Interactions of ovarian steroids with pituitary adenylate cyclase-activating polypeptide and GnRH in anterior pituitary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) releases LH and FSH from anterior pituitary cells. Although this effect is relatively weak, it has a strong sensitizing action on GnRH-induced gonadotropin secretion. Here we investigated the possibility that ovarian steroids, which are well-known modulators of LH secretion, interact with PACAP and GnRH in pituitary gonadotrophs. Rat pituitary cells were treated for 48 h with vehicle, 1 nmol/l estradiol, 1 nmol/l estradiol + 100 nmol/l progesterone or 48 h with 1 nmol/l estradiol and 4 h with 100 nmol/l progesterone. The cells were stimulated for 3 h with 1 nmol/l GnRH or 100 nmol/l PACAP. Estradiol treatment alone enhanced basal as well as GnRH- or PACAP-stimulated LH secretion. LH release was facilitated by additional short-term progesterone treatment. Long-term treatment with estradiol and progesterone led to reduced LH responses to GnRH and PACAP. Neither treatment paradigms affected cAMP production. However, estradiol treatment led to enhanced cAMP accumulation in quiescent or GnRH-stimulated cells. PACAP-induced increases of cAMP production were inhibited by estradiol treatment. After 7-h preincubation with 10 nmol/l PACAP, cells responded with enhanced LH secretion to GnRH stimulation. When steroid pretreatment was performed the responsiveness of gonadotrophs to low concentrations of GnRH was still increased. In contrast, at high concentrations of GnRH the sensitizing action of PACAP on agonist-induced LH secretion was lost in steroid-treated cells. There were no significant differences between the steroid treatment paradigms. It is concluded that estradiol but not progesterone acts as a modulator of adenylyl cyclase in gonadotrophs. The stimulatory effect of estradiol is thought to be involved in its sensitizing action on agonist-induced LH secretion. The inhibitory effect of estradiol on PACAP-stimulated adenylyl cyclase activities seems to be responsible for the loss of its action to sensitize LH secretory responses to GnRH.     

Short-term effects of IGF-I and estradiol on LH secretion from female rat gonadotrophs

OBJECTIVE: Growth factors and ovarian steroids modulate LH-secretion from pituitary gonadotrophs. Our previous studies demonstrated that long-term IGF-I treatment enhanced LH-secretion from female rat pituitary cells and estradiol facilitated this effect. The effects of estradiol on LH secretion are time-dependent. Short-term treatment inhibited, long-term treatment enhanced GnRH-induced LH-secretion in serum-containing medium. Here we tested the short-term actions of IGF-I and its interaction with estradiol and whether IGF-I is a prerequisite for the negative effect of short-term estradiol treatment in female rat pituitary cells. DESIGN: Pituitary cells were incubated with a series of increasing concentrations of estradiol (1 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM and 100 nM) for 4 h, IGF-I (10 pM, 100 pM, 1 nM and 10 nM) for 4 h and 14 h and their combinations for 4h in serum-free medium, and then stimulated with 1 nM GnRH during the last 3h of incubation. To clarify the role of IGF-I, cells were incubated simultaneously with estradiol, IGF-I and antibody against IGF-I. LH was measured by radioimmunoassay. RESULTS: Short-term IGF-I treatment did not modify basal or GnRH-induced LH-secretion. Short-term treatment with estradiol did not affect basal or GnRH-induced LH-secretion in serum-free medium. The addition of 100 pM IGF-I to serum-free medium established the negative effect of estradiol short-term treatment on GnRH-induced LH-secretion. The addition of IGF-I antibody fully abolished the negative effect of estradiol. CONCLUSIONS: In conclusion, effects of IGF-I on LH-secretion in female rat pituitary cells require long-term treatment. The negative effect of estradiol short-term treatment on GnRH-induced LH-secretion is dependent on serum-containing medium or the addition of 100 pM IGF-I to serum-free medium.     

Inhibitory actions of keoxifene on luteinizing hormone secretion in pituitary gonadotrophs

The effect of keoxifene (LY 156 758) on GnRH-stimulated LH release and its ability to antagonize estrogen actions were investigated in rat anterior pituitary cells. Estrogens exert either stimulatory or inhibitory effects on GnRH-induced LH secretion in rat pituitary cells depending on the incubation time with the steroid. When pituitary cells were treated for 24 h with 10(-9) M estradiol, the LH response to GnRH was clearly enhanced, and this effect was completely inhibited by 300 nM keoxifene. Short term treatment (4 h) of pituitary cells with 10(-9) M estradiol inhibits GnRH-stimulated LH release, and this effect was also blocked by keoxifene in a dose-dependent manner. In the absence of exogenous estrogen the treatment of pituitary cells for 4 h with increasing concentrations of keoxifene reduced the LH response to 10(-9) M GnRH only at very high concentrations (10(-5) M) of the antiestrogen. After treatment for 24 h, the inhibitory effect of keoxifene was evident at concentrations greater than or equal to 10(-8) M, with a reduction of GnRH-induced LH release by up to 60%. The effects of the antiestrogen were also analyzed in a dynamic culture system, in which pituitary cells grown on microcarrier beads were continuously perifused with medium and stimulated with GnRH in a pulsatile fashion. The LH response to a 2 min pulse of 10(-9) M GnRH was reduced in magnitude after 40 min of perifusion with 10(-9) M estradiol. When keoxifene (300 nM) was present at the same time, the LH response was identical to that observed in vehicle-treated cells. At the concentration of 300 nM, keoxifene per se did not change the responsiveness of the pituitary cells to the GnRH stimulus. These findings show that keoxifene is a potent antagonist of both positive and negative estrogen actions in the pituitary gonadotroph. In addition, after short term treatment with high concentrations or after long term treatment, keoxifene itself exerts an inhibitory effect on GnRH-induced LH secretion.     

Gonadal steroid modulation of signal transduction and luteinizing hormone release in cultured chicken pituitary cells

The mechanism whereby gonadal steroids modulate GnRH-stimulated LH secretion by primary cultures of chicken pituitary cells was investigated. Estradiol (10(-8) M), testosterone (10(-7) M), and progesterone (10(-7) M) inhibited LH release stimulated by GnRH (10(-7) M) by 56%, 61%, and 53%, respectively, and the inhibitory effects required prolonged preincubation (24-48 h) with the steroids. The steroids inhibited the spike (0-3 min) and plateau (9-30 min) phases of LH release to a similar degree. The ED50 values of estradiol, testosterone, and progesterone for inhibition of GnRH-stimulated LH release were 7 x 10(-11), 2 x 10(-9), and 1 x 10(-9) M, respectively. Estradiol, testosterone, and progesterone inhibited the maximal LH response to GnRH, but the ED50 of GnRH (4 x 10(-9) M) was not altered by steroid pretreatment. Steroid pretreatment did not cause a change in cellular LH content, suggesting that the steroids do not inhibit LH synthesis. Combinations of two or three of the steroids were not additive, suggesting that all three steroids affect GnRH-stimulated LH release via the same mechanism. In experiments investigating their mechanism of action, the steroids inhibited LH release stimulated by GnRH and Ca2+ ionophore A23187, but generally had no effect on the responses to phorbol ester (12-O-tetradecanoylphorbol-13-acetate), forskolin, K+, Bay K8644, or veratridine. Estradiol inhibited GnRH-stimulated 45Ca2+ efflux, but its inhibitory effect on GnRH-induced inositol phosphate production was not significant. Estradiol had no effect on binding of 125I-[His5,D-Tyr6]GnRH to a pituitary cell preparation. These findings suggest that the site of steroid modulation of GnRH action is distal to binding of GnRH to its receptor, and that the inhibitory effects are exerted at two intracellular sites: 1) the coupling events linking receptor activation to mobilization of Ca2+, and 2) a site distal to Ca2+ mobilization.     

Interaction between cortisol and arachidonic acid on the secretion of LH from ovine pituitary tissue.

In several species, glucocorticoids act directly on the pituitary gonadotroph to suppress the gonadotrophin-releasing hormone (GnRH)-induced secretion of the gonadotrophins, especially LH. A mechanism for this action of these adrenal steroids has not been established, but it appears that the glucocorticoids influence LH release by acting on one or more post-receptor sites. This study investigated whether glucocorticoids disrupt GnRH-induced LH release by altering the liberation of arachidonic acid from plasma membrane phospholipids, a component of GnRH-induced LH release. Using perifused ovine pituitary tissue, it was established that exposure of gonadotrophs to 1-1000 nmol cortisol/l for 4 h or longer significantly reduced GnRH-stimulated LH release with the maximal inhibitory effect being observed after 6 h of exposure to cortisol. This suppressive effect of cortisol could be reversed by administration of arachidonic acid, which in its own right could stimulate LH release from ovine pituitary tissue. Furthermore, the inhibitory effect of cortisol on GnRH-stimulated LH release could be directly correlated with decreased pituitary responsiveness to GnRH-stimulated arachidonic acid liberation, consistent with our hypothesis that glucocorticoids can suppress GnRH-induced secretion of LH by reducing the amount of arachidonic acid available for the exocytotic response of GnRH.     

Effects of gonadal steroids on gonadotropin secretion in males: studies with perifused rat pituitary cells

The feedback effects of testosterone (T) and estradiol (E2) on FSH and LH secretion were compared in dispersed pituitary cells from adult male rats perifused with pulses of GnRH. Cells were stimulated with 10 nM GnRH for 2 min every 1 h. T (10 nM) pretreatment for 24 h reduced the amplitude of FSH and LH pulses to 77 +/- 4% (mean +/- SE) and 47 +/- 3% of control values, respectively (P less than 0.01), whereas 6-h T treatment was without effect. By contrast, interpulse secretion of FSH was increased after 24 h T to 184 +/- 7% of the control value (P less than 0.01), but interpulse LH release was unchanged (104 +/- 5%). E2 (0.075 nM) treatment of pituitary cells reduced GnRH-stimulated FSH and LH release within 2 h to 75 +/- 2% and 73 +/- 3% of control values, respectively (P less than 0.01). E2 pretreatment for 24 h stimulated (P less than 0.025) GnRH-induced FSH (136 +/- 10%) and LH (145 +/- 8%) release and also increased (P less than 0.01) interpulse FSH (127 +/- 5%) and LH (145 +/- 8%) secretion. These data indicate that the suppression of FSH and LH secretion by T in males is due in part to a direct effect on the pituitary. The findings that T suppresses GnRH-stimulated FSH less than LH, and that T stimulates interpulse FSH, but not LH, provide evidence for differential regulation of FSH and LH secretion by T. The dissimilar actions of T on GnRH-stimulated pulses and interpulse gonadotropin secretion suggest that interpulse secretion is unrelated to stimulation by GnRH, although its physiological significance is unknown. Since E2, in physiological levels for males, increased pituitary FSH and LH secretion, the suppression of gonadotropin secretion by E2 in vivo in males may result from an effect on the hypothalamic pulse generator; however, additional studies are needed before extending these conclusions to higher mammals and men.     

Effects of inhibin from primate Sertoli cells on follicle-stimulating hormone and luteinizing hormone release by perifused rat pituitary cells

We used a pituitary cell perifusion system to investigate the time course and selectivity of the inhibin effect on pulsatile GnRH-stimulated LH and FSH release. Dispersed pituitary cells from 7- to 8-week-old male rats were perifused on a Cytodex bead matrix and stimulated with 10 nM GnRH for 2 min every hour for 8-11 h. The addition of a preparation of inhibin partially purified from primate Sertoli cells reduced pulsatile FSH release within 2 h. After removal of inhibin from the perifusion medium, the effect was reversed within 3 h. GnRH-stimulated LH release was also influenced by inhibin, although the decline in LH was less than that in FSH (80 +/- 3% vs. 68 +/- 4% of control; P less than 0.025). Smaller doses of inhibin suppressed GnRH-induced FSH secretion, but had no effect on LH release. Further, prolonged incubation of pituitary cells with inhibin at the higher dose reduced its FSH inhibitory effect and eliminated the effect on LH. These results indicate that inhibin can reduce both LH and FSH secretion in vitro, although the specificity and magnitude of the effect are a function of both the dose and duration of inhibin treatment. Further, the actions of inhibin and GnRH on the pituitary may be interrelated.     

Gonadotrophin surge-attenuating factor attenuates in-vitro LH secretion induced by gonadotrophin-releasing hormone from cultured ovine pituitary cells only during the breeding season.

Primary cultures of ovine pituitaries from adult ewes were used to investigate aspects of gonadotrophin surge-attenuating factor (GnSAF) bioactivity in human follicular fluid (hFF) from superovulated women. During the autumn and first half of the winter, LH secretion induced by gonadotrophin-releasing hormone (GnRH) was markedly reduced (43.5 +/- 5.2% of control GnRH-induced LH secretion) by incubation for 48 h with steroid-free hFF. For the rest of the year, treatment with the same batch of steroid-free hFF resulted in non-significant reduction or stimulation of GnRH-induced LH secretion (71.3 +/- 13.2 to 117.8 +/- 11.2% of control GnRH-induced LH secretion). Incubation of pituitary cells for 48 h with oestradiol (1 pmol/l to 1 mumol/l), progesterone (1 pmol/l to 1 mumol/l) or oestradiol and progesterone combined (1 pmol/l to 1 mumol/l) in a two-way titration for 48 h had no significant effect on GnRH-induced LH secretion (83.4 +/- 7.6 to 110.6 +/- 5.0% of control secretion). Separating hFF into fractions of different molecular mass by ultrafiltration demonstrated that GnSAF bioactivity was present in a form 10-30 kDa in size. Incubation for 48 h with these fractions had no significant effect on basal FSH secretion but significantly attenuated GnRH-induced LH secretion during the autumn. The same fractions had little effect on GnRH-induced LH secretion from pituitary cells collected during the summer. We conclude that ovine pituitaries display at least partial reduction in sensitivity to GnSAF outside the breeding season.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effects of oestradiol and progesterone on melittin- and gonadotrophin-releasing hormone-induced LH secretion.

It is well established that oestradiol and progesterone modulate gonadotrophin-releasing hormone (GnRH)-induced LH secretion from cultured rat pituitary cells. Short-term oestradiol and long-term progesterone treatment exert inhibition, while short-term progesterone and long-term oestradiol treatment lead to enhancement of GnRH-stimulated LH secretion. There are several lines of evidence to suggest that the steroid effects might be mediated via a mechanism involving modulation of the GnRH signal-transduction system. To evaluate the role of arachidonic acid, which serves as an intracellular signal transducer by itself or its lipoxygenase metabolites, in the mediation of oestradiol and progesterone actions, we examined their effects on melittin (activator of phospholipase (A2)-stimulated LH secretion. When pituitary cells from adult female rats were treated for 48 h with 1 nmol oestradiol/l or 1 nmol oestradiol/l plus 100 nmol progesterone/l, GnRH (1 nmol/l)-induced LH secretion was stimulated or inhibited respectively. However, melittin (10-300 nmol/l)-stimulated LH secretion remained unaffected after such treatment. Short-term treatment with oestradiol inhibited GnRH-induced LH secretion while progesterone treatment of oestradiol-primed cells led to a stimulatory effect. Interestingly, melittin-stimulated LH secretion was influenced in the same way after the short treatment paradigm. Perifusion studies were performed to assess the kinetics of these acute steroid actions further. Four separate perifusion chambers were continuously perifused with medium and stimulated for 2 min with 1 nmol GnRH/l or 1 mumol melittin/l every 50 min in a pulsatile fashion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol differentially modulates the exocytotic proteins SNAP-25 and munc-18 in pituitary gonadotrophs

Long-term treatment with estradiol increases LH secretion from female gonadotrophs. The mechanisms are not fully clarified yet. Our previous data indicated that sexual steroids might affect late steps in GnRH signal transduction such as exocytosis. The secretion of hormones from neuroendocrine cells requires the merger of secretory vesicles with the plasma membrane. This regulated exocytosis is mediated by specific proteins, which are present in the pituitary gland. Here, we examined whether two of these crucial exocytotic proteins, SNAP-25 and munc-18, are affected by estradiol in female gonadotrophs. Female rat anterior pituitary cells and alphaT3-1 cells, derived from a murine immortalized gonadotroph cell line, were treated with 100 pM estradiol for 48 h. LH secretion of anterior pituitary cells, additionally stimulated with eight consecutive pulses of 1 nM GnRH for 15 min at an interval of 1 h, was determined by RIA. Gene expression was measured by quantitative RT-PCR and protein expression by immunoblotting. Additionally, quantitative RT-PCR was performed in single rat gonadotrophs to ascribe effects exclusively to intact gonadotrophs. Pulsatile GnRH enhanced the mRNA expression of SNAP-25 and munc-18 in accordance with the LH secretory response with the greatest increase at the third pulse of GnRH. Estradiol treatment further increased GnRH-induced LH secretion at all GnRH pulses. SNAP-25 gene expression was significantly decreased at the fifth GnRH pulse and unaffected at basal after 48 h of estradiol treatment. In contrast, munc-18 mRNA levels were not significantly affected by estradiol at different GnRH-pulses in mixed anterior pituitary cells, whereas munc-18 gene expression was significantly increased at basal. In alphaT3-1 cells and single gonadotrophs, long-term estradiol treatment significantly reduced SNAP-25 protein and gene expression. In contrast, the protein and gene expression of munc-18 was significantly enhanced in both alphaT3-1 cells and single gonadotrophs. In conclusion, munc-18 and SNAP-25 were oppositionally influenced by estradiol. The results suggest that estradiol modulates the expression of exocytotic proteins in gonadotrophs and thus affects LH secretion.     

The effect of gonadotrophin surge-inhibiting factor on the self-priming action of gonadotrophin-releasing hormone in female rats in vitro.

The present study was designed to explore further the functional antagonism between gonadotrophin-releasing hormone (GnRH) and the ovarian factor, gonadotrophin surge-inhibiting factor (GnSIF). In all experiments, pituitary tissue was exposed to various amounts of GnSIF, after which the self-priming action of GnRH was studied. GnSIF was increased in vivo by FSH treatment and increased in vitro by adding various amounts of follicular fluid (FF) to cultured pituitary cells. Treatment with 3 or 10 IU FSH suppressed the initial LH response and delayed the maximally primed LH response to GnRH. Treatment with FSH was only effective in intact rats on days 1 and 2 of dioestrus. There was no difference in the rate of maximal LH release irrespective of treatment with either FSH or saline. Since FSH treatment was ineffective in long-term ovariectomized rats, it was concluded that the initial suppressive effect of FSH on LH release was mediated by GnSIF. Cycloheximide prevented the self-priming action of GnRH by inhibiting GnRH-induced protein synthesis. The initial protein synthesis-independent GnRH-stimulated LH release, which was already suppressed by FSH treatment, remained suppressed in the presence of cycloheximide. Pretreatment with GnRH in vivo increased the protein synthesis-independent GnRH-induced LH release during subsequent incubation of the glands. This increase did not occur after FSH treatment. Pituitary cells, cultured for 20 h in medium only, failed to elicit the self-priming effect of GnRH. Preincubation with FF maintained the self-priming effect. This was independent of the concomitant presence of various amounts of oestradiol. Preincubation with bovine FF suppressed the initial GnRH-stimulated LH release dose-dependently. Porcine FF, human FF and testicular extract suppressed the release of LH in a similar way. It was concluded that GnSIF suppresses the initial LH response to continuous GnRH stimulation. Increased levels of GnSIF caused by FSH treatment also delayed the primed LH release. The mechanism of functional antagonism between GnSIF and GnRH could give rise to the occurrence of the phenomenon of GnRH self-priming.     

Effects of bovine 35 kDa FSH-suppressing protein on FSH and LH in rat pituitary cells in vitro: comparison with bovine 31 kDa inhibin

The mode of action of a recently isolated gonadal protein, termed FSH-suppressing protein (FSP) or follistatin, on basal and gonadotrophin-releasing hormone (GnRH)-stimulated release of FSH and LH and on pituitary cell content of FSH and LH was examined in rat pituitary cell cultures and compared with the previously reported effects of inhibin. Pituitary cells were cultured for 3-9 days in the presence of graded doses of FSP and the basal release rates and changes in cell contents of FSH and LH determined during this period. FSP suppressed both the basal release rate and the cell content of FSH with median inhibitory concentrations (IC50) of 135 and 161 pmol/l respectively. The corresponding effects of FSP on LH basal release rate and LH cell content (IC50 = 200 pmol/l) were limited compared with the effects on FSH. The effect of FSP on GnRH-stimulated release of FSH and LH during 4 h was determined in cells which had been preincubated with FSP for 3 days, and the GnRH-stimulated release of FSH and LH analysed as a percentage of the respective gonadotrophin available for release. FSP antagonized GnRH action with dose-related increases in the GnRH median effective (stimulatory) concentrations for FSH and LH release (EC50 values = 56 and 400 pmol/l respectively) and a suppression in the maximum release of FSH and LH by excess GnRH (IC50 values = 142 and 150 pmol/l respectively). The effect of FSP on FSH cell content after 3 days in culture was insensitive to the neutralizing effects of an inhibin antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin-releasing hormone-stimulated luteinizing hormone secretion by perifused pituitary cells from normal, gonadectomized, and testicular feminized rats

To elucidate further the manner in which gonadal steroids influence the secretion of LH, we examined the effects of gonadectomy and the absence of functional androgen receptors on GnRH-induced LH release from dispersed rat anterior pituitary cells. Intact and gonadectomized (GNX) normal rats and androgen-resistant, testicular feminized (Tfm) animals from the King x Holtzman strain (a mutant strain that possesses defective androgen receptors) were used. Dispersed pituitary cells were perifused with Medium 199 during a 4-h equilibration period and then subjected to eight 2.5-min pulses of GnRH introduced at 30-min intervals at concentrations ranging from 0.03-100 nM. Basal LH secretion by cells from intact male and female rats was indistinguishable (P = 0.79) and was substantially lower (P less than 0.0001) than that by cells from GNX male and female animals. Basal LH secretion by cells from Tfm rats was significantly higher (P less than 0.01) than that by cells from intact animals, but lower (P less than 0.005) than that by cells from GNX animals. In response to GnRH, perifused pituitary cells from animals representing all experimental groups demonstrated concentration-dependent LH release. Pituitary cells from intact female rats showed an overall greater (P less than 0.05) response to GnRH than cells from intact male rats. Pituitary cells from Tfm rats demonstrated a greater GnRH-stimulated LH mean response than cells from intact male (P less than 0.0001) or intact female (P less than 0.0001) rats. Gonadectomy of male rats resulted in an overall GnRH-stimulated LH release similar to that exhibited by cells from gonadectomized female rats (P = 0.61). Cells from Tfm animals released more LH in response to GnRH than those from gonadectomized male and female rats (P less than 0.001). These data demonstrate that the release of LH in response to GnRH by pituitary cells from intact male rats (i.e. in the presence of androgen and functional androgen receptors) is less than that seen by cells from intact females rats. Since circulating levels of testosterone and estradiol are known to be elevated in the testicular feminized rat, the heightened GnRH-stimulated LH release by cells from such animals may reflect either the long term lack of androgenic influence and/or the combined effects of androgen resistance and elevated levels of circulating estrogens.     

Comparison of the facilitative roles of insulin and insulin-like growth factor I in the functional differentiation of granulosa cells: in vitro studies with the porcine model

The facilitative effects of insulin and IGF-I were compared in vitro with regard to induction of differentiated functions of porcine granulosa cells. The monolayers were maintained under serum-free conditions in the absence or presence of porcine FSH (20 micrograms/l), with or without graded doses of insulin or IGF-I. Concurrent treatment with IGF-I and FSH produced morphological differentiation and augmented LH/hCG receptor binding together with an enhancement in progesterone and estradiol secretion relative to treatment with FSH alone. IGF-I alone was incapable of exhibiting these effects. Insulin synergized with FSH to facilitate the granulosa cell functions except estradiol secretion. Maximal effective dose of IGF-I was 100 micrograms/l which is within the physiological concentration in vivo, whereas that of insulin was 1.0 mg/l, which is 1000-fold higher than the physiological level. Although the maximal effective doses of IGF-I and insulin produced a comparable increment in progesterone secretion and LH/hCG receptor induction, combined treatment with IGF-I and insulin did not prove additive. [125I]IGF-I binding revealed that specific IGF-I receptors with two classes of binding sites are present on porcine granulosa cells. No distinct differences were detected between IGF-I receptors of granulosa cells from small, medium and large follicles. Insulin was approximately 100-fold less active than IGF-I in competing for [125I]IGF-I binding. These findings suggest that porcine granulosa cells possess specific IGF-I binding sites which may mediate the cytodifferentiative actions of insulin-like peptides. Since IGF-I is more potent than insulin in amplifying the actions of FSH and maximally exerts the cytodifferentiative effects at the physiological concentration, it is likely that IGF-I plays the more important role in granulosa cell differentiation in synergy with FSH.     

Biphasic effects of estrogen on gonadotropin-releasing hormone-induced luteinizing hormone release in monolayer cultures of rat and monkey pituitary cells

The negative feedback effect of estrogen on LH secretion has been difficult to demonstrate in monolayer cultures of rat pituitary cells. The purpose of the present study was to establish the experimental conditions required for manifestation of this response and, in the process, to develop models for investigating the actions of steroids on pituitary cells. Both dynamic and static incubation systems were used. For perifusion experiments, trypsin-dispersed pituitary cells from rats at random stages of the estrous cycle were attached to glass coverslips with poly-L-lysine, incubated for 48 h, and then mounted in Sykes-Moore chambers. In each of these experiments, two chambers were perifused concurrently: one with medium containing 1.8 X 10(-10) M 17 beta-estradiol and the other with medium alone. GnRH (4.2 X 10(-9) M) was coinfused for 5 min out of every hour, and samples of perifusate were collected as 5-min fractions for assay of LH. Estrogen treatment significantly (P less than 0.01) suppressed LH release in response to the first five GnRH pulses compared to the control value. The inhibition was most pronounced early in the perifusion, but had disappeared by 6 h. These results demonstrate that estradiol exerts a potent but transient inhibition of GnRH-induced LH release in monolayer cultures of rat pituitary cells. In a subsequent set of experiments, we modified a static incubation system to assess sequentially the biphasic effects of estrogen on LH release by the same group of cells. Cultures of rat pituitary cells that had been established 42 h previously were treated simultaneously for 3 h with 17 beta-estradiol (3.7 X 10(-10) M) and various concentrations of GnRH (5 X 10(-10) to 1 X 10(-7) M) to measure the inhibitory effects of the steroid on LH secretion. This experiment was repeated on the same cells after 27 h of steroid exposure to estimate the facilitory actions of estrogen on LH release. The negative feedback of estrogen was demonstrable in static cultures of rat pituitaries provided that the period of estrogen exposure and duration of incubation were brief. Moreover, the results indicate that the same groups of cells can be used on consecutive days to investigate the inhibitory and stimulatory effects of estrogen on LH secretion. Experiments with cultures of monkey pituitary cells yielded similar results. Taken together, these findings indicate that cultured pituitary cells are responsive to the biphasic actions of estradiol and demonstrate the utility of two model systems for investigating these phenomena.     

Effects of a novel gonadotropin-releasing hormone antagonist (ORG 30850) on gonadotropin and prolactin secretion by rat pituitary cells in culture

The effect of a new GnRH antagonist (ORG 30850 ANT) on FSH, LH, and PRL secretion was studied using male rat pituitary cells in monolayer cell culture. In the absence of GnRH, ORG 30850 ANT did not alter spontaneous FSH and LH secretion into culture medium or the cell content of these hormones. In the presence of GnRH (10(-8) mol/l), ORG 30850 ANT significantly and dose-dependently inhibited FSH and LH secretion into culture medium while increasing their cell content. Conversely, in the presence of a single dose of ORG 30850 ANT, FSH and LH secretion rose significantly when subjected to increasing amounts of GnRH, whereas the hormonal cell content diminished. Furthermore, inhibition of GnRH-induced FSH and LH release by ORG 30850 ANT was not changed by pre-incubation with the GnRH antagonist regardless of the pre-incubation time. The inhibitory effect of the GnRH antagonist was observed early, with its peak occurring within 6 h of culture. These short-term studies indicate that ORG 30850 ANT specifically inhibits GnRH-induced gonadotropin release into culture medium, exerts no effect on the rate of gonadotropin production in the presence or absence of GnRH, competitively and reversibly inhibits the binding of natural GnRH to its receptors, and does not lead to any modifications in PRL secretion.     

Melatonin inhibits gonadotropin-releasing hormone-induced elevation of intracellular Ca2+ in neonatal rat pituitary cells.

GnRH stimulates LH release by increasing intracellular Ca2+ ([Ca2+]i). Melatonin is known to inhibit GnRH-stimulated LH release from neonatal rat pituitary cells. In the present report, the issue of whether melatonin acts through [Ca2+]i was addressed. [Ca2+]i was studied in cells in suspension, using Fluo-3 as a fluorescent indicator. In neonatal rat pituitary cells, melatonin inhibited the GnRH-induced [Ca2+]i increase in a dose-dependent manner; the GnRH-induced increase in [Ca2+]i was inhibited 40% by 100 nM melatonin. The relative potencies of several indoles as inhibitors of the GnRH stimulation of [Ca2+]i in neonatal pituitary cells (2-iodo-melatonin greater than melatonin greater than 6-hydroxymelatonin) correlate with their known potencies to inhibit LH release and with their binding affinity to high affinity melatonin receptors, which indicates that these receptors probably mediate the effects of melatonin. Further support for this interpretation comes from the observation that melatonin does not inhibit the GnRH effect on [Ca2+]i in cells obtained from adolescent rat pituitary glands, which lack melatonin receptors and are insensitive to melatonin as an inhibitor of GnRH-stimulated LH release. The possible involvement of an inhibitory G-protein was also investigated by studying the effects of pertussis toxin. Pretreatment with pertussis toxin antagonized the effects of melatonin on [Ca2+]i and LH release. This indicates that melatonin may inhibit the GnRH-induced increase in [Ca2+]i through a mechanism involving a pertussis toxin-sensitive G-protein. To examine the role of extracellular Ca2+ in this effect, the effects of melatonin were examined in a low Ca2+ medium. Under these conditions, the effect of melatonin was markedly reduced, which indicates that melatonin may act by inhibiting Ca2+ influx. These observations indicate that melatonin inhibits GnRH stimulation of [Ca2+]i in neonatal rat gonadotrophs, and this probably explains the inhibitory action of melatonin on GnRH stimulation of LH release.     

Acute facilitory action of progesterone on gonadotropin secretion of perifused rat pituitary cells

The purpose of this study was to investigate the kinetics and estrogen dependence of the facilitory progesterone action on LH and FSH secretion from pituitary cells under dynamic culture conditions. Anterior pituitary cells obtained from ovariectomized or intact adult Wistar rats were cultivated on Cytodex 1 microcarrier beads. The perifusion experiments were performed with four separate perfusion chambers. The cells of chambers I + II had been pretreated with medium containing vehicle (0.1% ethanol), those of chambers III + IV with medium containing 1 nmol/l estradiol for 48 h. After perfusion was started, each of the chambers was challenged with an initial 2-min GnRH (1 nmol/l) pulse. Chamber I was further perifused with medium containing vehicle, chamber II with medium containing vehicle + 100 nmol/l progesterone, chamber III with medium containing 1 nmol/l estradiol, and chamber IV with medium containing 1 nmol/l estradiol + 100 nmol/l progesterone. Three further GnRH pulses were administered at 50-min intervals to each of the chambers. In estradiol-primed cells from intact rats, progesterone induced a positive effect on LH and FSH secretion after 50 min of exposure to progesterone. After 100 min of progesterone treatment, LH and FSH release were enhanced to 420 and 500 per cent, respectively. When such cells were not primed with estradiol, a slight though insignificant positive action of progesterone on LH release was present after 50 and 100 min of treatment, whereas FSH secretion was not influenced. The facilitory effect of progesterone occurred only after 100 min when estradiol-primed cells from ovariectomized rats were used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells

The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 X 10(-10) M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (less than 15 microM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gln LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/microgram iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.     

Does cortisol inhibit pulsatile luteinizing hormone secretion at the hypothalamic or pituitary level?

Elevations in glucocorticoids suppress pulsatile LH secretion in sheep, but the neuroendocrine sites and mechanisms of this disruption remain unclear. Here, we conducted two experiments in ovariectomized ewes to determine whether an acute increase in plasma cortisol inhibits pulsatile LH secretion by suppressing GnRH release into pituitary portal blood or by inhibiting pituitary responsiveness to GnRH. First, we sampled pituitary portal and peripheral blood after administration of cortisol to mimic the elevation stimulated by an immune/inflammatory stress. Within 1 h, cortisol inhibited LH pulse amplitude. LH pulse frequency, however, was unaffected. In contrast, cortisol did not suppress either parameter of GnRH secretion. Next, we assessed the effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, duration, and frequency. Hourly pulses of GnRH were delivered to ewes in which endogenous GnRH secretion was blocked by estradiol. Cortisol, again, rapidly and robustly suppressed the amplitude of GnRH-induced LH pulses. We conclude that, in the ovariectomized ewe, cortisol suppresses pulsatile LH secretion by inhibiting pituitary responsiveness to GnRH rather than by suppressing hypothalamic GnRH release.