Effects of factors from ovarian follicular fluid and Sertoli cell culture medium on in-vivo and in-vitro release of pituitary gonadotrophins in the rat: an evaluation of systems for the assay of inhibin.

Inhibin-like activity is present both in testicular and ovarian fluids. Various methods can be used for the detection of this activity. Indirect methods, using organ weights as an endpoint, lack the specificity required for reliable estimation of inhibin-like activity. With in-vivo bioassay systems, using estimation of circulating concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in intact or gonadectomized, immature or adult, male or female rats, a suppression of FSH concentrations only is usually observed after injection of inhibin-like material. The largest suppression of FSH concentrations can be obtained in short-term gonadectomized adult female or 35-day-old male rats. Addition of inhibin-like activity to cultured pituitary cells specifically suppresses the spontaneous release of FSH from the cells. After stimulation of cultured pituitary cells with LH-releasing hormone (LH-RH), the release of both FSH and LH are suppressed when inhibin-like activity is present. From dialysis experiments it appears that the molecular weight of the inhibin-like material in follicular fluid is greater than 10 000. However, acid ethanol extracts of this fluid contain a factor with a molecular weight smaller than 10 000, which does not suppress the spontaneous release of FSH from cultured pituitary cells, but diminishes the LH-RH-stimulated release of both LH and FSH. Furthermore, both follicular fluid and Sertoli cell culture medium can stimulate the release of FSH and LH from pituitary cells when these are cultured without addition of fetal calf serum. These results suggest that gonadal fluids contain several non-steroidal factors which can influence the release of gonadotrophins from pituitary cells.     

Feedback control of FSH secretion in the male rat

Site of feedback control of FSH secretion in the male rat was studied by measuring changes in serum LH, FSH and hypothalamic LH-RH by radioimmunoassay in rats after castration and after 500 rad X-irradiation to the testis. The rise in serum LH and FSH in castrated animals was associated with a significant fall in hypothalamic LH-RH 16 and 24 days after castration. Serum FSH rose significantly after X-irradiation without a significant change in serum LH or hypothalamic LH-RH content up to 30 days after irradiation. When pituitary halves from X-irradiated animals were incubated in vitro in the presence or absence of synthetic LH-RH, there was a significant rise in FSH (but not LH) released in the incubation medium in the absence of added LH-RH. The response of the pituitaries to LH-RH was, however, not different between control and irradiated rats. It is concluded that the testicular FSH-inhibitory substance acts predominantly at the pituitary gland on the LH-RH independent release of FSH.     

Effect of pre-incubation with different concentrations of LH-RH on subsequent LH release caused by supramaximally active amounts of LH-RH; role of LH-RH-induced protein synthesis.

Anterior pituitary glands from intact diestrous female rats were incubated for two consecutive periods of 3 hours. During the first period various submaximally active amounts of luteinizing hormone-releasing hormone (LH-RH) were added to the media, whereas during the second period a supramaximally active concentration of LH-RH was present. When during the second incubation period protein synthesis was inhibited by cycloheximide, the amount of luteinizing hormone (LH) released during that period was positively correlated to the concentration of LH present during the first incubation period. This relationship was not seen when cycloheximide was absent, or when cycloheximide was present throughout both periods. Total LH was not affected by LH-RH; thus no effect of LH-RH on LH synthesis was observed. It is concluded that the amounts of protein synthesized by the pituitary glands in response to the different amounts of LH-RH during the first incubation period can constitute a limiting factor for the response to the supramaximally active amount of LH-RH added during the second incubation period.     

Comparison between the biological effects of LH-RH and buserelin on the induction of LH release from hemi-pituitary glands of female rats in vitro

The biological effects of LH-RH and the agonist [D-Ser(But)6-des Gly10]-LH-RH(1-9)-ethylamide (buserelin) were compared during 8 h of incubation with female rat hemi-pituitary glands. Similar dose-response relationships were found for LH-RH and buserelin as concerns the release of luteinizing hormone (LH) by pituitary glands from intact and ovariectomized rats. Also the LH secretion patterns from glands of intact rats were similar: an initial low response was followed by a fast increase (priming effect) after which the response declined again (desensitization). In a subsequent experiment pituitary glands from ovariectomized rats were first exposed to LH-RH or buserelin for 4 h and then further incubated in medium only. After discontinuation of the stimuli the rate of LH release decreased in all cases, but this decrease was significantly greater when the glands had been exposed to LH-RH. Short-term (1/2, 1 or 2 h) exposures to LH-RH or buserelin followed by an intervening period (1 1/2, 1 or 0 h, respectively) of incubation in medium only resulted in an almost similar, significant increase in the subsequent protein synthesis-independent LH response to LH-RH (priming effect). Only preincubation with LH-RH for 2 h was significantly more effective. The results demonstrate equal intrinsic activities for LH-RH and buserelin. Differences in the biopotencies for LH-RH and buserelin in vivo and in vitro may occur only after discontinuation of the external stimuli.     

Inhibin production by Sertoli cells in culture.

Studies on the secretion of inhibin and its mode of action were carried out in vitro, utilizing cell cultures. Isolated rat Sertoli cells secreted an inhibin-like heat-labile, non-dialysable substance, Sertoli Cell Factor (SCF), which could selectively suppress FSH secretion by rat anterior pituitary cells. SCF selectively suppressed the basal and GnRH-stimulated FSH release as well as the de-novo synthesis of FSH by acting directly on the pituitary cells. In 1 out of 5 experiments, SCF also suppressed the synthesis of LH, possibly by affecting the overall protein synthesis. Under similar culture conditions, Sertoli cells isolated from animals between 18 and 90 days of age secreted comparable amounts of SCF. In contrast, anterior pituitary cells from adult rats (60-90 days old) were considerably more sensitive to SCF than pituitary cells obtained from younger (18-33 days old) animals, suggesting that decline in circulating FSH level, occurring at approximately 35 days of age, may result from increased pituitary sensitivity to inhibin. Besides identifying the Sertoli cells as the site of inhibin production in the testis, these studies demonstrated direct action of inhibin at the pituitary cell level, resulting in suppression of FSH synthesis and release.     

Intracellular mechanism for the action of inhibin on the secretion of the follicular-stimulating hormone and luteinizing hormone induced by LH-RH in vitro

The FSH secretion-inhibiting action of inhibin in vitro under basal conditions and also in the presence of LH-RH is suppressed by the addition of MIX, a phosphodiesterase inhibitor. In the presence of LH-RH, inhibin reduces significantly the intracellular level of cAMP in isolated pituitary cells. In contrast, the simultaneous addition of MIX and inhibin raises the cAMP level, and this stimulation is comparable to the increase observed when MIX is added alone. These observations suggest that one mode of action of inhibin could be mediated by a reduction in cAMP within the pituitary gonadotropic cell.     

Monolayer cultures of dispersed cells from bovine anterior pituitary: responses to synthetic hypophysiotropic peptides.

Cells were dispersed from bovine anterior pituitary glands, by digestion with collagenase, and cultured. After 4 days the cell monolayers were incubated with fresh medium containing synthetic hypophysiotropic peptides for 2, 6, or 20 h, and hormone released into the medium was estimated by radioimmunoassay. After 2 h, thyroid releasing hormone (TRH) stimulated the release of thyroid-stimulating hormone (TSH) up to eightfold, and of prolactin (PRL) and follicle-stimulating hormone (FSH) about twofold at a minimal effective concentration of 1 ng/ml; enhanced growth hormone (GH) release was not apparent until 20 h, and release of luteinizing hormone (LH) and adrenocorticotrophic hormone (ACTH) was unaffected. Luteinizing hormone releasing hormone (LH-RH) enhanced release of LH maximally (three- to fourfold) during a 2 h incubation and was effective at 0.1 ng/ml; FSH release was significantly enhanced by about 50% above control level. Growth hormone release inhibiting hormone (GH-RIH)(somatostatin) showed significant effects only in the 20 h incubation; GH release was inhibited by 50% and release of PRL was slightly, but significantly, enhanced. Pituitary cell monolayers apparently permit maximal expression of releasing activities inherent in the hypothalamic hormones.     

Effects of acute ovariectomy on anterior pituitary gland follicle-stimulating hormone and luteinizing hormone secretion in the metestrous rat

Changes at the anterior pituitary gland level which result in follicle-stimulating hormone (FSH) release after ovariectomy in metestrous rats were investigated. Experimental rats were ovariectomized at 0900 h of metestrus and decapitated at 1000, 1100, 1300, 1500, 1700 or 1900 h of metestrus. Controls consisted of untreated rats killed at 0900 or 1700 h and rats sham ovariectomized at 0900 h and killed at 1700 h. Trunk blood was collected and the serum assayed for FSH and luteinizing hormone (LH) concentrations. The anterior pituitary gland was bisected. One-half was used to assay for FSH concentration. The other half was placed in culture medium for a 30-min preincubation and then placed in fresh medium for a 2-h incubation (basal FSH and LH release rates). The basal FSH release rate and the serum FSH concentration rose significantly by 4 h postovariectomy and remained high for an additional 6 h. The basal FSH release rate and the serum FSH concentration correlated positively (r=0.71 with 72 degrees of freedom) and did not change between 0900 and 1700 h in untreated or sham-ovariectomized rats. In contrast, the serum LH concentration and the basal LH release rate did not increase after ovariectomy. Ovariectomy had no significant effect on anterior pituitary gland FSH concentration. The results suggest that the postovariectomy rise in serum FSH concentration is the result, at least in part, of changes which cause an increase in the basal FSH secretion rate (secretion independent of the immediate presence of any hormones of nonanterior pituitary gland origin). The similarities between the selective rises in the basal FSH release rate and the serum FSH concentration in the ovariectomized metestrous rat and in the cyclic rat during late proestrus and estrus raise the possibility that an increase in the basal FSH release rate may be involved in many or all situations in which serum FSH concentration rises independently of LH.     

Effect of serotonin on the basal and gonadotrophin-releasing hormone-induced release of luteinizing hormone from rat pituitary glands in vitro

The effect of serotonin (5-HT) on the basal and gonadotrophin-releasing hormone (GnRH)-stimulated release of luteinizing hormone (LH) was studied in rat adenohypophysis in vitro. Anterior pituitary glands from ovariectomized rats were incubated for 1h in the presence of different doses of 5-HT (0.01 to 3 mumol/l). Serotonin added to the culture medium slightly dimished the basal release of LH and markedly inhibited the release of LH induced by GnRH. Responsiveness to GnRH (3 nmol/l) was significantly reduced, in a dose-dependent manner, by the simultaneous treatment of glands with 5-HT. Maximal inhibition to 65% of the response obtained with GnRH alone, was attained with 1 mumol/l 5-HT. The EC50 value was estimated to be about 1.9 X 10(-7) M. The inhibitory effect of 5-HT was evident within 30 min of incubation. Furthermore, 5-HT appear to exert a short-lasting action, since the rate of basal and GnRH-induced release of LH was reduced during the first hour of incubation, but after 2h the suppressive effects of 5-HT were no longer apparent. Methysergide, a serotonin receptor blocking agent, partially antagonized the inhibitory effect of 5-HT on LH release, either basal or GnRH-stimulated. This suggests that a receptor-mediated component may be involved in the mechanism of 5-HT action. The present results indicate that 5-HT can affect the release of LH by acting directly at the pituitary gland level.     

Effects of LH-RH on isolated pituitary gonadotropic cells.

Rat anterior pituitary glands were dissociated with Pronase and the cells were separated by velocity sedimentation at unit gravity. After 30 min of incubation of the enriched gonadotropic cells with LH-RH, there was a significant increase in LH and FSH in the incubation medium. LH-RH (100 ng/ml) and 10(-3) M cAMP both caused significant increases in LH in the incubation medium after 24 hr of incubation.     

Stimulatory effect of Sertoli cell culture medium on the FSH release by pituitary halves in vitro

The influence of the medium collected from cultured rat Sertoli cells on the spontaneous and LHRH-stimulated release of gonadotropins by incubated rat pituitary halves was examined. The homogeneity of the cultured population of Sertoli cells taken from 20-day-old rats ranged up to 98%. The cells in culture responded to FSH stimulation with characteristic morphological changes and with increased secretion of estradiol-17 beta. The hemi-pituitaries obtained from sexually mature male rats were incubated for 5 hours in the presence of Sertoli cell culture medium (SCCM) or its fractions obtained by use of ultrafiltration. The SCCM fraction deprived of MW less than 10 kD compounds exhibited a typical inhibin-like activity, whereas crude SCCM as well as its low-molecular-weight fraction stimulated the basal FSH release to about 150% and 175% of the control values, respectively. These fractions exerted an inhibitory effect on the LHRH-stimulated secretion of both LH and FSH. It is concluded that Sertoli cells cultured in chemically defined medium release, apart from inhibin, a non-steroidal, heat-labile substance of MW less than 10 kD which stimulates the basal secretion of FSH and LH and inhibits the LHRH-stimulated secretion of both gonadotropins from incubated rat hemi-pituitaries.     

Identification in human seminal fluid of an inhibin-like factor which selectively regulates FSH secretion.

Human seminal plasma obtained by centrifugation of human semen contains a factor capable of selectively inhibiting the secretion of FSH both in vivo (reduction of the levels of FSH in rats 24 h after castration) and in vitro (reduction of the FSH released by LH-RH in rat pituitary cell culture). This effect is not due to testosterone, oestradiol-17 beta or progesterone present in the active fractions. The factor has the characteristics of a protein in that its biological activity is destroyed by heat and trypsin digestion. It does not resemble androgen-binding protein. The biological action is not completely specific for FSH as inhibition of LH can be seen with doses usually higher than those which produce inhibition of FSH alone. There is no effect on TSH or prolactin levels in vitro. The factor clearly acts on the release and synthesis of gonadotrophins by gonadotrophs but an effect on the hypothalamus is not excluded. This factor fits the definition of inhibin.     

An increase of pituitary 3', 5' cyclic adenosine monophosphate produced by estradiol benzoate in vitro: possible implication of this increase in the secretion of luteinizing hormone.

In an attempt to study the site and mechanism of action of estrogen in producing positive feedback control, porcine anterior pituitary slices were incubated in vitro in the presence of estradiol benzoate (EB). EB elevated pituitary cyclic AMP concentration within 5 min and augmented pituitary release of luteinizing hormone (LH). The magnitude of increase of cyclic AMP and LH release was related to the doses of EB used. Also, luteinizing hormone releasing hormone (LH-RH) elevated pituitary cyclic AMP concentration and stimulated pituitary release of LH. The magnitude of increase of cyclic AMP and LH release was inversely related to the doses of LH-RH used. EB and LH-RH were additive in increasing cyclic AMP. Progesterone and clomiphene citrate interfered with an increase of pituitary cyclic AMP produced by EB, but did not significantly affect the basal level of pituitary cyclic AMP. Testosterone propionate, human chorionic gonadotropin and hexestrol were without effect on either basal or stimulated level of pituitary cyclic AMP. Since cyclic AMP and dibutyryl cyclic AMP (DBC) stimulated LH release, it is suggested that EB directly stimulates the release of LH by augmenting cyclic AMP synthesis in the anterior pituitary.     

Stimulatory and inhibitory effects of progesterone on FSH secretion by the anterior pituitary.

The purpose of this study was to investigate whether progesterone exerted progesterone receptor mediated direct effects on the anterior pituitary in the secretion of FSH and whether such effects were mediated through the 5 alpha-reduction of progesterone. Treatment of anterior pituitary dispersed cells for 48 h with 0.5 nM estradiol reduced the ED50 for gonadotropin releasing hormone (GnRH)-stimulated FSH release from 0.58 to 0.36 ng/ml and the ED50 for GnRH-induced LH release from 0.54 to 0.19 ng/ml. When dispersed pituitary cells were treated with 0.5 nM estradiol and exposed to various doses of progesterone for 1 to 6 h, the most consistent rise in basal and GnRH-stimulated FSH release was observed with the 50 nM dose of progesterone with a 3-h exposure period. All three doses of progesterone elevated basal LH and GnRH-stimulated LH was increased by the 50 and 100 nM doses of progesterone during the 3-h period of treatment. Using the 50 nM dose of progesterone, basal and GnRH-stimulated LH was increased after 2, 3 and 6 h of progesterone treatment. When the period of exposure of progesterone was extended to 12, 36 or 48 h, there was a significant inhibition of GnRH-stimulated FSH release. GnRH-stimulated LH release was inhibited at 36 and 48 but not 12 h after progesterone treatment. These studies showed that the effect of progesterone administered for periods of 1 to 6 h enhanced the secretion of LH and FSH whereas progesterone administered for periods beyond 12 h inhibited FSH and LH release by dispersed pituitary cells in culture. These results are similar to those observed in vivo after progesterone treatment. Furthermore estrogen priming of the dispersed pituitary cells was necessary to observe the effects of progesterone. The progesterone antagonist RU486 prevented the progesterone-induced rise in GnRH-stimulated FSH release. Furthermore the 5 alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane- 17 beta-carboxamide also prevented the progesterone-induced rise in GnRH-stimulated FSH release in estrogen-treated dispersed pituitary cells. These results indicate that the anterior pituitary is a major site of action of progesterone in the release of FSH and that 5 alpha-reduction of progesterone plays an important role in FSH release.     

Dopamine inhibition of prolactin release but not synthesis in the male Syrian hamster: in vitro studies

The hypothalamic mechanisms controlling prolactin (PRL) cell function in the male Syrian hamster are unclear. Equally unclear is the role of dopamine (DA) in regulating lactotrophic cell activity in long photoperiod-exposed hamsters particularly with respect to PRL synthesis and release. The synthesis of PRL, as measured by the incorporation of 3H-leucine into newly synthesized PRL, by anterior pituitary glands from male hamsters is linear over a five h incubation period. Approximately two-fold more 3H-PRL remained in the pituitary glands than in the medium by the end of the incubation period. The incubation of hamster hemipituitaries with DA at concentrations of either 5 X 10(-7) M or 5 X 10(-5) M, resulted in a 77% to 83% inhibition of the release of immunoreactive PRL into the medium as compared with controls. Similarly, the release of 3H-PRL into the medium was inhibited by 71% to 76% as compared with controls; however, the synthesis of PRL was virtually the same among the experimental and control groups. These results suggest that DA may be an important regulator of short-term PRL release but not synthesis in the long photoperiod-exposed male hamster.     

Site of the in vivo stimulatory effect of prostaglandins on LH release

A possible direct effect of prostaglandins E₁ and E₂ (PGE₁ and PGE₂) on luteinizing hormone (LH) release at the pituitary level was studied using anterior pituitary cells in primary culture, a system approximately 10-fold more sensitive to stimulation of LH release than previously used hemipituitaries. No effect of PGE₁ or PGE₂ could be detected on the time course of basal or LH-RH-stimulated LH release or on the LH responsiveness to LH-RH. This absence of a direct effect of PGEs at the pituitary level on LH release was confirmed by experiments using female rats under Surital anesthesia in the afternoon of proestrus. After intravenous injection, under these conditions, 15(S)-15-methyl PGE₂ was 3–5 times more potent than PGE₂ to increase plasma LH levels while PGE₁ had about 50% the potency of PGE₂. Injection of sheep anti-LH-RH serum one hour before PGE₁ or PGE₂ injection not only lowered basal plasma LH levels but prevented the rise induced by PGEs. These data indicate clearly that the increased plasma LH levels observed after PGE injection are secondary to a stimulation of LH-RH release while PGEs do not appear to have a significant effect on LH release at the pituitary level.     

A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat

Experiments were undertaken to investigate if changes occur at the level of the anterior pituitary gland to result in selective follicle-stimulating hormone (FSH) release during late proestrus in the cyclic rat. At 1200 h proestrus, prior to the preovulatory luteinizing hormone (LH) surge in serum and the accompanying first phase of FSH release, serum LH and FSH concentrations were low. At 2400 h proestrus, after the LH surge and shortly after the onset of the second or selective phase of FSH release, serum LH was low, serum FSH was elevated about 4-fold, pituitary LH concentration was decreased about one-half and pituitary FSH concentration was not significantly decreased. During a two hour $\text{in}$$\text{vitro}$ incubation, pituitaries collected at 2400 h released nearly two-thirds less LH and 2.5 times more FSH than did pituitaries collected at 1200 h. Addition of luteinizing hormone releasing hormone (LHRH) to the incubations caused increased pituitary LH and FSH release. However, the LH and FSH increments due to LHRH in the 2400 h pituitaries were not different from those in the 1200 h pituitaries. The results indicate that a change occurs in the rat anterior pituitary gland during the period of the LH surge and first phase of FSH release which results in a selective increase in the basal FSH secretory rate. It is suggested that this change is primarily responsible for the selective increase in serum FSH which occurs during the second phase of FSH release.     

Parellel inhibition of LH-RH-induced cyclic AMP accumulation and LH and FSH release by LH-RH antagonists in vitro.

The relative potencies of seven antagonists of LH-RH to inhibit LH-RH-induced cyclic AMP accumulation and LH and FSH release were measured using rat hemipituitaries in vitro. At appropriate concentrations, [Des-His2, D-Ala6] LH-RH, [Des-His2, D-Ala6, des-Gly-NH210] LH-RH ethylamide, [Des-His2, D-Leu6] LH-RH, [D-Phe2] LH-RH, [Des-His2, Des-Gly-NH210] LH-RH propylamide, [D-Phe2, D-Leu6] LH-RH and [D-Phe2, D-Phe6] LH-RH led to parallel inhibition of cyclic AMP accumulation and LH and FSH release. [D-Phe2, D-Leu6] LH-RH and [D-Phe2, D-Phe6] LH-RH can inhibit 50% of LH-RH action at molar ratios of 100 and 30, respectively. These findings of parallel changes of cyclic AMP levels and LH and FSH release add strong support to the already obtained evidence for a mediator role of the adenylate cyclase system in the action of LH-RH in the anterior pituitary gland.     

Investigation into the hypogonadism of the obese mouse (genotype ob/ob)

Features of the reproductive axis in the genetically hypogonadal, obese mouse (genotype, ob/ob) were examined at 5-8 months of age and compared with those of wild-type litter mates. Hypothalamic concentrations of dopamine and 5-hydroxytryptamine were normal. Those of 5-hydroxyindoleacetic acid, noradrenaline and LH-RH were raised. LH-RH was biologically active. Pituitary concentration of LH was normal, but that of FSH was raised. Serum concentrations of LH and FSH, compared with those of wild-type animals, were normal and low, respectively. Gonad and accessory sex organs weights were reduced. These findings suggest that the release of FSH but not LH is defective in the ob/ob mouse. Preliminary in-vitro experiments indicated that the pituitary gland responded normally or even supernormally towards LH-RH in its release of LH. The defect in the reproductive axis of the obese mouse may be due to inadequate release of LH-RH although an insensitivity of the pituitary gland towards LH-RH in its release of FSH cannot be excluded.     

Follicle-stimulating hormone within and secreted from anterior pituitaries of female golden hamsters during the estrous cycle and after ovariectomy

Anterior pituitary (AP) glands were removed from groups of female golden hamsters at 0900 h on estrus (E), diestrus I (DI), and diestrus II (DII) and at 1200 h and 1500 h on proestrus (P12 and P15), as well as at 0900 h from ovariectomized hamsters (OVX). Hemipituitaries were incubated in culture medium with or without 10(-8) M luteinizing hormone-releasing hormone (LHRH) for 3 h at 37 degrees C to determine the magnitude of basal and LHRH-stimulated follicle-stimulating hormone (FSH) release. All samples were assessed for FSH activity by radioimmunoassay and radioreceptor assay. In a second set of experiments, AP were removed from E, DII, and OVX hamsters and bisected. One hemipituitary was homogenized in 10 mM Tris-HCl and the other half was incubated for 3 h. Follicle-stimulating hormone forms present within pituitary extracts or secreted into medium were separated by an isoelectric focusing technique, chromatofocusing. Basal FSH release was lowest in AP collected on DII and P12, higher in AP collected on E and DI, and highest in AP from OVX. Luteinizing hormone-releasing hormone-stimulated release of FSH was highest in AP obtained on DII and P12, lower in AP collected on E and DI, and lowest in AP from OVX. Radio-receptor-to-radioimmunoassay ratio of secreted FSH was greatest when basal FSH secretion was low and LHRH sensitivity was high (DII and P12) and least when basal FSH secretion was high and LHRH sensitivity low (E and after OVX).(ABSTRACT TRUNCATED AT 250 WORDS)