine serum concentration of LH, FSH and PRL, and pituitaries were processed for RT-PCR analysis of total (A+B) progesterone receptor (PR) mRNA, immunocytochemistry of PR and incubation. Pituitaries from each of the five groups were incubated in DMEM, with or without 20 nM of the antiprogestin at the receptor ZK299, for 3 h with: 10^–8M 17β-estradiol, 10^–6M PPT, 10^–6M DPN, PPT+DPN or medium alone, respectively, to determine LH, FSH and PRL secretion, and, when challenged with two pulses of 15 min 1 h apart of 10^–8M gonadotropin-releasing hormone (GnRH) (GnRH self-priming). EB, PPT and PPT+DPN treatments increased PR mRNA and the number and intensity of nuclei immunoreactive (IR) for PR in gonadotropes, and reduced the number of gonadectomy cells. Like E, PPT alone or in combination with DPN stimulated PRL secretion, increased basal and GnRH-stimulated LH and FSH secretion and induced GnRH self-priming in the absence of ZK299 in the incubation medium. DPN alone had only a significant E-like effect on gonadectomy cells and IR-PR, but not on GnRH self-priming. In addition, while DPN lacked an agonistic action on peripheral tissue and serum pituitary reproductive hormones concentration, EB, PPT and PPT+DPN induced similar uterine ballooning and vaginal cornification, and increased and decreased, respectively, serum concentrations of PRL and gonadotropins. Overall, these results indicate that most of these E actions on the pituitary are exerted through the ERα isoform. The finding that activation of ERβ with its selective DPN agonist had an estrogenic effect on IR-PR nuclei, but not on GnRH self-priming, a characteristic ERα-mediated effect of E, suggests that the biological action of E at the pituitary may involve both isoforms of ER.

Effects of leptin on gonadotropin secretion in juvenile female rat pituitary cells.

Leptin is an adipocyte-derived hormone, which is the product of the obese gene and it is thought to play important roles in pubertal development and maintenance of reproductive function in the female. In a study using adult male or female rats, it was found that leptin stimulated the secretion of gonadotropin directly from the pituitary in a dose-related manner. However, there is no study in juvenile female rats before puberty. In this study, we cultured pituitary cells from 4-, 6- and 8-week-old female Wistar rats with leptin (0-10(-7)mol/l) and gonadotropin-releasing hormone (GnRH) (0 or 10(-8) mol/l). Basal or GnRH-stimulated secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and their synthesis within cells were determined by radioimmunoassay (RIA). Leptin induced bell-shaped dose--response curves of basal LH and FSH secretion from cultured cells of every age-group of rats studied. The most effective concentration of leptin on the basal secretion of LH and FSH from 6- and 8-week-old cultured pituitary cells was 10(-10) mol/l. This leptin concentration was consistent with circulating physiological serum leptin levels at each age. As for juvenile 4-week-old pituitary cells, the most effective concentration was 10(-11) mol/l which was lower than that of 6- and 8-week-old rats. It was consistent with the circulating serum leptin levels of 4-week-old rats. Also, the synthesis and the GnRH-stimulated secretion of LH and FSH were effectively controlled by leptin at concentrations similar to the serum leptin levels of given ages. Leptin induced pituitary cells to synthesize and secrete both LH and FSH regardless of the presence or absence of GnRH. The concentration of leptin that induced the greatest synthesis and secretion of gonadotropins from pituitary cells changed around the pubertal period. The most effective leptin concentrations in each experiment were similar to the physiological serum leptin level at each animal age. These results indicate that leptin stimulates gonadotrophs not only in the pubertal and the mature period but also in the juvenile period before puberty. It is also conceivable that leptin may modulate the sensitivity of gonadotrophs until the appearance of GnRH stimulation, and may be the factor that brings about puberty onset.

Antiprogestin RU486 blunts the stimulatory effect of galanin on LH secretion from rat pituitaries in vitro in an estrous cycle stage-dependent manner

Galanin (GAL), a neuroactive peptide detected in the hypothalamus and anterior pituitary, stimulated in a dose (0.1 and 1 μM)-dependent manner luteinizing hormone (LH) secretion from metestrous and proestrous rat pituitaries incubated in culture medium devoid of progesterone (P). GAL had no effect on follicle-stimulating hormone (FSH). Antiprogestin RU486 (10 nM) decreased non-stimulated (basal) secretion of LH and FSH only in pituitaries from proestrous rats and blunted the stimulatory effects of GAL on LH secretion in both metestrous and proestrous pituitaries. These findings are consistent with the hypothesis that GAL-mediated signal transduction interacts with estrogen-dependent P receptor at the pituitary level to stimulate LH secretion.

Joint basal and pulsatile hypersecretory mechanisms drive the monotropic follicle-stimulating hormone (FSH) elevation in healthy older men: concurrent preservation of the orderliness of the FSH release process: a general clinical research center study.

To appraise the neuroendocrine mechanisms that underlie a selective (monotropic) elevation of serum FSH concentrations in healthy older men, we sampled blood in 11 young (ages 21-34) and 8 older men (ages 62-72) men every 2.5 min overnight. Serum FSH concentrations were quantitated in an automated, high-sensitivity, chemiluminescence-based assay. Rates of basal and pulsatile FSH secretion were estimated by deconvolution analysis, and the orderliness of the FSH release process via quantitated the approximate entropy statistic. Statistical analysis revealed that healthy older men manifest dual neuroendocrine hypersecretory mechanisims; specifically, a 2-fold increase in the basal (nonpulsatile) FSH secretion rate, and a concurrent 50% amplification of FSH secretory burst mass (and amplitude). The regularity or orderliness of ad seriatim FSH release is preserved in older individuals. We postulate that higher basal FSH secretion in older men is a consequence of reduced testosterone negative feedback, whereas amplified FSH secretory burst mass reflects net enhanced stimulation of gonadotrope cells by endogenous FSH secretagogues (e.g. GnRH and/or activin). The foregoing specific mechanisms driving heightened FSH secretion in older men contrast with the lower-amplitude pulsatility and more disorderly patterns of LH release in the same individuals. Thus, the present data illuminate an age-dependent disparity in the disruption of FSH neuroregulation in the aging male.

Characterization of a rat anterior pituitary cell bioassay

We have described the protocols and characterization of a pituicyte culture, which became established as a reliable and reproducible bioassay for the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The bioassay was used to measure the bioactivity of factors that inhibit and stimulate gonadotrophin secretion. The protocol that was used involved the culling of female Wistar rats (200 to 250 g weight), at random stages of their cycle, and dispersal of their pituicytes in a concentration of 0.4 x 10(6) cells.ml-1.well-1 in serum-free medium (Dulbecco's modified Eagle's medium/Ham's F12 mixture, supplemented with insulin and transferrin) in Falcon 3047 24-well culture plates. After 24 h of pre-culture, the medium was changed and the cells cultured for a further 48 h. The supernatant was removed and assayed for basal secretion of FSH and LH. The cells were then stimulated with 10(-8) M GnRH for 4 h and the supernatant assayed for gonadotrophin-releasing hormone (GnRH)-stimulated FSH and LH secretion. All samples were assayed as pairs of duplicates (i.e. quadruplicate samples) which were randomly added to the plates to minimize plate effects. Random number tables were used to achieve this randomization.

Significant role of 5 alpha-reductase on feedback effects of androgen in rat anterior pituitary cells demonstrated with a nonsteroidal 5 alpha-reductase inhibitor ONO-3805.

A 5 alpha-reductase inhibitor with nonsteroidal structure, ONO-3805, has been used to study the role of 5 alpha-reductase on positive and negative feedback effects of androgen on gonadotropin secretion in the rat anterior pituitary gland. Initially, the potential of ONO-3805 to inhibit the formation of 5 alpha-dihydrotestosterone (DHT) was evaluated. The activity of 5 alpha-reductase in rat anterior pituitary gland was approximately one-fourth of that in rat prostate gland or epididymis, with a Michaelis' constant (Km) value for testosterone of 5-6 x 10(-7) M. When homogenates of rat anterior pituitary glands were incubated with 14C-testosterone (14C-T) in the presence of > or = 10(-7) M ONO-3805, the formation of labeled DHT and its metabolite 5 alpha-androstane-3 alpha,17 beta-diol was inhibited by > 90%. The inhibition pattern was non-competitive, and the inhibition constant (Ki value) derived from Lineweaver-Burk plots was 3.9 x 10(-11) M. Dispersed pituitary cells (1-2 x 10(5)/ml) were cultured for 48 hours and then further incubated with or without androgen and/or the inhibitor for 72 hours (basal secretion). After this incubation, the media were saved, and 10 nM of luteinizing hormone-releasing hormone (LH-RH) with the same concentration of androgen and/or the inhibitor as used in the 72-hour incubation was added to the cultures for 6 hours (LH-RH-induced secretion). Secreted follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were assayed by radioimmunoassay (RIA). Both testosterone (T) and DHT stimulated 72-hour basal FSH secretion from cultured pituitary cells in a dose-dependent fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of corticotropin-releasing factor or cortisol on follicle-stimulating hormone secretion by isolated pig pituitary cells

This study investigated the direct effects of hydrocortisone (HS), corticotropin-releasing factor (CRF), and adrenocorticotropin (ACTH) on basal and gonadotropin-releasing hormone (GnRH)-stimulated secretion of follicle-stimulating hormone (FSH) from dispersed pig pituitary cells in vitro. Pig pituitaries were dispersed into cells with collagenase, DNAase, and hyaluronidase and then cultured in McCoy's 5a medium containing horse serum (10%) and fetal calf serum (2.5%) pretreated with dextran-coated charcoal for 3 days. Cells were preincubated with steroids, CRF, or ACTH before GnRH was added. HS did not affect basal FSH secretion after 72 h of incubation. Treatment of pituitary cells with increasing concentrations (0.001–800 μg/ml) of HS for 72 h resulted in a dose-dependent decrease in GnRH-stimulated FSH release. HS pretreatment did not cause a change in cellular FSH content. Increasing duration (6–72 h) of treatment with HS (200 μg/ml) led to a time-dependent decrease in GnRH-stimulated FSH release, achieving statistical significance by 12 h. Porcine ACTH had no influence on basal and GnRH-stimulated FSH secretion. CRF decreased GnRH-stimulated FSH secretion in a dose-dependent manner, and the inhibitory effect required preincubation (6–18 h) with CRF. HS inhibited the FSH secretory responses to phospholipase C, melittin, and 8-bromo-cAMP but did not affect the response to 1,2-dioctanoyl- sn-glycerol and ionophore A23187. These results indicate that both cortisol and CRF can act directly on pig pituitary to inhibit FSH responsiveness to GnRH.

Role of gonadal negative feedback on the gonadotrophin responses to gonadotrophin-releasing hormone (GnRH) in ram lambs from two lines of sheep selected for their luteinizing hormone response to GnRH

Divergent selection has resulted in two lines of lambs (high and low) that have a 5-fold difference in their ability to release luteinizing hormone (LH) in response to 5 micrograms of gonadotrophin-releasing hormone (GnRH). Baseline gonadotrophin concentrations, the gonadotrophin responses to a GnRH challenge and the concentrations of testosterone and oestradiol were compared in lambs which were castrated at birth and intact lambs from both selection lines at 2, 6, 10 and 20 weeks of age. The pattern of LH and follicle-stimulating hormone (FSH) secretion was similar in the two lines, but differed between the intact and the castrated lambs. Basal LH and FSH secretion were significantly higher in the castrates than in the intact lambs from both selection lines. The high-line lambs had significantly higher basal FSH concentrations at all ages tested and significantly higher basal LH concentrations during the early postnatal period. The magnitude of the gonadotrophin responses to GnRH differed significantly between the intact and the castrated lambs within each line, the amount of gonadotrophins secreted by the castrated lambs being significantly greater. The removal of gonadal negative feedback by castration did not alter the between-line difference in either LH or the FSH response to the GnRH challenge. Throughout the experimental period, the concentration of testosterone in the intact lambs was significantly greater than in the castrated lambs in both selection lines, but no significant difference was seen in the concentrations of oestradiol. No significant between-line differences were found in the peripheral concentrations of testosterone or oestradiol in the intact lambs from the two selection lines. Therefore, despite similar amounts of gonadal negative feedback in the selection lines, there were significant between-line differences in basal gonadotrophin concentrations, at 2 and 6 weeks of age, and in the LH and FSH responses to an exogenous GnRH challenge, at all ages tested. Removal of gonadal negative feedback did not affect the magnitude of the between-line difference in the response of the lines to GnRH stimulation. The results indicate that the effects of selection on gonadotrophin secretion are primarily at the level of the hypothalamo-pituitary complex.

Chronic Inhibitory Effect of Follicle-Stimulating Hormone (FSH)-Suppressing Protein (FSP) or Follistatin on Activin- and Gonadotropin-Releasing Hormone-Stimulated FSH Synthesis and Secretion in Cultured Rat Anterior Pituitary Cells*

The effects of bovine FSH-suppressing protein (FSP) or follistatin on activin- and GnRH-stimulated FSH synthesis and secretion have been studied using cultured pituitary cells from adult male Sprague-Dawley rats. Exposure to FSP (0.001-10 nM) for 3 days dose-dependently suppressed basal FSH secretion (IC50 = 146 +/- 21 pM., mean +/- SE), cellular content (IC50 = 269 +/- 8 pM) and total FSH (IC50 = 181 +/- 25 pM), with no effect on LH. Activin (0.3 nM) increased FSH secretion 2.1-fold, cellular content 1.3-fold, and total FSH 1.9-fold during a 3-day incubation, but these increases were dose-dependently inhibited by concomitant treatment with 35-kDa bovine FSP (0.1-3 nM), with complete inhibition occurring at concentrations between 1 and 3 nM. The 31- and 39-kDa forms of bovine FSP also antagonized the actions of activin. GnRH (1 nM) increased FSH secretion 1.8-fold and total FSH 1.6-fold during a 3-day incubation, effects that were dose-dependently inhibited by concomitant treatment with 35-kDa bovine FSP. The highest tested concentration of FSP (3 nM) suppressed GnRH-stimulated FSH secretion and total FSH to 59 and 57%, respectively, of the levels found in untreated cultures. All three forms of bovine FSP produced a significant inhibition of FSH secretion and total FSH stimulated by GnRH. FSP also suppressed FSH secretion and total FSH in response to activators of protein kinase C including 100 nM phorbol 12-myristate 13-acetate (43 and 59%, respectively) and 100 nM mezerein (40 and 60%, respectively). Finally, treatment of cultured pituitary cells with 35-kDa FSP at 1 and 3 nM for 3 days resulted in 21 and 24% decreases in GnRH binding sites, respectively. It is concluded that (i) FSP inhibits not only the secretion but also the synthesis of FSH induced by activin and GnRH in long-term culture, and (ii) FSP may cause its inhibitory effects on GnRH by suppression of the protein kinase C system, and possibly by reduction of GnRH binding sites.

A Possible Dual Mechanism of the Anovulatory Action of Antiprogesterone RU486 in the Rat1

The purpose of these experiments was to investigate the mechanism of the anovulatory action of antiprogesterone RU486 (RU486) in rats by studying its effects on follicular growth, secretion of gonadotropins and ovarian steroids, and ovulation. Rats with 4-day estrous cycles received injections (s.c.) of either 0.2 ml oil or 0.1, 1, or 5 mg of RU486 at 0800 and 1600 h on metestrus, diestrus, and proestrus. At the same times, they were bled by jugular venipuncture to determine serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17 beta-estradiol (E), and progesterone (P). On the morning of the day after proestrus, ovulation and histological features of the ovary were recorded. Rats from each group were killed on each day of ovarian cycle to assess follicular development. Rats treated similarly were decapitated at the time of the ovulatory LH surge and blood was collected to measure LH. The serum levels of LH increased and those of FSH decreased during diestrus in rats treated with RU486. Neither E nor P levels differed among the groups. Treatment with RU486 caused both a blockade of the ovulation and an increase in ovarian weight in a dose-dependent manner. At the time of the autopsy (the expected day of ovulation), rats treated with 1 mg RU486 had ovaries presenting both normal and post-ovulatory follicles and unruptured luteinized follicles. Rats treated with 5 mg RU486 presented post-ovulatory follicles without signs of luteinization. The number of follicles undergoing atresia increased in rats treated with RU486. Rats treated with 5 mg RU486 exhibited a significant decrease in ovulatory LH release. The mechanism by which RU486 produces the ovulatory impairment in rats seems to be dual: first, by inducing inadequate follicular development at the time of the LH surge and second, by reducing the amount of ovulatory LH released. The physiological events-decreased basal FSH secretion and follicular atresia-that result from use of RU486 cannot be elucidated from these experiments and should be investigated further.

Blockade of the Selective Increase in Serum Follicle-Stimulating Hormone Concentration in Immature Female Rats and its Effects on Ovarian Follicular Development1

We investigated whether administration of monosodium L-glutamate (MSG) to neonatal female rats would block the selective increase in serum follicle-stimulating hormone (FSH) concentration in immature rats in an attempt to provide a model in which to study the importance of the selective FSH rise on ovarian follicular development. In two separate experiments, s.c. injections of MSG (4 mg/g BW) on Days 1, 3, 5, 7 and 9 after birth blocked the selective increase in serum FSH concentration observed on Days 7 and 15 without blocking basal FSH secretion. Serum luteinizing hormone (LH) levels were unaffected in the first experiment and changed little in the second. MSG-treated rats had smaller ovaries on Days 15 and 23. The ovaries of MSG-treated rats on Day 15 showed decreased follicular growth as evidenced by a decrease in the number and percentage of follicles with diameters greater than 50 microns, in the number of follicles with greater than 1 layer of granulosa cells, and in the number of follicles beyond the primary stage of follicular development. These differences between MSG-treated rats and controls all but disappeared by Day 23. The results demonstrate that neonatal administration of MSG blocks the selective increase in serum FSH concentration in immature female rats and suggest that this selective increase in serum FSH levels plays a role in the normal acceleration of ovarian follicular development but is not needed for the development of preovulatory follicles by the sixth week after birth.

Neonatal treatment of male rats with a gonadotropin-releasing hormone antagonist results in altered function of the pituitary-testicular axis in adult age.

In most mammals, pituitary-testicular hormone secretion is very active during the perinatal period, but the physiological significance of this function for later pituitary-gonadal interactions and sexual maturation is largely unknown. Short-term neonatal treatment with gonadotropin-releasing hormone (GnRH) antagonist results in delayed sexual maturation and infertility in male rats. We have now extended our earlier findings and studied in more detail the pituitary-gonadal function in adult rats after such neonatal treatment. In this study, the pituitary-testicular activity of newborn male rats was temporarily blocked by treatment with a GnRH antagonist analogue (N-Ac-4-Cl-D-Phe1, 4-Cl-D-Phe2, D-Trp3, D-Phe6, des-Gly10-GnRH-D-alanylamide; Organon 30039; 2 mg/kg s.c. twice daily) on Days 1-5 of life. Timing of puberty was slightly delayed in the treated rats (average: 2 days, p less than 0.05), as determined by the age of the balano-preputial separation. In adult rats (90-110 days), only 3 of the 17 rats treated neonatally with GnRH antagonist were fertile (14 of 17 controls, p less than 0.01), despite normal circulating androgen levels. Pituitary and serum follicle-stimulating hormone (FSH) levels were slightly but consistently elevated (20-30%; p less than 0.05) in antagonist-treated animals, whereas luteinizing hormone (LH) levels (both immunoreactive and bioactive) were unaffected. The pituitary contents of GnRH receptors were increased in antagonist-treated animals 85 +/- 6.6 (mean +/- SEM, n = 19) vs. 58 +/- 4.1 fmol/gland in controls (n = 20; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Similar effects of inhibin and cycloheximide on gonadotropin release in superfused pituitary cell cultures1

The actions of two inhibin preparations and cycloheximide on gonadotropin release were investigated in superfused pituitary cell cultures. Pituitary cells isolated from 18-day-old male rats were grown in Matrigel-coated superfusion chambers in chemically defined medium. After stationary culture for 4 days, the cell monolayers were superfused at a constant speed (0.25 ml/min) and were intermittently stimulated (6 min/h) with 10 nM gonadotropin-releasing hormone (GnRH). Groups of cultures were exposed to the test substances for varying time periods during stationary culture and/or during superfusion. Inhibitory effects of both inhibin preparations on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in response to GnRH pulses were observed after 2 h of exposure and became maximal after about 6 h. Basal secretion of FSH between GnRH pulses was also suppressed, whereas the basal interpulse secretion of LH was not changed. When exposure to inhibin was discontinued, the secretion of both FSH and LH progressively increased and returned to control values by approximately 6 h. Cycloheximide (500 ng/ml) affected gonadotropin release with dynamics similar to those observed for the inhibin preparation. These data support the hypothesis that inhibition of gonadotropin synthesis may be an important step in the molecular mechanism of action by which inhibin regulates gonadotropin release.

Increases in the basal secretion rate of follicle-stimulating hormone (FSH) accompany age-associated changes in serum FSH levels on estrus.

This laboratory has recently reported that by 5-6 months of age, alterations in the secretion and production of follicle-stimulating hormone (FSH) occur in virgin female rats which precedes the age-related disruption of estrous cycles and attenuation of preovulatory gonadotropin surges. Specifically, circulating immunoreactive FSH levels are higher on estrus in rats 5 months and older compared to levels measured in 2- to 3-month-old rats. Therefore, the present study was conducted to explore a possible mechanism for this age-related increase in FSH levels. At 1400 hr on proestrus, estrus and diestrus-1, groups (n = 6-12 rats/group) of 3- and 7-month-old, cyclic rats were decapitated, trunk blood was collected, and anterior pituitary glands were bisected and placed in incubation flasks containing 1 ml media (medium 199). Following a 30-min preincubation period, hemipituitary fragments were incubated for an additional 2 hr. Media and serum FSH levels were quantified by RIA. Levels of FSH were twofold higher in the serum of 7-month-old rats than 3-month-old rats on estrus. Similarly, the basal secretion rate (BSR) of FSH (expressed as ng FSH/ml/2 hr) was significantly (P less than 0.05) higher from incubated hemipituitary fragments of 7-month-old estrous rats than from fragments obtained from younger estrous rats (7 month: 1637 ng/ml/2 hr vs 3 months: 1253 ng/ml/2 hr). Neither the serum FSH levels nor the BSR of FSH differed between age groups on proestrus or diestrus-1. These results show that age-associated increases in circulating FSH levels on estrus may be attributed to an enhanced basal secretion of FSH from the pituitary gland.

Isolation and partial characterization of follistatin: a single-chain Mr 35,000 monomeric protein that inhibits the release of follicle-stimulating hormone.

A Mr 35,000 protein with follicle-stimulating hormone release-inhibitory activity was isolated from porcine ovarian follicular fluid by heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and multiple steps of high-performance liquid chromatography. The isolated molecule is highly enriched in cysteines and is composed of a single polypeptide chain. In addition, it has no sequence homology with the previously characterized follicular fluid inhibins, which are heterodimeric proteins of Mr 32,000 with follicle-stimulating hormone release-inhibiting activity. This protein specifically inhibits the basal secretion of follicle-stimulating hormone, but not that of luteinizing hormone, in the rat anterior pituitary monolayer culture system with a half-maximal effective dose of 2.5-6.0 ng/ml. Another form of the molecule of Mr 32,000 present in much lower concentration in follicular fluid was also isolated. It may differ from the Mr 35,000 form in glycosylation or carboxyl-terminal truncation. We suggest that this compound be called "follistatin" to signify its structural difference from inhibin.

Age-associated increases in serum follicle-stimulating hormone levels on estrus are accompanied by a reduction in the ovarian secretion of inhibin.

In light of recent data demonstrating age-related alterations in the secretion and production of follicle-stimulating hormone (FSH) during the secondary FSH surge on estrus, the following study was conducted to determine the effects of age on periovulatory inhibin secretion. Ovarian venous blood was collected from groups of ether-anesthetized 3- and 7-month-old rats exhibiting 4-day estrous cycles at the following times: 1200 and 2400 h on proestrus and 1600 h on estrus. Following a 10-min collection period, a terminal blood sample was obtained from the abdominal aorta. Peripheral serum concentrations of luteinizing hormone (LH), FSH, estradiol-17 beta (E2), progesterone (P) and testosterone (T) were measured by RIA. Inhibin activity in ovarian venous serum (OVS) was assessed by the ability of OVS to suppress basal FSH secretion from dispersed pituitary cells during a 24-hour culture period. At 1200 h on proestrus, serum FSH (and LH) levels were higher in 7-month-old rats than in younger rats while the FSH-suppressing activity of OVS did not differ between age groups at this time. Bioassayable inhibin activity substantially declined between 1200 and 2400 h on proestrus in both groups. By 1600 h on estrus, serum FSH levels and inhibin secretion were higher and lower, respectively, in the older age group compared to 3-month-old rats. Significant increases in inhibin secretion between 2400 h on proestrus and 1600 h on estrus were observed only in younger rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A direct pituitary action of progesterone on basal secretion of follicle-stimulating hormone in ovine cell culture: dependence on ovaries in vivo.

Progesterone (P4) inhibits FSH production by 60-70% in cell cultures of pituitaries from normal ewes. This communication reports that P4 is much less effective in inhibiting FSH production in cultures prepared from pituitaries of ovariectomized ewes. P4 (10(-7) M) inhibited 61 +/- 5% of basal FSH secretion in cultures from intact anestrous ewes. When cultures were prepared from pituitaries collected on day 9, 21, or 42 after ovariectomy, P4 inhibited FSH secretion by only 46 +/- 3%, 16 +/- 2%, and 10 +/- 2%, respectively. One group of ewes received implants of 17beta-estradiol (E2) at the time of ovariectomy. When cultures were prepared from these ewes 35 days after ovariectomy, P4 inhibited FSH secretion 56 +/- 5%, not significantly different (P greater than 0.05) from inhibition in normal cultures. Furthermore, under similar culture conditions, E2 and a porcine ovarian inhibin preparation inhibited FSH secretion regardless of the length of time after ovariectomy. These in vitro results suggest that ovariectomy causes a time-dependent decrease in pituitary responsiveness to P4 in vivo. Since E2 can maintain P4 sensitivity in pituitaries from ovariectomized ewes, E2 may be the only ovarian factor required to maintain pituitary responsiveness to P4 in vivo.