Gonadotrope oestrogen receptor-alpha and -beta and progesterone receptor immunoreactivity after ovariectomy and exposure to oestradiol benzoate, tamoxifen or raloxifene in the rat: correlation with LH secretion

The selective oestrogen receptor modulator (SERM) tamoxifen (TX) has agonist/antagonist actions on LH secretion in the rat. Whereas in the absence of oestrogens TX elicits progesterone receptor (PR)-dependent GnRH self-priming, it antagonizes oestrogen-stimulatory action on LH secretion. The aim of these experiments was to explore whether TX treatment-induced differential expression of oestrogen receptor (ER)alpha and ERbeta in the gonadotrope may determine its agonist effect on LH secretion. In the first experiment, basal LH secretion, GnRH-stimulated LH secretion and PR-dependent GnRH self-priming were determined in incubated pituitaries from ovariectomized (OVX) rats treated with oestradiol benzoate (EB), TX or raloxifene (RX). Cycling rats in metoestrus or pro-oestrus were used as basic controls. As in pro-oestrus, pituitaries from OVX rats treated with EB exhibited GnRH-stimulated LH secretion, immunohistochemical PR expression and GnRH self-priming. While RX had no effect on these parameters, TX induced PR expression and GnRH self-priming. GnRH self-priming was absent in pituitaries incubated with the antiprogestin ZK299. In the second experiment, we evaluated the immunohistochemical expression of ERalpha and ERbeta in gonadotropes of cycling rats and OVX rats treated with EB, TX or RX. We found that while ERalpha expression was similar in all six groups, ERalpha expression was oestrous cycle dependent. Moreover, ERalpha expression in gonadotropes of TX-treated rats was as high as that found in pro-oestrus, while ERalpha expression in the gonadotropes of RX-treated rats was lower than in metoestrous or pro-oestrous pituitaries. These results suggest that, in the absence of the cognate ligand, TX, unlike RX, may regulate LH secretion through the ERalpha subtype in gonadotropes.     

Oestradiol-17beta inhibits tamoxifen-induced LHRH self-priming blocking hormone-dependent and ligand-independent activation of the gonadotrope progesterone receptor in the rat

In the rat, administration of tamoxifen (TX) in the absence of oestrogen (E) induces LHRH self-priming, the progesterone receptor (PR)-dependent property of LHRH that increases gonadotrope responsiveness to itself. The oestrogen-dependent PR can be phosphorylated/activated by progesterone (P4) and, in the absence of the cognate ligand, by intracellular LHRH signals, particularly cAMP/protein kinase A. We have recently found that oestradiol-17beta (E2), acting on a putative membrane estrogen receptor-alpha in the gonadotrope, inhibits this agonist action of TX. This study investigated the mechanism by which E2 inhibits TX-elicited LHRH self-priming using both incubated pituitaries from TX-treated ovariectomized (OVX) rats and anterior pituitary cells from OVX rats cultured with TX. It was found that (1) in addition to the inhibitory effect on TX-elicited LHRH self-priming, E2 blocked P4 and adenylyl cyclase activator forskolin augmentation of LHRH-stimulated LH secretion, and (2) E2 did not affect the increasing action of TX on gonadotrope PR expression or pituitary cAMP content. Furthermore, inhibition of protein phosphatases with okadaic acid suppressed E2 inhibition of TX-elicited LHRH-induced LH secretion, while stimulation of protein phosphatases with ceramide blocked TX-induced LHRH self-priming. Together, these results indicated that membrane ER-mediated E2 inhibition of the TX-stimulated LHRH self-priming pathway involves a blockade of gonadotrope PR phosphorylation/activation, but not a deficient response of PR to phosphorylases. Results also suggested that the inhibitory effect of E2 on TX-induced LHRH self-priming is exerted through modulation of cellular protein phosphatase activity in the gonadotrope.     

Effects of progesterone (P) and antiprogestin RU486 on LH and FSH release by incubated pituitaries from rats treated with the SERM LY11701 8-HCl and/or recombinant human FSH

The aim of the present study was to investigate the role of the estrogen (ES) background on the effects of P or its antagonist RU486 on basal and LHRH-stimulated LH and FSH secretion. To do this, pituitaries collected from: intact rats in proestrus; rats injected with the ES antagonist LY11701 8-HCl; rats injected with recombinant-human FSH (r-hFSH) to stimulate ovarian hormonogenesis; and rats injected with both LY11701 8-HCl and r-hFSH were incubated with or without LHRH (10 nM) in the presence of P (100 nM) or RU486 (10 nM). RU486 decreased basal and LHRH-stimulated release of LH and FSH and LHRH self-priming in pituitaries from control rats, while P increased both pituitary responsiveness and LHRH self-priming. These effects were absent in pituitaries from rats treated either with the ES antagonist or r-hFSH, which, in the absence of P or RU486 in the incubation medium, reduced gonadotropin release. Because r-hFSH did not increase E2 serum concentration significantly, the putative FSH-dependent ovarian non-steroidal gonadotropin surge inhibiting factor (GnSIF) might be the hormonal cause of the reduced secretion of LH and FSH. Combined treatment with LY117018-HCl and r-hFSH had additive inhibitory effects on gonadotropin release. These results indicate that ES-inducible P receptor (PR) in the pituitary can be activated in a ligand-independent manner by intracellular messengers giving rise to enhanced basal and LHRH-stimulated gonadotropin secretion. The results also suggested that the r-hFSH-stimulated ovarian bioactive entity GnSIF and RU486 may share a similar mechanism of action involving pituitary PR.     

The In Vitro Inhibitory Action of Antiprogestin RU486 on LH and FSH Secretion in the Absence of Progesterone in Rats Is Estrogen-Dependent

Previous in vivo findings show that in the virtual absence of progesterone (P), the antiprogestin RU486 reduces LH and FSH secretion in proestrous rats, indicating that activation of P receptor (PR) can occur in the absence of the cognate ligand. The present study investigates, in vitro, whether or not the inhibitory effect of antiprogestin RU486 on gonadotropin secretion in the absence of P is estrous cycle dependent, and whether its specific expression in proestrus mirrors the high estrogen (E₂) background.In the first experiment we investigated the effect of RU486 (10 nM) and/or LHRH (10 nM) on LH and FSH secretion in incubated pituitaries collected on each day of the estrous cycle of the rat. In the second experiment, we determined the effect of RU486 and/or LHRH on preovulatory LH and FSH release by pituitaries from female rats that were ovariectomized (OVX), treated with the antiestrogen LY117018-HCL (Eli Lilly & Co.), or injected with 20 g of estradiol benzoate (EB). The third experiment investigated the effect of RU486 and/or LHRH on LH and FSH release by pituitaries collected from intact or EB-treated (0.1 mg/kg over three consecutive days) male rats.RU486 reduced both basal and LHRH-stimulated LH and FSH secretion in proestrous pituitaries from normal 4-day cyclic rats. By contrast, in diestrous pituitaries, RU486 increased both parameters of LH secretion but was without effect on FSH release. RU486 was also without effect in pituitaries collected from rats in estrus or metestrus, or from OVX or antiestrogen-treated rats. Moreover, EB injection or treatment induced the full inhibitory effect of RU486 in pituitaries from female and male rats, respectively.The above results suggested that P occupancy of the receptor is not required for the formation or function of the active receptor and hence for preovulatory LH and FSH secretion, and that this form of PR activation at pituitary level is E₂-dependent and not genetically determined.     

Tamoxifen induces gonadotropin-releasing hormone self-priming through an estrogen-dependent progesterone receptor expression in the gonadotrope of the rat

Tamoxifen (TX) is an antiestrogen with varying levels of antagonist/agonist activity on the reproductive axis of the rat. It has been reported that TX, in contrast to other selective estrogen receptor modulators (SERMs), increases the content of cytosolic estrogen receptors (ER) in the gonadotrope and induces gonadotropin releasing hormone (GnRH) self-priming in the absence of E. GnRH priming is believed to be a consequence of E-dependent progesterone receptor (PR) activation. The purpose of this study was to determine whether TX induces PR expression in the gonadotrope in an E-dependent manner, and whether the blockade of PR activation affects TX-dependent GnRH self-priming in ovariectomized (OVX) rats. Chronic OVX rats were injected (sc) over 3 days with 25 microg estradiol benzoate (EB), 3 mg TX, 0.5 mg RU58668, a 'pure' anti-E (aE), 2 mg RU38486, an anti-P at the receptor (aP), TX+aE and TX+aP. Controls were given 0.2 ml oil. While EB and TX increased mRNA for both PR A+B and PR B expression and the number and intensity of nuclei immunoreactive (IR) for PR in the gonadotrope, the aE and aP given alone had no effect on either PR mRNA levels or nuclear PR-IR. The aE reduced the effect of TX on PR expression (mRNA and nuclear IR) while the aP slightly reduced nuclear PR-IR only. In addition, pituitaries from each of the seven groups were incubated with: 10(-8)M E(2), 10(-7)M TX, 10(-8)M aE, 10(-8)M aP, TX+aE, TX+aP or medium alone, respectively. Pituitaries were tested for GnRH self-priming (two pulses of 15 min 1 h apart) and the secretion of LH and PRL determined by specific RIAs. Pituitaries from rats treated with EB and incubated with E(2) had increased basal and GnRH-stimulated luteinizing hormone (LH) and prolactin (PRL) secretion and GnRH self-priming. TX reduced basal and stimulated LH secretion, increased PRL secretion and induced a robust GnRH self-priming. All these effects of TX were blocked by the aE, while the aP blocked GnRH self-priming only. In conclusion, tamoxifen induced PR expression (mRNA and nuclear IR) in the gonadotrope in an E-dependent manner, while activation of these PR through intracellular signaling of GnRH induced GnRH self-priming.     

Antiprogestins RU486 and ZK299 suppress basal and LHRH-stimulated FSH and LH secretion at pituitary level in the rat in an oestrous cycle stage-dependent manner

We have previously shown that administration of antiprogestin (AP) type II RU486 to ovariectomized (OVX) rats on the morning of pro-oestrus decreases the magnitude of preovulatory gonadotrophin surge. This suggests that the effect of RU486 on LHRH-dependent gonadotrophin release may be independent of its ability to block progesterone actions. The aim of the present research was to study the possible site of RU486 action and to determine whether the gonadotrophin suppressive effect of APs RU486 and ZK299 is dependent on the oestrogen background. Intact or OVX rats in the morning of pro-oestrus were injected s.c. with 4 mg of RU486 or ZK299 (AP type I) at 0900 h on pro-oestrus. At 1830 h, serum concentration of FSH and LH and median eminence (ME) content of LHRH were determined. In the second experiment, the effect of RU486 and ZK299 on pituitary responsiveness to LHRH (100 ng, i.p.) and ME content of LHRH at 1830 h pentobarbital-blocked intact or OVX rats was evaluated. In the last study, the anterior pituitary release of FSH and LH from pro-oestrus or metoestrus donors incubated with or without LHRH (1, 10 or 100 nM) in the presence or absence of APs (20 nM) was evaluated. Both APs reduced serum FSH and LH levels at 1830 h on pro-oestrus in intact and OVX rats. The suppressive effect on gonadotrophin release brought about by AP treatment was also evidenced in PB-blocked intact and OVX rats. This suggested that the inhibitory effect of APs occurred, at least in part, at pituitary level. Furthermore, in the absence of the natural ligand, APs significantly reduced basal and LHRH-stimulated FSH and LH release from pro-oestrous but not from metoestrus pituitaries. In conclusion, these experiments have shown, both 'in vivo' and 'in vitro', that APs RU486 and ZK299 have suppressive effects at pituitary level on basal and LHRH-stimulated FSH and LH secretion, regardless of their antiprogestagenic activity, in pro-oestrus but not in metoestrus.     

The integrated action of oestrogen receptor isoforms and sites with progesterone receptor in the gonadotrope modulates LH secretion: evidence from tamoxifen-treated ovariectomized rats

The specific role of each oestrogen receptor (ER) isoform (alpha and beta ) and site (nucleus and plasma membrane) in LH release was determined in ovariectomized (OVX) rats injected over 6 days (days 15-20 after OVX) with a saturating dose (3 mg/day) of tamoxifen (TX), a selective ER modulator with nuclear ERalpha agonist actions in the absence of oestrogen. This pharmacological effect of TX was demonstrated by the fact that it was blocked by the selective ERalpha antagonist methyl-piperidinopyrazole. Over the past 3 days of the 6-day TX treatment, rats received either 25 microg/day oestradiol benzoate (EB), 1.5 mg/day selective ERalpha agonist propylpyrazole triol (PPT) and the selective ERbeta agonist diarylpropionitrile (DPN), or a single 3 mg injection of the antiprogestin onapristone (ZK299) administered on day 20. Blood samples were taken to determine basal and progesterone receptor (PR)-dependent LH-releasing hormone (LHRH)-stimulated LH secretion and to evaluate LHRH self-priming, the property of LHRH that increases gonadotrope responsiveness to itself. Blood LH concentration was determined by RIA and gonadotrope PR expression by immunohistochemistry. Results showed that i) EB and DPN potentiated the negative feedback of TX on basal LH release; ii) DPN reduced TX-induced PR expression; iii) EB and PPT blocked TX-elicited LHRH self-priming and iv) ZK299 reduced LHRH-stimulated LH secretion and blocked LHRH self-priming. These observations suggest that oestrogen action on LH secretion in the rat is exerted at the classic ERalpha pool and that this action might be modulated by both ERbeta and membrane ERalpha through their effects on PR expression and action respectively.     

Biological role of pituitary estrogen receptors ERalpha and ERbeta 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, alpha and beta, both activated by E. The relative contribution of alpha and beta forms in E regulatory actions is largely unknown. To this end, 2-week-old ovariectomized (OVX) rats were injected over 3 days with 25 microg estradiol benzoate (EB), 1.5 mg of propylpyrazole triol (PPT), a selective ERalpha agonist, 1.5 mg of the selective ERbeta 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(-8)M 17beta-estradiol, 10(-6)M PPT, 10(-6)M 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(-8)M 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 ERalpha isoform. The finding that activation of ERbeta with its selective DPN agonist had an estrogenic effect on IR-PR nuclei, but not on GnRH self-priming, a characteristic ERalpha-mediated effect of E, suggests that the biological action of E at the pituitary may involve both isoforms of ER.     

The role of estrogen-dependent progesterone receptor in protein kinase C-mediated LH secretion and GnRH self-priming in rat anterior pituitary glands

The aim of the present study was to explore the involvement of pituitary progesterone receptor (PR) in PKC-mediated LH secretion and LHRH self-priming and the role of the estrogen (E) environment. Eight randomly selected hemipituitaries from adult female rats in proestrus or from 2 weeks ovariectomized (OVX) rats were incubated, in the absence of progesterone (P), over 3 h in Dulbecco's modified Eagle's medium (DMEM). In the first experiment, hemipituitaries were incubated continuously with: medium alone, GnRH (10 nM), the PKC stimulator PMA (100 nM), the PKC inhibitor staurosporine (100 nM), the antiprogestin at the receptor RU486 (10 nM), LHRH+staurosporine, GnRH+RU486 or PMA+RU486. In the second experiment, hemipituitaries were incubated, one h apart, with GnRH to determine the GnRH self-priming and this was compared with the priming effect of PMA. Also, the effect of staurosporine and RU486 during the induction period (1st h) on GnRH and PMA priming was evaluated. Medium was aspirated at the end of each h to determine LH accumulation and to evaluate GnRH self-priming. Both GnRH and PMA stimulated LH secretion. Staurosporine and RU486 reduced basal and GnRH-stimulated LH secretion, and RU486 reduced PMA-stimulated LH secretion from proestrus pituitaries. The stimulating effect of GnRH and PMA on LH secretion and the inhibitory action of staurosporine and RU486 on basal or stimulated LH secretion were significantly reduced in OVX-rats. Both GnRH and PMA induced GnRH priming. Staurosporine during the induction h reduced GnRH self-priming while RU486 reduced both GnRH self-potentiation and PMA priming. The magnitude of these inhibitory effects was blunted in OVX-rats. These results showed that PKC signaling pathway in the gonadotrope mediates, at least in part, basal and GnRH-stimulated LH secretion and GnRH self-priming. Also, the results are suggestive of an interaction of PKC signaling pathway with E-dependent PR in a ligand-independent activation manner in the gonadotrope.     

A paradoxical inhibitory effect of oestradiol-17beta on GnRH self-priming in pituitaries from tamoxifen-treated rats

Two-week ovariectomized (OVX) rats were injected over three days with 25 microg oestradiol benzoate (EB), 3 mg tamoxifen (TX) and 0.2 ml oil and their pituitaries were harvested for incubation experiments. Pituitaries from EB- and TX-treated OVX rats exhibited GnRH self-priming when incubated with their corresponding ligand. However, incubation of pituitaries with different ligands yielded divergent results: when pituitaries from EB-treated rats were incubated with 10(-7) M TX they displayed GnRH self-priming, whereas incubation of pituitaries from TX-treated rats with 10(-8) M oestradiol-17beta (E2) blocked GnRH self-priming. Further studies to analyse the latter finding revealed that: (a) E2 inhibited TX-induced GnRH self-priming in a dose-dependent manner while 10(-8) M oestradiol-17alpha did not; (b) co-incubation of E2 with the pure anti-oestrogen ICI 182,780, but not with the selective oestrogen receptor modulator TX, reversed the E2 inhibitory effect; (c) the oestrogen receptor (ER)-alpha selective agonist propylpyrazole triol, but not the ERbeta selective agonist diarylpropionitrile, mimicked the inhibitory effect of E2; (d) the analogue membrane-impermeable conjugated E2-BSA also inhibited TX-induced GnRH self-priming; and (e) a 15-min exposure of the pituitaries to E2 was sufficient to inhibit the GnRH self-priming elicited by TX. Although other explanations may exist, altogether these results suggested that E2, via an ER different from classical ER, inhibits the GnRH self-priming elicited by TX.     

Absence of gonadotropin surges and gonadotropin-releasing hormone self-priming in ovariectomized (OVX), estrogen (E2)-treated, progesterone receptor knockout (PRKO) mice.

It is well known that estrogen (E2) stimulates expression of progesterone receptors (PRs), thereby inducing responsiveness of several tissues to the actions of progesterone (P). Recent studies have also suggested, however, that biological actions previously ascribed to E2 alone may also be mediated by activation of E2-induced PRs, even independently of signal changes in P concentrations. In the present experiments, the progesterone receptor knockout (PRKO) mice were used to assess the role of PR activation in the positive feedback actions of E2 on gonadotropin release. Ovariectomized (OVX) PRKO mice were tested for their capacity to mount primary gonadotropin surges in response to exogenous E2, and to exhibit a GnRH self-priming effect in response to sequential injections of the decapeptide. Wild-type (WT) and PRKO mice were OVX, treated with both 17beta-estradiol and estradiol benzoate (EB), and then killed at 1900 h on day 7 postOVX. Plasma LH RIA revealed that WT mice exhibited surges in response to the E2 treatment; the PRKO mice, however, showed no elevation in plasma LH above untreated controls. Instead, plasma LH levels in E2-treated, OVX PRKO mice decreased significantly in comparison to untreated OVX PRKO mice, suggesting that E2 can exert a negative feedback influence on LH release in PRKO mice, despite the absence of positive feedback effects. A slight but significant rise in plasma FSH was observed in E2-treated OVX WT mice in comparison to untreated controls: an effect not seen in E2-treated OVX PRKO mice, reinforcing the observation that estrogen's positive feedback effects are compromised in PRKO mice. In a second experiment, E2-treated OVX WT and PRKO mice were given either one or two pulses of GnRH 60 min apart, and killed 10 min later. The WT mice were found to exhibit a robust GnRH self-priming effect, as WT mice receiving two GnRH pulses displayed LH responses approximately 2-fold greater than those receiving only one pulse. By contrast, PRKO mice receiving two GnRH pulses exhibited no additional increase in plasma LH levels. We conclude that PR activation is obligatory for expression of the GnRH self-priming effect as well as for generation of E2-induced LH and FSH surges. The extent to which failure of LH surge secretion in PRKO mice is due to the absence of GnRH self-priming, lack of hypothalamic GnRH surges, and/or defects in other processes remains to be determined. These observations clearly demonstrate, however, that the presence of PR is an absolute requirement for the transmission of E2-induced signals leading to gonadotropin surges.     

Inhibin suppresses luteinizing hormone (LH)-releasing hormone self-priming: direct action on follicle-stimulating hormone secretion and opposition of estradiol-enhanced LH secretion.

Previous studies have suggested that the ovary produces a factor that maintains the pituitary in a state of low LHRH responsiveness that must be overcome by the self-priming action of LHRH. To determine the role of inhibin in maintaining low LHRH responsiveness in pituitaries of diestrous female rats, endogenous inhibin was passively immunoneutralized in vivo, and the pituitaries were removed 18-20 h later and examined for LHRH responsiveness in vitro. Pituitaries from diestrous control rats produced the biphasic pattern of gonadotropin secretion that typifies LHRH self-priming: an initial low secretory response to LHRH (lag phase), followed by a protein synthesis-dependent transition to an enhanced rate of secretion with continued LHRH exposure (primed phase). Immunoneutralization of endogenous inhibin [antiserum (AS) treated] resulted in an increased rate of LH secretion during the lag phase, while no change was observed in the primed phase rate of LH secretion. FSH secretion from pituitaries of AS-treated rats was increased during the lag phase to a rate of secretion similar to that observed during the primed phase of FSH secretion from control pituitaries, and it was increased further during the primed phase of secretion. These results suggest that inhibin is at least partially responsible for the low secretion of LH observed during the lag phase response to LHRH exposure and is totally responsible for the lowered rate of FSH secretion during the lag phase. The observation that the enhanced rate of gonadotropin secretion observed with AS-treated pituitaries during the lag phase was resistant to inhibition of protein synthesis provides further evidence that a partial transition from the lag to the primed phase had already occurred. Pituitaries from ovariectomized rats were also examined in order to place the contribution of inhibin in perspective with the total ovarian influence on pituitary responsiveness to LHRH. Unexpectedly, LH secretion during the lag phase was similar to the low secretion rate of diestrous control pituitaries, and the higher primed rate of secretion failed to fully develop, suggesting that an additional ovarian factor was required to induce and maintain pituitary responsiveness to LHRH in terms of LH secretion. FSH secretion from the ovariectomized rats was similar to that observed from pituitaries of AS-treated rats, thus further supporting the concept that inhibin is fully responsible for the suppression of FSH secretion in response to LHRH. Plasma from the AS-treated rats revealed a 2-fold increase in estradiol levels compared with diestrous control rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of LH-releasing hormone antagonist or lesions of the medial basal hypothalamus on periovulatory gonadotrophin release in female rats

Follicle-stimulating hormone release on the morning of oestrus was examined by using two different techniques which eliminate LH-releasing hormone (LHRH) stimulation of the pituitary gland. Cyclic female rats were given a potent LHRH antagonist (ALHRH) or were subject to electrolytic lesions of the medial basal hypothalamus (MBH) before or after the pro-oestrous phase of FSH release. Administration of ALHRH at 14.00, 15.30 and 17.00 h or lesioning of the MBH between 11.30 and 13.00 h on pro-oestrus entirely blocked the preovulatory LH surge and both phases of FSH release. Ovulation was abolished in all of these animals. However, when ALHRH was given at 20.30, 22.00 and 23.30 h or lesions of the MBH made between 20.00 and 21.30 h on pro-oestrus after the pro-oestrous FSH and LH surges had occurred, the oestrous phase of FSH release was indistinguishable from that of saline-treated control rats. Ovulation occurred in all of these animals, and the mean number of ova shed was eight/rat. The conclusions are that (1) the pro-oestrous phase of FSH release is dependent upon the hypothalamic hormonal stimulation by LHRH and (2) the oestrous phase of FSH release is entirely independent of direct LHRH stimulation, or any hypothalamic stimulus.     

Progesterone enhances the surge of luteinizing hormone by increasing the activation of luteinizing hormone-releasing hormone neurons

The ability of progesterone (P) to enhance the surge of LH in the rat is well documented, but whether its primary site of action is on the pituitary or brain is unclear. To determine whether P can alter the activation of LHRH neurons, 1) intact female rats were treated with the P antagonist RU486 (5 mg) at 1230 h on proestrus and killed at specified times during the afternoon and evening for comparison of plasma LH levels and cFos expression in LHRH neurons with untreated proestrous rats. RU486 treatment greatly reduced both the magnitude of the LH surge and the degree of cFos induction (numbers of cells expressing cFos and intensity of cFos staining) in LHRH neurons during proestrus. 2) Ovariectomized (OVX) rats were primed with estradiol benzoate (EB, 1 microgram) and then were treated with EB alone (50 microgram) or EB plus P (5 mg). Treatment with EB without P resulted in significantly lower peak LH levels and a reduced cFos response in LHRH neurons than the EB-P treated rats. These data suggest that the actions of P eventuate in an enhanced activation of LHRH neurons that may be responsible for the increased magnitude of the LH surge.     

The self-priming action of LHRH is under negative FSH control through a factor released by the ovary: observations in female rats in vivo

Female rats were treated with Metrodin (highly purified urinary FSH from menopausal women) or saline during the oestrous cycle. On the day of pro-oestrus they were anaesthesized with phenobarbital and received four repetitive LHRH injections 1 h apart. This treatment with FSH suppressed the unprimed LH response to the first LHRH injection. During the subsequent injections the maximal LHRH self-priming was delayed by 3 h till the fourth LHRH stimulation. At this time, LH release in response to LHRH was equally as high as shown in the saline controls after the second LHRH injection. Ovariectomized rats did not show the self-priming effect and FSH treatment was ineffective in suppressing LHRH-induced LH release. Administration of FSH followed by an additional 4- or 24-h period before LHRH stimulation were equally effective in suppressing the unprimed LH release and delaying (up to 3 h) the maximal priming of LH release by LHRH. Even 4-20-fold increased amounts of LHRH did not affect the suppressed unprimed release of LH after FSH treatment. Treatment with FSH did not change oestradiol and progesterone levels. It was concluded that FSH treatment suppresses the unprimed LHRH-induced LH release and delays maximal LHRH self-priming by enhancing the release of an ovarian factor.     

Modulation of pituitary gonadotropins and prolactin secretion by testosterone in vitro.

Investigations were undertaken to study the differential modulation of LH, FSH and PRL secretion by testosterone (T) using whole pituitary (PI) or pituitary-hypothalamus coincubates (PHC) as in vitro constructs. PI and PHC from intact and castrated rats were incubated with or without T thrice, for 24 h each, (24 h x 3, total incubation period 72 h). The spent media was replenished every 24 h. At the end of 72 h, a few of the pituitary glands were challenged with 10 nM LHRH for 4 h. The spent media and pituitary glands were analyzed for LH, FSH and PRL using specific RIAs. Incubation of PI or PHC from intact rats with T stimulated the release of LH and FSH but inhibited the release of PRL. T had no effect on the intrapituitary contents of LH but inhibited intrapituitary contents of FSH and PRL, as compared to controls incubated without T. Castration increased intrapituitary contents of LH and FSH with concomitant decrease in PRL levels. Incubation of PI or PHC from castrated rats with T inhibited intrapituitary contents of LH to intact pituitary levels, while PRL levels were further reduced instead of being ameliorated. It is concluded that PI or PHC can be used as convenient in vitro models to monitor the effect of castration or of T modulation of pituitary and hypothalamus functions. T does not affect the synthesis of LH at the gonadotroph level but facilitates the regulation of intracellular LH and FSH levels. It is postulated that T inhibits the synthesis of FSH/PRL at the gonadotroph/lactotroph levels.     

Effect of destruction of the dorsal anterior hypothalamus on follicle-stimulating hormone secretion in the rat

The role of the paraventricular nucleus-dorsal anterior hypothalamus (PVN-DAHA) in the control of anterior pituitary gland secretion of FSH and LH in castrated male and female rats was examined. Bilateral radiofrequency lesions of the PVN-DAHA in chronically ovariectomized (OVX) rats lowered plasma FSH levels by 33% (P less than 0.005) compared to values in unoperated and sham-operated control rats; plasma LH concentrations were unaltered. RIA of median eminence (ME) LHRH concentrations in these animals revealed no differences among the three experimental groups. Other categories of diencephalic destruction did not result in this pattern of selectively reduced FSH release. Bilateral radiofrequency destruction of the PVN-DAHA also attenuated by 50% (P less than 0.025 to P less than 0.005) the progesterone-induced surge of FSH in estrogen-primed OVX rats. Progesterone-induced LH release was unaffected by PVN-DAHA lesions. Other lesion categories failed to show the same result. Bilateral ablation of the PVN-DAHA in male rats resulted in a selective diminution of the postcastration rise of plasma FSH beginning 48 h postcastration (P less than 0.05 to P less than 0.005) and persisting for 14 days (P less than 0.005) after orchidectomy, thus revealing the time course and permanence of this procedure on plasma FSH levels. The postcastration rise of plasma LH levels was not affected by PVN-DAHA lesions. The concentration of ME LHRH was the same among orchidectomized male rats whether they bore PVN-DAHA lesions, sham lesions, or no lesions. In summary, destruction of the PVN-DAHA was found to reduce significantly the elevation of plasma FSH, but not LH, in the OVX rat and the estrogen-progesterone-stimulated OVX rat. PVN-DAHA lesions also attenuated the postcastration rise of FSH, but not that of LH, in the male. The failure of lesions of the PVN-DAHA to alter ME LHRH concentrations in the face of decreased FSH release does not prove that LHRH release is totally unaffected by this procedure. This finding is, however, consistent with the concept that diminished FSH secretion could be the result of a deficiency of a hypothalamic releasing factor (FSH-releasing factor?) other than that of the LHRH decapeptide.     

Prolonged sensitisation of pituitary glands in vitro to repeated administration of luteinising hormone-releasing hormone: effects of pulse frequency, ovariectomy, estradiol and progesterone

Anterior pituitary gland fragments were obtained from female Wistar-derived rats on dioestrus or pro-oestrus and perifused in Biogel columns in vitro. They were subjected at the beginning of each of 5 h of perifusion to a volley of 6 1-min pulses of luteinising hormone-releasing hormone (LHRH, 10 nM), given 4 min apart. Luteinising hormone (LH) was measured by radioimmunoassay in sequential 2-min fractions of the perifusate. Pituitary glands removed at 14.00 h on dioestrus showed a characteristic pattern of sensitisation followed by desensitisation to the repeated volleys of LHRH, whereas tissues removed at 10.00 or 14.00 h on pro-oestrus showed no evidence of desensitisation over the 5-hour period, the response to each LHRH volley being greater than the preceding one. Estradiol (E, 3-100 pg/ml) added to the medium from the start of perifusion had no significant effect on the pattern of response from tissues removed on pro-oestrus, but the highest concentration significantly enhanced the response of dioestrus pituitaries to all but the last of the LHRH volleys. Progesterone (P, 1-50 ng/ml) added to the medium produced a dose-related inhibition of the response of pro-oestrous tissues to the LHRH volleys. Groups of animals were ovariectomised (OVX) on dioestrus and used for experiment the next morning. OVX at 10.00 h on dioestrus produced the pattern of response characteristic of dioestrus the next morning, but with much higher levels of LH release, which were unaltered by the addition of E to the medium. OVX at 17.00 h on dioestrus produced an entirely different pattern of response the next day, with high basal and moderate LHRH-induced LH release, and no evidence of changes in sensitivity of the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin induces gonadotrophin release through stimulation of LH-releasing hormone release from the median eminence

The effects of serotonin (5-HT) on the release of gonadotrophins and LH-releasing hormone (LHRH) were examined in an in-vitro perifusion system using median eminences and/or anterior pituitaries obtained from male or pro-oestrous female rats. Animals were killed by decapitation between 12.00 and 13.00 h. A serial double-chamber perifusion system was employed. Three types of experiments were performed. In the first, median eminences were placed in the first chamber and one anterior pituitary in the second chamber. In the second group, only the anterior pituitary was perifused. In the third group, only five median eminences were perifused. In the first and second experiments, LH, FSH and prolactin were determined in the perifusion efflux by radio-immunoassay (RIA). In the third experiment, LHRH was determined by RIA. Addition of 5-HT (final concentrations 0.06, 0.6 and 6.0 mumol/l) into the first chamber containing the median eminences stimulated the release of LH and FSH from the pituitary, but did not affect the levels of prolactin in the effluent in the same experiment (pro-oestrous rats). The stimulatory effect of 5-HT was blocked by the addition of cyproheptadine (l mumol/l) in the perifusion fluid. The introduction of 5-HT (0.6 mumol/l) into the tube connecting the first and second chambers did not modify the release of LH, nor did 5-HT added to the pituitaries perifused alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

The depressing (negative) effect of oestradiol benzoate on the in vitro secretion of LH and FSH by the pituitary gland of the LRH-pretreated long-term ovariectomized rat changes into the augmentative (positive) effect after discontinuation of the LRH-pretreatment

The effect of pretreatment in vivo with oestradiol benzoate on in vitro secretion of LH and FSH was studied in long-term ovariectomized (OVX) rats both at the end of a 5-day continuous in vivo pretreatment with LRH and 4-days after cessation of such LRH pretreatment. Rats were on day 0 sc implanted with osmotic minipumps which released LRH at the rate of 250 ng/h. Control rats were implanted with a piece of silicone elastomer with the dimensions of a minipump. On days 2 and 4 the rats were injected with either 3 micrograms EB or with oil. On day 5 part of the rats were decapitated and the in vitro autonomous (i.e. non-LRH-stimulated) and 'supra-maximally' LRH-stimulated release of LH and FSH was studied using a perifusion system. From other rats the minipumps were removed on day 5 and perifusion was performed on day 9. On the 5th day of the in vivo LRH pretreatment the pituitary LH/FSH stores were partially depleted; the pituitaries of the EB-treated rats more so than those of the oil-injected rats. EB alone had no significant effect on the content of the pituitary LH- and FSH stores. On day 9, i.e. 4 days after removal of the minipumps, the pituitary LH and FSH contents had increased in both the oil- and the EB injected rats, but had not yet recovered to control values. In rats not subjected to the 5-days pretreatment with LRH EB had a positive effect on the supra-maximally LRH-stimulated secretion of LH and FSH as well as on the non-stimulated secretion of LH. EB had no effect on the non-stimulated secretion of FSH.(ABSTRACT TRUNCATED AT 250 WORDS)