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.     

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 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.     

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.     

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)     

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.     

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.     

Adenosine 3',5'-monophosphate primes the secretion of follicle-stimulating hormone

We investigated whether cAMP acts as a mediator for LHRH in either its immediate FSH release action or its self-priming action. Pituitary pieces from cyclic female rats were superfused in vitro in the presence of Bu2cAMP, 8-bromo-cAMP, or forskolin or used as controls. For pituitary pieces from proestrous rats, the first significant increase in the baseline FSH secretion rate occurred after approximately 90 min of exposure to elevated cAMP resulting from forskolin treatment. By comparison, in the same system LHRH caused a 3-fold increase in FSH secretion during a 10-min exposure to the peptide. In contrast to its ineffectiveness as a secretagogue, cAMP elevation resulted in a several-fold augmentation of both LHRH- and elevated K+-stimulated FSH secretion from pituitary pieces from proestrous, but not estrous, rats; for these experiments, superfusion with a cAMP analog or forskolin for varying times preceded a 10-min pulse of either 8 nM LHRH or 47 mM K+. Augmentation of K+-stimulated secretion was evident after 30 min of cAMP elevation. Priming of LHRH-stimulated FSH secretion required 30-90 min of pretreatment with cAMP; longer exposures to cAMP analogs or forskolin were coincident with greater potentiation. Cycloheximide prevented Bu2cAMP augmentation of LHRH-stimulated FSH secretion. These data show that cAMP does not mimic the FSH release action of LHRH, but does augment LHRH- or K+-stimulated FSH secretion with characteristics that lead us to suggest that cAMP mediates, at least in part, the self-priming function of LHRH.     

Progesterone antagonizes the ability of porcine ovarian inhibin to sensitize ovine pituitary cell culture to luteinizing hormone-releasing hormone: dependence on ovaries in vivo

Progesterone (P4) and a porcine follicular preparation of inhibin (MGRA-IV) have opposite actions on regulation of the ability of LHRH to release LH in ovine pituitary cell culture. Both P4 and inhibin change the response to LHRH. The ability of inhibin to sensitize cultures to LHRH (126-273%) was greatly inhibited (up to 100%) in the presence of P4 (10(-7) M). The inhibitory action of P4 on LHRH-stimulated and inhibin-sensitized LHRH-stimulated LH secretion in ovine pituitary cell culture was dependent on the presence of ovaries in vivo. P4 inhibited 68% of LHRH-stimulated LH secretion in pituitary cultures from intact ewes. However, when cultures were prepared from pituitaries collected on days 9, 21, and 42 after ovariectomy, P4 inhibited LHRH-stimulated LH secretion by only 36%, 13%, and 0%, respectively. Ovariectomy had no effect (P greater than 0.05) on the sensitizing action of inhibin on LHRH-stimulated LH secretion, but ovariectomy did cause a time-dependent decline in the inhibitory action of P4 on inhibin-sensitized LHRH-stimulated LH secretion. Furthermore, when cultures were prepared from pituitaries collected from ewes ovariectomized for 35 days but treated with estradiol implants, both LHRH-stimulated and inhibin-sensitized LHRH-stimulated LH secretion were inhibited as well by P4 as in pituitary cultures from intact ewes. These results suggest that although P4 can completely inhibit the sensitizing action of inhibin on LHRH-stimulated LH secretion, its inhibitory action is dependent on the presence of ovaries or estradiol in vivo.     

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)     

Tamoxifen but not other selective estrogen receptor modulators antagonizes estrogen actions on luteinizing hormone secretion while inducing gonadotropin-releasing hormone self-priming in the rat

Selective estrogen receptor modulators (SERMs) are compounds which may function as agonists or antagonists depending upon the target tissue. This study compares the actions of different SERMs on luteinizing hormone (LH) secretion, and on gonadotropin-releasing hormone (GnRH) self-priming in the rat. To do this, 4-day cyclic rats were injected twice, on day 2 (metestrus) and day 3 of the estrous cycle, with one of the following SERMs: 0.25 mg ICI 182,780, 3 mg tamoxifen (TX), LY139481-HCl or LY117018-HCl, or 0.5 mg RU58668. Control rats were given subcutaneous injections of 0.2 ml oil. On the morning of day 4 (proestrus in controls), rats from each group were either injected intraperitoneally with pentobarbital (40 mg/kg) for in vivo study or decapitated and their pituitaries collected for incubation (in vitro study). Additionally, pituitaries taken on each day of the estrous cycle from control rats as well as on day 4 from SERM-treated rats were processed for immunohistochemical determination of the estrogen receptor-alpha (ERalpha) gonadotrope. The plasma concentration or accumulation of LH in the medium was determined after 1 h (basal secretion). Thereafter, an intravenous bolus of GnRH (50 ng/0.5 ml/100 g BW) or 10(-8) M GnRH was injected or added to the medium, respectively. After 1 h of GnRH exposure, blood or medium were taken, and another challenge of GnRH was made. At the end of the 3rd h of the experiment, blood or medium samples were taken again and the LH plasma concentration or accumulation in the medium were determined. All SERM treatments reduced uterus weight and decreased basal and stimulated LH secretion. Also, on day 4, rats treated with any SERM other than TX showed vaginal smears infiltrated by leukocytes and a reduction in GnRH self-priming. TX-treated rats exhibited cornified vaginal smears and an estrogenic effect on GnRH self-priming. Moreover, 15-min exposure to two consecutive GnRH (10(-8) M) challenges 1 h apart in incubated pituitaries with estradiol (E(2), 10(-8) M), TX (10(-7) M), E(2) + TX, or medium alone form ovariectomized rats injected for 3 days with estradiol benzoate (25 microg), TX (3 mg), estradiol benzoate + TX, or 0.2 ml oil, respectively, showed that TX increased GnRH self-priming, as did E(2), whereas it reduced the E(2)-sensitizing effect on GnRH-stimulated LH secretion and cancelled the E(2)-dependent GnRH self-priming. All SERMs prevented the physiological nucleocytoplasmic shuttling of ERalpha exhibited during proestrus in control rats, and TX, in addition, induced a significantly larger number of gonadotropes displaying strong cytosolic immunosignals corresponding to ERalpha than the rest of the experimental groups. Overall, data from this study indicated that, in contrast to the general antagonistic effect of the tested SERMs, TX seemed to display both selective agonist and antagonist activity at the gonadotrope level and on GnRH self-priming of LH secretion respectively.     

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.     

Autoregulation of follicle-stimulating hormone secretion in the hamster: evidence for control at the level of the anterior pituitary gland

Pituitary tissue obtained from proestrous, phenobarbital-treated hamsters was placed in organ culture, and the release rates of FSH and LH were monitored. Addition of LHRH to the culture medium increased gonadotropin release rates. Preincubation of pituitaries in medium that contained 1 microgram purified FSH ( NIADDK rat FSH-I-5) resulted in enhanced basal and LHRH-induced FSH release rates. Full expression of FSH hypersecretion by pituitary tissue occurred after 3-h exposure to purified FSH. This phenomenon appears to require adequate tissue calcium concentrations. Further, exposure of pituitaries to purified FSH slightly but significantly enhanced basal but not LHRH-stimulated LH release compared with that in untreated controls. Experiments that employed labeled FSH, reduced exposure time to purified FSH, or decreased calcium concentration in the medium proved that the increased FSH secretory rate was not due to contamination of the medium with the purified FSH used to stimulate the pituitary. These studies suggest that FSH has the ability to influence its own secretion by an action at the level of the anterior pituitary gland.     

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.     

Kinetic characteristics of LH and FSH responses to LHRH in incubated pituitaries from ovariectomized or ovariectomized, estrogen-implanted rats

Incubated pituitary halves from ovariectomized, estrogen-implanted female rats were shown to be much more sensitive to LHRH than pituitaries from castrated, nontreated animals. LHRH in a concentration of 1,885 pg/ml increased the release of LH and FSH from 7.3 +/- 0.9 and 0.91 +/- 0.13 ng/h/hemipituitary respectively to 21.4 +/- 1.9 and 1.97 +/- 0.18 ng/h in animals implanted with the steroid. In contrast, 5,000 pg/ml of LHRH increased LH secretion from 3.4 +/- 0.3 to 8.4 +/- 0.4 ng/h in ovariectomized, nontreated animals. In pituitaries from both steroid and nontreated animals a highly significant dose response for LH and FSH secretion to the actual concentration of LHRH measured in each incubation tube by radioimmunoassay was observed. When expressed as percent of the corresponding control release, maximal stimulation of LH and FSH was comparable. Pituitaries from implanted animals provided a very sensitive bioassay for LHRH, in which amounts of the peptide lower than 100 pg/ml were detected. The apparent responsiveness to LHRH of pituitaries from estradiol-treated rats was found to be over 20 times greater than that of pituitaries from nontreated castrates.     

Blockade of the spontaneous midcycle gonadotropin surge in monkeys by RU 486: a progesterone antagonist or agonist?

Preovulatory ovarian secretion of progesterone (P4), several hours before the onset of the typical midcycle gonadotropin surge, occurs in humans and monkeys. We investigated the potentially obligatory role of preovulatory P4 secretion in stimulating the midcycle LH surge by administering a potent P4 antagonist, RU 486(17 beta-hydroxy-11 beta-[4-dimethylaminophenyl-1]17 alpha-[prop-1-ynyl]estra-4,9-dien-3-one), to sexually mature, normally ovulatory cynomolgus monkeys on days 10-12 of the menstrual cycle (n = 18). Monkeys were randomized to receive RU 486 alone (5 mg/day, im; group I); RU 486 plus dexamethasone (1 mg/day, im; group II); dexamethasone alone (group III); or vehicle (ethanol; 0.5 ml; group IV). Before drug treatment, the follicular phases were quite similar among groups. The administration of RU 486 blocked (delayed) the expected gonadotropin surge, despite rising estrogen concentrations (greater than 250 pg/ml). The expected LH surge was delayed by RU 486 (n = 5) or RU 486 with dexamethasone (n = 3) until 36 +/- 7 (+/- SEM) and 27 +/- 8 days in groups I and II, respectively. In contrast, groups III (n = 3) and IV (n = 5) had timely midcycle surges after the administration of dexamethasone or vehicle alone (4 +/- 2 and 6 +/- 2 days, respectively). The intermenstrual interval was lengthened by RU 486 administration in both group I and II animals (61 +/- 6 and 54 +/- 6 days) compared to controls (30 +/- 2; P less than 0.0001). In summary, RU 486 effectively blocked imminent midcycle gonadotropin surges, delayed subsequent folliculogenesis, and significantly extended the menstrual cycle length. If RU 486 acted as a pure P4 antagonist, then P4 is necessary for timely midcycle gonadotropin surges to occur. However, recent evidence showing agonistic properties of RU 486 (in the virtual absence of P4) at both endometrial and pituitary levels may favor a P4-like (agonistic) blockade of the estrogen-induced FSH/LH surges by RU 486.     

Gonadal steroids modulate pulsatile luteinizing hormone secretion by perifused rat anterior pituitary cells

Recent studies have shown that LH secretion is pulsatile and that LH pulse characteristics are affected by the prevailing steroid environment in both male and female rats. In the present study, a cell perifusion system was used to examine the effects of testosterone (T) and 17 beta-estradiol (E) on LHRH-stimulated pulsatile LH secretion. T inhibited LH secretion, increasing the EC50 for LHRH, while E stimulated secretion, lowering the EC50. Steroid effects were independent of both LHRH pulse amplitude and frequency. E also affected the pattern of LH secretion by facilitating both LHRH self-priming and desensitization to LHRH. These results show that steroids can affect pulsatile LH secretion by actions exerted at the pituitary level and that steroids can induce both quantitative and qualitative changes in LH secretion in the presence of an invariant LHRH stimulus. These results help to elucidate the mechanisms underlying steroid feedback in vivo, since reduction in pituitary responsiveness to LHRH may play an important role in T feedback, while facilitation by E of both self-priming and desensitization may serve to increase the magnitude and shorten the duration of the proestrous 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.     

Acute inhibitory effects of 17 beta-estradiol are observed on gonadotropin secretion from perifused pituitary fragments of metestrous, but not proestrous, rats

The in vivo suppression of LH by 17 beta-estradiol (E2) has been documented frequently. However, the demonstration of a direct inhibitory action of E2, in contrast to a stimulatory action, on the secretion of LH from the anterior pituitary has been inconsistent. The aim of this study was to determine if E2 can suppress either basal (unstimulated) or GnRH-stimulated gonadotropin secretion directly at the level of the anterior pituitary gland. Anterior pituitaries were obtained from metestrous and proestrous females rats at 0900 h, and trunk blood was collected for serum measurements of LH, FSH, E2, and progesterone (P). Each anterior pituitary was cut into eighths and placed into a microchamber for perifusion. Pituitary fragments were perifused at a rate of 10 ml/h using medium 199 (without phenol red) that contained E2 (1 nM) or ethanol as a control. Six pulses of GnRH (peak amplitude, 50 ng/ml; duration, 2 min) were administered one per h starting at 60 min. Fractions of perfusate were collected every 5 min for measurement of LH and FSH. The total amounts of LH and FSH secreted during the 1-h interval after each GnRH pulse or corresponding basal hour were calculated. Both basal and LH and FSH responses to GnRH were significantly greater from pituitaries of proestrous compared to metestrous rats. The selective suppression of LH secretion by in vitro treatment with E2 was demonstrated using pituitaries from metestrous rats receiving GnRH pulses, but not using pituitaries from proestrous rats. Thus, a negative feedback effect of E2 on LH secretion was observed only in pituitaries from donors with low serum levels of E2 and high P, but not from donors with high serum levels of E2 and low P. We believe that the in vivo steroid environment determined the subsequent responses to in vitro treatment with E2 on GnRH-stimulated gonadotropin secretion from the isolated pituitary gland.