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.     

Antiprogesterone RU486 induces dissociation of LH and FSH secretion in the cyclic rat: effect of anti-inhibin serum.

Administration of the antiprogesterone RU486 to 4-day cyclic rats from metoestrus to pro-oestrus increases serum levels of LH while decreasing levels of FSH. If it is assumed that there is only one gonadotrophin-releasing hormone, there is no direct explanation for the decrease in FSH concentrations. The purpose of these experiments was to investigate the effect of RU486 on gonadotrophin secretion in cyclic rats during periods when the secretion of LH and FSH diverges. RU486 blunted the transient increase in FSH concentration on the afternoon of metoestrus and the compensatory ovarian hypertrophy on the next day of oestrus in unilaterally ovariectomized 4-day cyclic rats. In addition, bilateral ovariectomy reversed the effect of RU486 on the basal secretion of FSH. RU486 induced an increase in basal LH concentrations. Since ovarian inhibin decreases the basal release of FSH, and decreases in peripheral inhibin seem to be responsible for the transient rise in FSH during the oestrus cycle, the effect of RU486 on serum levels of LH and FSH during dioestrus in rats injected with a sheep anti-inhibin serum (AIS) were further evaluated. Treatment with AIS increased FSH levels in oil-treated rats without altering the levels of LH. In contrast, the effects of AIS on FSH secretion were blunted in RU486-treated rats. The results suggest that inhibin might be involved in the RU486-induced decrease of FSH secretion in cyclic rats.     

Hormone concentrations and ovulatory response in rats treated with antiprogestagens

Administration of antiprogestagens (2 mg/day) to female rats for 21 days induces high serum prolactin levels. These levels stimulate luteal progesterone production and an increase in ovarian weight. Compared with RU486 (mifepristone) the increase in prolactin is less after treatment with ZK299 (onapristone), an antiprogestagen with lower antiglucocorticoid activity. To study whether cyclic ovulations occur in rats treated with antiprogestagens, 5-day cyclic rats were given daily injections of RU486 or ZK299 (2 mg) from metoestrus (day 1) to pro-oestrus. This treatment advanced the forthcoming ovulation by 1 day; however, the ovulation rate was low. Injection of 10 IU human chorionic gonadotrophin on the afternoon of pro-oestrus (day 3) increased the ovulation rate, but not to the level found in oil-treated rats. Serum LH concentrations measured from metoestrus to oestrus at 10.00 and 17.00 h were higher in antiprogestagen- than in oil-treated rats from day 2 (17.00 h) onwards. A low preovulatory LH surge was found in antiprogestagen-treated rats on the afternoon of pro-oestrus (day 3). Ovarian histology at the day of oestrus (day 4) confirmed the presence of a low LH surge as, besides ruptured follicles, unruptured follicles with dispersion of cumulus cells were present. The pro-oestrus surge of prolactin was also advanced by 24 h. The magnitude, however, was not different from that in oil-treated rats at day 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Antiprogesterone RU486 increases serum immunoreactive inhibin levels and LH:FSH and testosterone:oestradiol ratios in cyclic rats.

Since administration of the antiprogesterone RU486 to cyclic rats results in a dissociation of basal LH and FSH secretion we studied its effects on peripheral levels of inhibin, oestradiol and testosterone throughout the oestrous cycle. Cyclic rats were given RU486 (2 mg) twice daily (09.00 and 17.00 h) on metoestrus, dioestrus and pro-oestrus. Oil-treated rats were used as controls. Serum concentrations of immunoreactive inhibin in oil-treated rats increased from metoestrus to pro-oestrus and decreased at oestrus. RU486-treated rats had serum inhibin concentrations significantly increased over oil-treated rats at dioestrus and pro-oestrus, but not at oestrus. At both pro-oestrus and oestrus serum concentrations of LH, testosterone and oestradiol were significantly raised in RU486-treated rats compared with oil-treated controls. In contrast, serum FSH concentrations in RU486-treated rats were decreased on both days. Ovaries from RU486-treated rats showed an increased testosterone content at pro-oestrus, mainly in the interstitial tissue. The results of the present study demonstrate that RU486 has a stimulatory effect on inhibin secretion, and offer an explanation for the decrease in basal serum FSH levels. The low FSH secretion on the morning of oestrus in spite of the low levels of inhibin suggests that progesterone is involved in FSH secretion at this time.     

The ovary-mediated FSH attenuation of the LH surge in the rat involves a decreased gonadotroph progesterone receptor (PR) action but not PR expression

Hyperstimulation of ovarian function with human FSH (hFSH) attenuates the preovulatory surge of LH. These experiments aimed at investigating the mechanism of ovarian-mediated FSH suppression of the progesterone (P(4)) receptor (PR)-dependent LH surge in the rat. Four-day cycling rats were injected with hFSH, oestradiol benzoate (EB) or vehicle during the dioestrous phase. On pro-oestrus, their pituitaries were studied for PR mRNA and protein expression. Additionally, pro-oestrous pituitaries were incubated in the presence of oestradiol-17beta (E(2)), and primed with P(4) and LH-releasing hormone (LHRH), with or without the antiprogestin RU486. After 1 h of incubation, pituitaries were either challenged or not challenged with LHRH. Measured basal and LHRH-stimulated LH secretions and LHRH self-priming were compared with those exhibited by incubated pituitaries on day 4 from ovariectomized (OVX) rats in metoestrus (day 2) injected with hFSH and/or EB on days 2 and 3. The results showed that: i) hFSH lowered the spontaneous LH surge without affecting basal LH and E(2) levels, gonadotroph PR-A/PR-B mRNA ratio or immunohistochemical protein expression; ii) incubated pro-oestrous pituitaries from hFSH-treated rats did not respond to P(4) or LHRH, and lacked E(2)-augmenting and LHRH self-priming effects and iii) OVX reversed the inhibitory effects of hFSH on LH secretion. It is concluded that under the influence of hFSH, the ovaries produce a non-steroidal factor which suppresses all PR-dependent events of the LH surge elicited by E(2). The action of such a factor seemed to be due to a blockade of gonadotroph PR action rather than to an inhibition of PR expression.     

Interdependence of oestradiol and 5 alpha-dihydrotestosterone in modulating LH and FSH secretion in rats

The effects of oestradiol, 5 alpha-dihydrotestosterone (DHT) and oestradiol plus DHT on pituitary responsiveness to LHRH were studied. Rats ovariectomized for 2 weeks were infused s.c. (by osmotic minipump) with LHRH at 250 ng/h for 6 days. Control rats received a sham s.c. pump. On day 3, silicone elastomer implants containing oestradiol or DHT were implanted s.c. and on day 6 the effects of these in-vivo treatments on pituitary LH and FSH content and on in-vitro (perifusion) LH and FSH secretion following maximal LHRH stimulation (1 microgram/ml perifusion medium) were assessed. Luteinizing hormone-releasing hormone alone decreased pituitary LH/FSH content and, in response to acute LHRH challenge in vitro, the absolute rate of LH/FSH release, but not LH/FSH release expressed as a fraction of pituitary content. Oestradiol alone increased pituitary LH/FSH content and LHRH-induced LH/FSH release in vitro, both absolutely and as a fraction of pituitary LH/FSH. Oestradiol exacerbated the decrease in pituitary LH/FSH caused by LHRH pretreatment in vivo, and decreased the absolute rate of LHRH-stimulated LH/FSH release in vitro, but increased this rate when it was expressed as a fraction of pituitary LH/FSH. In both LHRH-treated and control rats, DHT increased pituitary LH/FSH content, did not change the absolute rate of LH/FSH release in response to acute LHRH challenge in vitro, but decreased the rate of LH/FSH release expressed as a fraction of pituitary LH/FSH content.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Differential regulation of pulsatile luteinizing hormone (LH) and follicle-stimulating hormone secretion in ovariectomized rats disclosed by treatment with a LH-releasing hormone antagonist and phenobarbital

Although pulsatile LH release in ovariectomized (OVX) rats appears to be controlled by pulsatile discharges of LHRH, the neuroendocrine regulation of episodic FSH release remains to be explored. The main objective of the present study is to compare and contrast the effects of a potent LHRH antagonist (ALHRH) and a central nervous system depressant, phenobarbital (PhB), on pulsatile LH and FSH release in OVX rats. Three to 4 weeks after ovariectomy, blood samples were obtained at 10-min intervals for 3 h, after which LHRH was injected and sampling continued for an additional hour. In control OVX rats, periodic increases in plasma LH and FSH levels occurred approximately every 30 to 60 min, respectively. Treatment of OVX rats with PhB several hours earlier resulted in a suppression of mean plasma levels and pulse frequencies of both LH and FSH. Interestingly, PhB suppressed the pulse amplitude of LH, but not of FSH. Phenobarbital increased pituitary LH responses to LHRH, but did not alter the FSH responses. When ALHRH was given to OVX rats 24 h before blood sampling, mean plasma LH levels as well as LH pulse frequency and amplitude were severely diminished. In striking contrast, ALHRH did not affect the frequency or amplitude of FSH pulses. However, mean plasma FSH levels were suppressed to 31% of levels measured in control OVX rats. These results demonstrate that in contrast to LH secretion, FSH secretion in OVX rats appears to be regulated by two distinct neuroendocrine mechanisms: an LHRH-dependent mechanism controlling the nonepisodic component of FSH secretion (baseline secretion) and a LHRH-independent mechanism controlling pulsatile FSH release.     

Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-induced LH secretion only under conditions leading to preovulatory LH surges.

We recently demonstrated that neuropeptide Y (NPY) potentiates the ability of pulsatile LHRH infusions to restore LH surges in pentobarbital (PB)-blocked, proestrous rats. In the present study we determined if specific endocrine conditions are necessary for the expression of these direct pituitary effects of NPY. Facilitatory actions of NPY were examined in the absence of gonadal feedback [ovariectomy (OVX)], in the presence of negative gonadal feedback (metestrus), after estrogen priming of the pituitary gland [OVX plus 30 micrograms estradiol benzoate (EB) 2 days before experiments], and after treatments which evoke preovulatory-like LH surges (OVX plus EB and 5 mg progesterone or P the morning of experiments). Rats received jugular catheter implants the day before experiments. On the day of experiments, hourly blood samples were taken from 1100-2100 h. At 1330 h, rats received injections of PB to block endogenous LHRH release, or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15 ng/pulse) or saline, along with concurrent pulses of NPY (1 or 5 micrograms/pulse) or saline. Plasma samples were analyzed by LH RIA. In all cases, pulsatile administration of 15 ng LHRH resulted in plasma LH levels that were significantly elevated above saline-treated, PB-blocked controls. Only in the case of EB+P-treated rats did coadministration of 5 micrograms NPY along with LHRH significantly enhance LHRH-stimulated LH secretion (P < 0.001). NPY had no effect on LHRH-stimulated LH secretion in OVX, OVX + EB-treated, or metestrous rats. Pulsatile administration of either dose of NPY alone did not stimulate LH release in any of the four groups examined. These results demonstrate that the facilitatory effects of NPY on LHRH-stimulated LH secretion can be manifest only under the endocrine conditions required to produce full, preovulatory-like LH surges, i.e. after estrogen and P treatment.     

Dynamics of ovarian inhibin secretion during the oestrous cycle of the rat

Plasma and ovarian concentrations of inhibin were determined at 3-h intervals throughout the 4-day oestrous cycle of rats by a radioimmunoassay (RIA) based on a bovine RIA. Plasma concentrations of LH, FSH, progesterone, testosterone, oestradiol-17 beta, and pituitary contents of FSH and LH were also determined during the cycle. Plasma levels of inhibin showed a marked increase on the morning of oestrus and the afternoon of metoestrus, and a further increase was noted on the morning of pro-oestrus. These increases in plasma levels of inhibin were probably due to the following three events in the ovary, follicular recruitment on the morning of oestrus, selection of follicles on the day of metoestrus, and final maturation of follicles for ovulation on the morning of pro-oestrus with an increase in oestradiol-17 beta secretion. A striking decrease in inhibin secretion occurred during the process of ovulation after the preovulatory gonadotrophin surge on the afternoon of pro-oestrus. Basal levels of plasma FSH gradually decreased from metoestrus to pro-oestrus as plasma levels of inhibin increased. There was a significant inverse relationship between plasma levels of FSH and inhibin throughout the oestrous cycle (r = -0.51). The present findings suggest that changes in the plasma levels of inhibin during the oestrous cycle provide a precise indicator for follicular recruitment, selection and ovulation, and that changes in concentrations of oestradiol-17 beta in the plasma are associated with follicular maturation.     

Hypothalamic luteinizing hormone releasing factor and corticotrophin releasing activity in relation to pituitary and plasma hormone levels in male and female rats.

Hypothalamic corticotrophin releasing (CR) activity and LH-releasing factor (RF) content, and pituitary and plasma LH, FSH and ACTH were measured in adult male and female Wistar rats maintained under 14 h light per day. Hypothalamic LH-RF and pituitary and plasma hormones were estimated by radioimmunoassay while CR-activity was assessed by the amount of ACTH released from hemipituitaries in vitro. Two experiments were carried out on male animals. In the first, some of the animals were kept in a room, distant from the animal house, in which the lighting was reversed with respect to the external environment. In animals exposed to the reversed lighting regime, hypothalamic LH-RF content and pituitary gonadotrophin concentrations were significantly lower than the values in male rats kept in the animal house where they were in close proximity to female rats. In the second experiment, which was carried out on animals which had all been kept in the animal house, there was no significant differences between the LH-RF contents measured at 3-4 h intervals throughout the day. Pituitary LH and FSH contents, but not concentrations, were significantly increased at 12.00 h. There was little differences between the experiments in CR-activity, plasma ACTH concentrations and profiles of pituitary ACTH content and concentration. As expected there was a diurnal rhythm in plasma corticosterone concentrations (determined by competitive protein-binding assay) with the peak occurring between 15.00 and 18.00 h. The profiles of plasma and pituitary ACTH were similar to that of plasma corticosterone. Corticotrophin releasing activity dropped significantly between 12.00 and 16.00 h, but remained steady at the other times. In female rats there were no significant differences between hypothalamic LH-RF content throughout the 4-day cycle. During pro-oestrus the mean LH-RF content rose to teach a high level at 18.00 h at which time plasma LH concentration had risen sharply to a level consistent with the peak of the preovulatory surge. Plasma FSH concentration also rose significantly between 15.00 and 18.00 h of pro-oestrus. At metoestrus and dioestrus, plasma FSH levels were lower in the morning than in the evening. These results suggest that (1) there is no diurnal rhythm in hypothalamic LH-RF, (2) there may be a diurnal rhythm in pituitary gonadotrophin content in the male and in plasma FSH concentration on the days of metoestrus and dioestrus in the female, (3) if a surge of LH-RF does occur on the afternoon of pro-oestrus, the rate of LH-RF synthesis exceeds its release, and (4) the mechanism which regulates gonadotrophin secretion in the male may be affected by factors in the environment other than daylength. The results provide further evidence for the view that the diurnal rhythm of corticosterone secretion is under hypothalamo-hypophysial control.     

Pituitary responsiveness to LHRH during pregnancy in the rat: effect of progesterone

The LH and FSH responses to a standard infusion of LHRH were studied on days 8, 12, 15, 17, 19, 21 and 22 after conception as well as on day 23, i.e. after parturition. Groups of rats were also killed on days 8, 15, 19, 22 and 23 and on the day of pro-oestrus of the 4-day cycle for the assay of progesterone, 20 alpha-dihydroprogesterone (DHP), oestradiol-17 beta, LH and FSH. Finally, the post-partum surges of LH and FSH were compared with those at pro-oestrus in 4-day cyclic rats. The LH and FSH responses to LHRH were relatively low on days 8 and 12, twice as high on days 15, 17 and 19, had increased further on day 21 and reached maximal values on day 22. The gonadotrophin responses were low on day 23. The post-partum surges of LH and FSH were much higher than the pro-oestrous surges. Pituitary contents of LH and FSH were higher than on pro-oestrus of the 4-day cycle. On day 23, however, the pituitary contents had declined by 60-80%. No apparent relationship was found between plasma concentrations of LH and FSH and LHRH responsiveness during pregnancy. Concentrations of oestradiol-17 beta on day 22 were higher than on all other days of pregnancy, but lower than on pro-oestrus of the 4-day cycle. Concentrations of progesterone were high until day 19 and low on days 22 and 23; the concentration of DHP was low until day 19 and very high on days 22 and 23.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma gonadotrophin concentrations, pituitary gonadotrophin content and pituitary responsiveness to LHRH in rats treated with LHRH and oestradiol benzoate

Suppression of gonadotrophin secretion during prolonged treatment with a high dose of LHRH was studied. Long-term ovariectomized rats were infused for 6 days with various doses of LHRH (25, 50, 100, 250 or 500 ng/h) with or without simultaneous treatment with oestradiol benzoate (OB; 3 micrograms/s.c. injection); a further group was treated with OB only. The effects of these treatments were studied on plasma concentrations of LH and FSH, the pituitary content of LH and FSH and on LH and FSH secretion in vitro (perifusion) in the unstimulated state and following maximal LHRH stimulation (1 microgram LHRH/ml perifusion medium). Administration of LHRH caused a dose-dependent reduction in plasma concentrations of LH and FSH and depleted the pituitary LH/FSH stores. Treatment with OB lowered the plasma concentration of LH to about 30% and that of FSH to about 65% of the control values. Administration of OB plus a low dose of LHRH (25 or 50 ng/h) markedly stimulated the secretion of LH but not of FSH, so that the plasma concentrations of LH were fully restored to the control value. With higher rates of LHRH infusion, OB caused no enhancement of the plasma concentrations of LH and FSH. The experiments in vitro revealed a sensitizing, i.e. stimulatory effect, of OB on LHRH-stimulated LH and FSH secretion. However, the higher the rate of infusion of LHRH the smaller the sensitizing effect. Interpolation from dose-response curves showed that an LHRH infusion rate of about 150 ng/h (which would establish a plasma concentration of LHRH of about 68 pmol/l) would have no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro basal and GnRH-stimulated secretion of gonadotrophins reflects long-lasting modulatory effects, and peripheral levels are not predicted by pituitary responsiveness to GnRH

OBJECTIVE: Production of the appropriate pattern of gonadotrophin levels is crucial to proper functioning of the female reproductive system. We aimed to establish whether the pituitary has invariant secretory characteristics when isolated from in vivo controls. We aimed to obtain information during both the rising and declining phases of the gonadotrophin surge. DESIGN: This study investigated factors that are directed at the pituitary by isolating it from the acute influences of the in vivo environment and studying gonadotrophin secretion in vitro. METHODS: Pituitaries of adult female rats were collected at selected times during the day of pro-oestrus and incubated in vitro, and at the same time blood was collected. Peripheral levels of LH and FSH were measured over the whole day of pro-oestrus, basal in vitro secretions of LH and FSH from pituitaries were measured, GnRH-stimulated LH and FSH secretion were assessed, and the responsiveness of LH and FSH secretion to GnRH were calculated. RESULTS: Peripheral levels of LH peaked at 1800 h (P<0.02) followed by a subsequent decline. In contrast, although FSH had a peak at 1800 h (P<0.01), serum levels were also high at the end pro-oestrus. The profile of basal LH and FSH secretion from the pituitary in vitro, in the absence of added secretagogue, resembled that of the peripheral blood levels of each gonadotrophin. Pituitaries collected at 1800 h secreted most LH (P<0. 02). FSH secretion was low early on the day of pro-oestrus and then increased to and was maintained at high levels in the last quarter of the day (P<0.01).When the pituitaries were stimulated with GnRH the patterns of LH release and FSH release approximated those observed for basal release. Responsiveness of the pituitaries to GnRH was calculated by determining the ratio of GnRH-stimulated release to basal release. However, low levels of gonadotrophin were secreted even from pituitaries which were highly responsive as determined from consideration of percentage increase in secretion induced by GnRH. CONCLUSIONS: The secretory activity was dependent on the time of day the pituitaries were collected. Since the secretion occurred after the tissue had been removed from the direct influence of the in vivo environment, the variations in secretion must reflect long-lasting components of the mechanism that regulate gonadotrophin concentrations. There were changes in both LH and FSH responsiveness to GnRH stimulation over the day of pro-oestrus. Delineation of the time courses and changing predominance of multiple processes is needed to assist understanding the mechanisms underlying the female reproductive cycle.     

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.     

Control of follicle-stimulating hormone secretion by steroid-free bovine follicular fluid in the ovariectomized rat

The effects of steroid-free bovine follicular fluid (bFF) and sodium phenobarbitone on spontaneous LH releasing hormone (LHRH)-induced secretion of FSH and LH were studied in ovariectomized rats. Luteinizing hormone releasing hormone was administered by infusion to rats anaesthetized with phenobarbitone. Bovine follicular fluid reduced FSH release and synthesis. Luteinizing hormone release remained unaffected after bFF treatment. Phenobarbitone reduced both FSH and LH release. The observed suppressive effects of bFF and phenobarbitone on FSH secretion were additive, suggesting that the basal release of FSH has an LHRH-dependent and an LHRH-independent component. Furthermore, bFF did not affect pituitary responsiveness of LH secretion to LHRH and reduced the responsiveness of FSH secretion only when administered some time before the LHRH challenge. The present observations support the view that in the ovariectomized rat the pituitary gland is the only site of action of inhibin-like activity as present in bFF.     

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.     

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.