Effects of antibody to oestrogen or of ovariectomy on the incorporation of [35-S]methionine into brain protein and on gonadotrophin levels during the oestrous cycle in the rat.

The incorporation of [35S]methionine into protein of the anterior pituitary and discrete brain areas was measured following the administration of antibodies to oestrogen, ovariectomy, or adrenalectomy on the afternoon of dioestrus. The antibody to oestrogen deleted the circadian rhythms of methionine incorporation normally observed in the various brain areas together with the peaks of incorporation normally observed in the median eminence area and anterior pituitary on the evening of pro-oestrus. The peaks of incorporative activity normally observed in the preoptic area and amygdala (relative to the putamen) at 03.00 h on the day of pro-oestrus were also deleted. Administration of the antiserum on the morning of pro-oestrus failed to alter the pattern of methionine incorporation normally observed on the evening of pro-oestrus. Ovariectomy performed at 16.00 h of dioestrus blocked the preovulatory rise of luteinizing hormone (LH) (as did the antibody to oestrogen) and inhibited the peak of methionine incorporation normally observed in the anterior pituitary on the evening of pro-oestrus. However, for the peak in the median eminence to be inhibited, ovariectomy had to be performed on the morning of the preceeding oestrus. Adrenalectomy alone, or adrenalectomy with ovariectomy, performed on the afternoon of dioestrus did not affect the levels of methionine incorporation in the brain or anterior pituitary at 18.00 h on the day of pro-oestrus. Animals which had been ovariectomized and injected with 2-5 mug oestradiol benzoate on the morning of oestrus showed significantly increased levels of methionine incorporation in all the brain areas and the anterior pituitary at 18.00 h of expected pro-oestrus. The administration of antibody to oestrogen to a similar group of animals on the afternoon of expected dioestrus inhibited the rise at 18.00 h of expected pro-oestrus. The apparent discrepancy between the results obtained with ovariectomy and the antiserum appeared to be due to the ability of the antiserum to neutralize the activity of oestrogens retained by the tissues. The present results suggest that the changes in incorporation of methionine into protein in the brain and anterior pituitary are brought about by the action of endogenous oestrogen: there appears to be a steady summative effect on the median eminence throughout the oestrous cycle together with a short-lived effect occurri4g during pro-oestrus and affecting the anterior pituitary.     

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.     

Gonadotrophin-releasing activity of neurohypophysial hormones: I. Potential for modulation of pituitary hormone secretion in rats

Neurohypophysial hormones have been implicated in the control of anterior pituitary function, and oxytocin has been shown to stimulate gonadotrophin excretion and ovarian follicular development in certain species. To determine the role of neurohypophysial peptides in the control of gonadotrophin release, their actions on LH and FSH secretion were analysed in rats in vivo and in vitro. In adult female rats, administration of oxytocin during early pro-oestrus advanced the spontaneous LH surge and markedly increased peripheral LH levels at 15.00 h compared with control animals. In cultured pituitary cells from adult female rats, oxytocin and vasopressin elicited dose-related increases in LH and FSH release. Such responses were not affected by a potent gonadotrophin-releasing hormone (GnRH) antagonist that abolished GnRH agonist-induced release of LH and FSH. Oxytocin did not enhance GnRH agonist-stimulated gonadotrophin release to the same extent as it increased basal secretion, but at low concentrations of GnRH agonist the effects were additive. The gonadotrophin responses to oxytocin and vasopressin were inhibited by the specific neurohypophysial hormone antagonists, [d(CH2)5D-Ile2,Ile4,Arg8]vasopressin and [d(CH2)5Tyr (Me),Arg8]vasopressin. These results provide direct evidence that neurohypophysial hormones can stimulate gonadotrophin secretion through a receptor system distinct from the GnRH receptor. Such a mechanism could represent a complementary hypothalamic control system for long-term modulation of LH and FSH secretion by exerting a basal or tonic influence on gonadotrophin production.     

Effects of active and passive immunization with LH-RH on gonadotrophin secretion and reproductive function in female rats.

In order to get information about the physiological role played by LH-RH in the regulation of tonic and phasic secretion of gonadotrophins, and about the existence of FSH-RH distinct from LH-RH, we attempted to neutralize endogenous LH-RH by passive and active immunization with LH-RH in female rats. Anti-LH-RH serum prevented pre-ovulatory gonadotrophin surges at proestrus and ovulation, and it suppressed gonadotrophin increase induced by oestradiol benzoate or progesterone in ovariectomized rats. Elevated serum gonadotrophin concentrations in long-term ovariectomized rats were lowered by anti-LH-RH serum injection. The decrease in FSH levels was less than that in LH levels. Serum FSH rose without significant changes in serum LH level for 6 h after ovariectomy on pro-oestrus or dioestrus. The post-ovariectomy rise of FSH was not suppressed by the anti-LH-RH serum which was enought to inhibit serum LH to undetectable levels. Active immunization with LH-RH resulted in decreasing LH levels but failed to alter FSH levels in both serum and pituitary. Seven out of 10 rats immunized with LH-RH became constantly di-oestrous. The weights of anterior pituitary, ovary and uterus of the LH-RH immunized rats were significantly smaller than those of BSA immunized controls. Ovaries of LH-RH immunized rats contained few fresh corpora lutea. These results indicate that LH-RH plays a significant role in the control of both plastic and tonic secretion of LH and FSH. The existence of FSH-RH distinct from LH-RH or mechanism which specially controls the basal FSH secretion is indicated.     

Evidence that norepinephrine and epinephrine systems mediate the stimulatory effects of ovarian hormones on luteinizing hormone and luteinizing hormone-releasing hormone

Results from previous investigations have suggested an important role for central epinephrine (EPI) systems in mediating the stimulatory effects of ovarian hormones on LH release in ovariectomized female rats. The purpose of these experiments was 1) to test whether selective inhibition of EPI synthesis blocks the sequential accumulation and decline of LHRH concentrations in the median eminence that precedes the ovarian hormone-induced LH surge and 2) to test whether the stimulatory ovarian hormone regimen enhances the activity of EPI systems in the hypothalamus. Ovariectomized rats were treated with estradiol, followed 2 days later by progesterone. Animals were treated before progesterone administration with saline, one of the EPI synthesis inhibitors [SK&F 64139 (2,3-dichloro-tetrahydroisoquinoline HCl) or LY 78335 (dichloro-alpha-methylbenzylamine)], or the dopamine-beta-hydroxylase inhibitor FLA-63 (bis-4-methyl-1-homopiperazinyl thiocarbonyl disulfide), which inhibits NE and EPI synthesis. The catecholamine synthesis inhibitors blocked or delayed the afternoon LH surge. FLA-63 completely prevented the accumulation of LHRH in the median eminence that preceded the rise in LH release. However, selective EPI synthesis inhibition with SK&F 64139 only partially prevented this increase in LHRH. A second EPI synthesis inhibitor, LY 78335, delayed both the LH surge and the rise in LHRH. In a second experiment, the administration of estradiol and progesterone to ovariectomized rats increased the alpha-methyltyrosine-induced depletion of hypothalamic EPI, suggesting increased activity in this system during the LH surge. Further experiments localized this effect to the medial basal hypothalamus. The depletion of both NE and EPI after synthesis inhibition was also enhanced during an earlier period, approximating the time of LHRH accumulation. These results suggest that the ovarian hormones activate both NE and EPI systems to stimulate the early afternoon rise of LHRH in the median eminence and to induce the subsequent LH surge.     

Influence of gonadotrophins on cAMP formation in theca cells isolated from pre-ovulatory rat follicles

Pre-ovulatory follicles were obtained from immature PMSG treated rats at various times on the day of pro-oestrus. From these follicles the theca capsules were mechanically isolated and subsequently incubated in the presence or absence of LH and/or FSH. The accumulation of cyclic adenosine 3'5'-monophosphate (cAMP) in tissue plus medium was determined. In certain experiments the rats were injected with LH, FSH or saline 2 h prior to sacrifice. When the theca cells were isolated from rats sacrificed before the endogenous gonadotrophin surge, FSH added in vitro did not significantly stimulate cAMP formation, while LH had a small but significant stimulatory effect. When isolated 2-5 h after the endogenous gonadotrophin surge the theca cells responded with a marked increase in cAMP formation when exposed to LH in vitro, whereas FSH in vitro still had no stimulatory effect. Theca cells, isolated before the endogenous gonadotrophin surge, from rats pre-treated with a single ip injection of FSH 2 h prior to sacrifice, responded to a subsequent in vitro exposure to LH with a marked increase in cAMP levels. The results indicate that the FSH component of the endogenous gonadotrophin surge increases the sensitivity of the theca cells to LH - i.e. in vivo exposure to FSH seems to be essential for the development of responsiveness of the cAMP system to LH in the theca cells from pre-ovulatory rat follicles.     

Effects of testosterone, FSH, and LH on oestradiol and progesterone secretion by preovulatory cumulus oophorus complexes of the rat.

Female Wistar rats exhibiting a regular 4-day oestrous cycle were included in this study. They were killed in succession on the day of pro-oestrus at 11.00, 18.00, and 22.00 h. From ovarian preovulatory follicles cumulus oophorus complexes (COCs) were isolated and subsequently cultured with or without testosterone (T), T plus FSH, or T plus LH. In control cultures COCs isolated at all investigated hours released similar amounts of oestradiol. T stimulated this basal secretion and the effect was usually enhanced in the presence of FSH or LH. In control cultures the amount of released progesterone was greatest when expanded COCs were isolated (22.00 h). T present in culture media diminished the amount of secreted progesterone. However, when T was added with FSH or LH a distinct stimulatory effect was observed, except in cultures with T plus FSH set up at 22.00 h. Previously, gonadotrophins alone did not effect progesterone secretion. The results suggest that T can regulate steroid, and especially progesterone secretion by COCs. Until the preovulatory gonadotrophin surge T can inhibit luteinization of COCs, while afterwards, acting synergestically with gonadotrphins (especially with LH), T can stimulate progesterone production in the cumulus granulosa cells.     

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)     

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.     

Hormonal control of oocyte meiosis, ovulation and luteinization in mammals.

The ovulatory process can be regarded as a series of biochemical and morphological changes ultimately leading to the release of a mature oocyte and the transformation of the Graafian follicle into the corpus luteum. This process involves acute changes in steroidogenesis, resumption of oocyte meiosis, and finally rupture of the follicular wall and luteinization of the granulosa cells. Normally, all of these changes are induced synchronously by the pre-ovulatory LH surge. Experimentally, however, these changes in steroidogenesis, oocyte maturation and follicular rupture can be dissociated from each other showing that the LH effect is mediated via different cellular messengers. The gonadotrophins act in an orderly sequence to induce follicular maturation. The granulosa cells increase their number of LH receptors and respond to LH with increased stimulation of cyclic AMP accumulation and progesterone secretion. Concomitantly, they decrease in their FSH receptors and their response to FSH diminishes in terms of ability to stimulate cyclic AMP accumulation. The ovulatory process is associated with increased uptake of LH by the follicle; when granulosa cells are obtained from pre-ovulatory follicles and cultured they luteinize spontaneously. Steroid hormones modulate the actions of gonadotrophins on follicular maturation. In addition, there are non-steroidal factors in follicular fluid which regulate follicular maturation: an oocyte maturation inhibitor keeps the oocyte in meiotic arrest; a luteinizing inhibitor prevents the granulosa cells from luteinizing prior to follicular rupture; a folliculostatin inhibits FSH release from the pituitary gland. The functional activity and the lifespan of the corpus luteum depend on adequate pre-ovulatory as well as post-ovulatory gonadotrophic stimulation. Its lifespan may also be regulated by an LH binding inhibitor.     

Detection of chorionic gonadotrophin like activity in infertile cycles with a short luteal phase

Three of 24 infertile women with a short luteal phase transiently showed chorionic gonadotrophin (HCG) like immunoreactivity in serum as measured by the HCG-beta subunit radioimmunoassay. The plasma progesterone concentration was elevated above the lower normal postovulatory level in 2 of these women who also had elevated basal body temperature at the time HCG was detected. Positive HCG reaction coincided with the pre-ovulatory surge of luteinizing hormone (LH) in one patient, but in 2 other cases the LH concentration was low suggesting absence of LH interference. Our results suggest that some infertile women may conceive during their cycles with a short luteal phase and maintain pregnancy until HCG becomes detectable.     

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.     

Changes in the hypothalamic contents of LHRH-I and -II and in pituitary responsiveness to synthetic chicken LHRH-I and -II during the progesterone-induced surge of LH in the laying hen

The contents of LHRH-I and -II in the anterior hypothalamus and posterior hypothalamus (including the mediobasal hypothalamus and median eminence) were measured at 90, 180 and 360 min after the i.m. injection of laying hens with progesterone. Whilst no changes were observed in the content of LHRH-I in the anterior hypothalamus, LHRH-I in the posterior hypothalamus tended to fall at 90 and 180 min after injection of progesterone in hens maintained on 16 h light:8 h darkness (16L:8D) and 8L:16D respectively. Pretreatment of laying hens with tamoxifen significantly increased the hypothalamic contents of LHRH-I and -II, raised the basal plasma concentration of LH and modified the LH response to progesterone injection. In hens in which tamoxifen prevented an increase in the plasma concentration of LH after progesterone injection, the content of LHRH-I in the posterior hypothalamus remained unchanged. In contrast, in hens in which progesterone stimulated a steep increase in LH within 90 min, there was a pronounced and significant fall in LHRH-I content of the posterior hypothalamus. No change in the hypothalamic content of LHRH-II was observed during the progesterone-induced surge of LH until plasma concentrations had attained maximal values or started to decline. Then, in hens maintained on 16L:8D, a significant fall in the content of LHRH-II in the anterior hypothalamus was found at both 180 and 360 min after injection with progesterone. Tests in vitro and in vivo of the responsiveness of the pituitary gland to synthetic LHRH-I and -II revealed no change at 90 min after injection of laying hens with progesterone, when plasma concentrations of LH were increasing, but a pronounced reduction when plasma LH concentrations were maximal or falling. These results suggest that LHRH-I mediates in the progesterone-induced increase in the plasma concentration of LH. Although the subsequent decline in plasma LH was associated with a reduced responsiveness of the pituitary gland to LHRH, a significant correlation between the contents of LHRH-I and -II in the anterior hypothalamus and a fall in the hypothalamic content of LHRH-II when plasma LH was maximal or declining allows the possibility of an involvement of this peptide in the neuroendocrine events preceding ovulation.     

Evidence that the dopaminergic incerto-hypothalamic tract has a stimulatory effect on ovulation and gonadotrophin release

2 micrograms dopamine (DA) were injected into particular hypothalamic sites containing dopaminergic nerve terminals, on the morning of pro-oestrus in cyclic rats, in which ovulation was blocked by administration of 35 mg/kg pento-barbitone Na, in the afternoon of pro-oestrus. DA in a specific area of the zona incerta (ZI) (medial area at A 5.4 mm; de Groot atlas), and medial anterior hypothalamus (AH) overcame the pentobarbitone block and induced ovulation. Injections into the median eminence and preoptic area were ineffective, as were injections of 5-hydroxytryptamine and nor-adrenaline into the ZI. Injection of 2 micrograms haloperidol and lesions in the ZI inhibited ovulation in otherwise untreated cyclic rats. Lesions induced constant dioestrus for 14 +/- 0.7 days, but the animals were not pseudopregnant. 2 micrograms DA were also injected into the ZI of ovariectomised rats primed with 2 micrograms oestradiol benzoate 48 h before; this treatment stimulated a rise in plasma LH approximately 40 min later. These findings indicate that the dopaminergic incerto-hypothalamic tract which has nerve terminals in the ZI and AH has a stimulatory role in the control of gonadotrophin release.     

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.     

Progesterone modulation of the luteinizing hormone surge: regulation of hypothalamic and pituitary progestin receptors

We have investigated the possible role of hypothalamic and pituitary progestin receptors (PR) in modulation of the estradiol-induced LH surge by progesterone in the immature rat. Rats (28 days old) that received Silastic implants containing estradiol in oil at 0900 h had LH surges approximately 32 h later. Progesterone implants were inserted concurrently with estradiol capsules or 24 h later, leading to inhibition or facilitation of the LH surge, respectively. Cytoplasmic and nuclear PR were measured by in vitro exchange assays, using near-saturating concentrations of [3H]R5020 (3H-labeled promegestone; 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), 1, 8, 24, and 32 h after insertion of progesterone or blank implants. Kd values of complexes between [3H]R5020 and PR were 0.5-1 nM (cytoplasmic), and 2-3 nM (nuclear). The sedimentation rates of these complexes in sucrose gradients were 7-8S (cytoplasmic) and 3-4S (nuclear). In rats treated concurrently with estradiol and progesterone for 1 or 8 h, cytoplasmic PR were depleted to 40-60%, and this was accompanied by slight increases in nuclear PR. In control rats treated with estradiol and blank implants, there was no induction of either cytoplasmic or nuclear PR in the hypothalamus-preoptic area for up to 48 h; however, in the pituitary and uterus of these animals, PR increased significantly in both compartments (2- to 3-fold at 24 h, 3- to 5-fold at 32 h, and 4- to 7-fold at 48 h). Administration of progesterone either to inhibit or facilitate LH surges almost completely blocked the inductive effect of estradiol on cytoplasmic PR, but the absence of PR from the cytosol could not be accounted for by their presence in the nucleus. In the hypothalamus-preoptic area of estradiol-treated control rats, neither cytoplasmic nor nuclear PR increased significantly for up to 48 h. The low levels of specific [3H]R5020 binding in pituitary and uterine cytosols from progesterone-treated rats appeared to be due mainly to a decrease in the number of binding sites, rather than to an effect on binding affinities. The 7-8S peak of cytoplasmic PR was considerably reduced in rats treated for 48 h with estradiol and 24 h with progesterone. These results are consistent with the notion that hypothalamic and pituitary PR are involved in modulation of the LH surge by progesterone and point primarily to a pituitary site of action for progesterone facilitation.     

Steroids and pituitary responsiveness in female, androgenized female and male rats.

Spontaneous gonadotrophin release and the gonadotrophin response to LH releasing factor (RF) were studied in pro-oestrus, androgenized female and male rats. The animals were either intact or gonadectomized (about 32 h previously) and treated with various steroids. The gonadotrophin response (especially LH) was much lower in intact males and androgenized females than in pro-oestrous females. Oestrogen plus progesterone increased plasma gonadotrophin concentrations and responses in ovariectomized rats, but inhibited the increase in the plasma gonadotrophin concentration and the LH response which followed castration in males. As in the normal female, ovariectomy decreased the LH response but increased the plasma FSH concentration and response in the androgenized female; oestrogen and progesterone had relatively little effect. Apart from reducing the postcastration rise in plasma FSH, testosterone had no significant effect in gonadectomized male or female animals. These results show that the effect of steroids on the gonadotrophin response to LH-RF as well as the spontaneous secretion of gonadotrophin depends upon sexual differentiation of the hypothalamo-hypophysial system. Studies with various metabolites of progesterone indicated that the facilitatory action of this steroid could be due, in part, to a 5alpha-reduced derivative.     

Oestradiol receptors in the rat anterior pituitary gland during the oestrous cycle: quantitation of receptor activity in relation to gonadotrophin releasing hormone-mediated luteinizing hormone release.

Specific oestradiol binding to a receptor in nuclear and cytosol fractions of the rat anterior pituitary gland and pituitary responsiveness to gonadotrophin releasing horomone (GnRH) during the oestrous cycle have been studied. To accomplish this, both unoccupied and occupied oestradiol-binding sites in the nucleus and total cell were measured during the oestrous cycle. The concentration of unoccupied and occupied sites and total oestradiol binding in the cytosol fluctuated during the cycle. At pro-oestrus, the concentration of cytosol receptor was diminished by about 40% and replenishment occurred during oestrus. On the other hand, a profound increase is concentrations of cellular and nuclear receptors occurred at pro-oestrus. Administration of GnRH significantly LH release at all stages of the cycle. The maximum stimulation of LH release by GnRH was observed at 13.00 h of pro-oestrus. From these studies, it is concluded that pituitary responsiveness to exogenous GnRH during pro-oestrus parallels the changes in the content of oestrogen receptors in the cytosol and nucleus.     

Evidence for a dopaminergic component in the series of neural events that lead to the pro-oestrous surge of LH

The possible participation of dopamine in the neural events that lead to the pro-oestrous surge of luteinizing hormone (LH) was investigated utilizing a dopaminergic ergoline derivative (lergotrile mesylate). Administration of reserpine (2.0 mg/kg, ip) to rats on the day of pro-oestrus depleted brain dopamine and norepinephrine and prevented the LH surge and ovulation. Administration of legotrile mesylate prior to or at the same time as reserpine prevented the inhibitory effects of reserpine on LH release and on ovulation in about half of the animals. When lergotrile mesylate was given on the morning of pro-oestrus, the LH surge was advanced. The results indicate that there is a dopaminergic component in the series of neural events that precede the surge of LH on pro-oestrus, and that the dopaminergic stimulus precedes the LH surge by about 4--5 h.     

CHANGES IN PLASMA LEVELS OF PROLACTIN, IN RELATION TO THOSE OF FSH, OESTRADIOL, ANDROSTENEDIONE AND PROGESTERONE AROUND THE PREOVULATORY SURGE OF LH IN WOMEN

Plasma samples were obtained at 8 hourly intervals around the preovulatory surge of LH in three groups of women with spontaneously ovulatory menstrual cycles, in order to clarify the hormonal events around the time of ovulation. In 21 of 25 women in whom samples were collected every 8 hours the start of the LH surge occurred between midnight and 0800. In 16 of these women the concentration of LH, FSH and progesterone was measured every 8 hours around the pre-ovulatory surge of LH. A progressive increase in progesterone started with the onset of the LH surge, with a transient fall after 32-40 h at a time coincident with that of ovulation. In 10 women oestradiol, androstenedione and prolactin were measured 8 hourly around the pre-ovulatory surge of LH beginning at 0800 h. Prolactin showed a sustained increase in levels beginning at the start of and lasting for the duration of the pre-ovulatory LH surge; oestradiol levels did not rise around this time, and declined by 24 h after the onset of the LH surge. These results suggest that (1) the pre-ovulatory LH surge begins between midnight and 0800 h in the majority of women, (2) luteinization of the granulosa cells within the pre-ovulatory follicle occurs in response to the LH surge, (3) the increase in prolactin at the time of the LH surge is not directly related to increasing levels of oestradiol but may be due to a decrease in hypothalamic inhibition of prolactin secretion which occurs coincident with the release of LHRH associated with the preovulatory LH surge.