GnRH-induced LH Research Articles

Action of leptin on in vitro luteinizing hormone release in the European sea bass (Dicentrarchus labrax).

The discovery of leptin has sparked a rapidly growing number of publications concerning the role of leptin in the regulation of body adiposity, feeding, and reproductive system in mammals. To date, there have been no reports on the presence of leptin-related peptide, and functional studies on the role of leptin remain limited in fishes. We investigated the effect of mouse recombinant leptin on basal and sea bream (sb) GnRH-induced LH release from dispersed pituitary cells obtained from male European sea bass (Dicentrarchus labrax) at different stages of sexual development. The potential interaction of leptin with the porcine neuropeptide Y (pNPY), known to play a dual role in feeding and reproduction in vertebrates, was also investigated. High doses of leptin (10(-8)-10(-6) M) and/or pNPY (0.1 and 1 nM) had different effects on LH release at various stages of sexual development. Porcine NPY alone was weakly effective on basal LH release, but it enhanced LH release induced by leptin (10(-6) M) in late prepuberty but not in early postpuberty. Additive or inhibitory effects of leptin were observed on sbGnRH-induced LH release depending on sbGnRH dose and stage of sexual development. The direct action of leptin on LH release at the pituitary level in sea bass suggests that leptin is a regulator of the reproductive system in fishes.

Inhibition of P450 Aromatase Enhances Gonadotropin Secretion in Early and Midpubertal Boys: Evidence for a Pituitary Site of Action of Endogenous E

In early pubertal boys, E concentrations are very low. We studied the role and site of action of endogenous E in the regulation of gonadotropin secretion in early and midpubertal boys by inhibiting the action of E with a potent and specific P450 aromatase inhibitor, letrozole. A total of 35 boys who were referred to us because of suspicion of delayed puberty were included in the study. The boys were in either early or midpuberty, and they composed 3 groups: 10 boys did not receive any treatment, 12 boys received T alone, and 13 boys received T and letrozole. In the untreated group during the 5-month follow-up, no changes were observed in 17β-E2, T, basal gonadotropin, or inhibin B concentrations or in the GnRH-induced gonadotropin responses. In the T-treated group during the 5-month treatment, the T concentration increased by 55% (P < 0.05), and the 17β-E2 concentration increased by 130% (P < 0.02). Concurrently, basal gonadotropin concentrations were suppressed, but the GnRH-induced gonadotropin responses and the inhibin B concentration remained unchanged. In the T- plus letrozole-treated group during the 5-month treatment, an increase in T concentration of 606% was observed (P < 0.001), but the 17β-E2 concentration remained unchanged. The changes in the 17β-E2 concentration within 5 months in the untreated and the T- plus letrozole-treated groups were different (P < 0.02), indicating significant inhibition of endogenous E synthesis during letrozole treatment. During the T plus letrozole treatment, basal gonadotropin concentration, the GnRH-induced LH response, and inhibin B concentration increased, and the GnRH-induced FSH response did not change significantly. Serum nocturnal gonadotropin pulses were determined in 5 boys treated with T and in 5 boys treated with T plus letrozole. In the T- plus letrozole-treated group, the nocturnal LH pulse amplitude increased, and the LH pulse frequency and interpulse interval remained unchanged. In conclusion, in early and midpubertal boys, suppression of the action of E by the P450 aromatase inhibitor increased LH concentration, LH pulse amplitude, and the GnRH-induced LH response, which indicates that in boys during early and midpuberty, endogenous E regulates LH secretion at the site of the pituitary.

Inhibition of P450 aromatase enhances gonadotropin secretion in early and midpubertal boys: evidence for a pituitary site of action of endogenous E.

In early pubertal boys, E concentrations are very low. We studied the role and site of action of endogenous E in the regulation of gonadotropin secretion in early and midpubertal boys by inhibiting the action of E with a potent and specific P450 aromatase inhibitor, letrozole. A total of 35 boys who were referred to us because of suspicion of delayed puberty were included in the study. The boys were in either early or midpuberty, and they composed 3 groups: 10 boys did not receive any treatment, 12 boys received T alone, and 13 boys received T and letrozole. In the untreated group during the 5-month follow-up, no changes were observed in 17beta-E2, T, basal gonadotropin, or inhibin B concentrations or in the GnRH-induced gonadotropin responses. In the T-treated group during the 5-month treatment, the T concentration increased by 55% (P < 0.05), and the 17beta-E2 concentration increased by 130% (P < 0.02). Concurrently, basal gonadotropin concentrations were suppressed, but the GnRH-induced gonadotropin responses and the inhibin B concentration remained unchanged. In the T- plus letrozole-treated group during the 5-month treatment, an increase in T concentration of 606% was observed (P < 0.001), but the 17beta-E2 concentration remained unchanged. The changes in the 17beta-E2 concentration within 5 months in the untreated and the T- plus letrozole-treated groups were different (P < 0.02), indicating significant inhibition of endogenous E synthesis during letrozole treatment. During the T plus letrozole treatment, basal gonadotropin concentration, the GnRH-induced LH response, and inhibin B concentration increased, and the GnRH-induced FSH response did not change significantly. Serum nocturnal gonadotropin pulses were determined in 5 boys treated with T and in 5 boys treated with T plus letrozole. In the T- plus letrozole-treated group, the nocturnal LH pulse amplitude increased, and the LH pulse frequency and interpulse interval remained unchanged. In conclusion, in early and midpubertal boys, suppression of the action of E by the P450 aromatase inhibitor increased LH concentration, LH pulse amplitude, and the GnRH-induced LH response, which indicates that in boys during early and midpuberty, endogenous E regulates LH secretion at the site of the pituitary.

Relationship between follicle size and gonadotrophin surge attenuating factor (GnSAF) bioactivity during spontaneous cycles in women

We have previously demonstrated that follicles < or =11 mm diameter from women undergoing IVF contain higher concentrations of gonadotrophin surge attenuating factor (GnSAF) bioactivity than large follicles from the same ovaries. To determine whether this finding is relevant to spontaneous cycles, follicular fluid aspirated from 37 follicles between 3 and 25 mm in diameter from 14 pairs of ovaries from regularly cycling women undergoing total abdominal hysterectomy and bilateral salpingoophorectomy for benign gynaecological disease was pooled into size categories (3 + 4, 5 + 6, 7 + 8, 9 + 10, 11 + 12, 14 + 15, 18 and 25 mm). These pools were bioassayed for GnSAF and inhibin-A, inhibin-B and activin-A concentrations were determined. Follicles of 5 + 6 mm diameter contained the highest concentrations of GnSAF bioactivity (reducing GnRH-induced LH secretion to 38 +/- 8% of control, P < 0.001), while those of 25 mm diameter contained one quarter of this concentration (reducing GnRH-induced LH secretion to 72 +/- 2% of control, P < 0.05). GnSAF bioactivity was closely related to follicle size (r = -0.836, P < 0.01), but not to inhibin-A, inhibin-B or activin-A concentrations. The finding that small follicles contain high concentrations of GnSAF bioactivity, which fall as folliculogenesis progresses during spontaneous cycles, support the hypothesis that GnSAF has a role in preventing the premature onset of the LH surge in women.

Endotoxin Inhibits Pituitary Responsiveness to Gonadotropin-Releasing Hormone

Immune/inflammatory challenges powerfully suppress reproductive neuroendocrine activity. This inhibition is generally considered to be centrally mediated via mechanisms that regulate GnRH secretion. The present study provides two lines of evidence that bacterial endotoxin, a commonly used model of immune/inflammatory challenge, also acts to inhibit pituitary responsiveness to GnRH. In the first experiment, pulsatile secretion of GnRH into pituitary portal blood and LH into peripheral blood were monitored in ovariectomized ewes treated with a low dose of endotoxin. Although this treatment only marginally suppressed GnRH pulsatile secretion, it markedly disrupted LH pulsatility. In extreme cases, the low dose of endotoxin blocked LH pulses without inhibiting endogenous GnRH pulses, thereby uncoupling GnRH and LH pulsatile suppression. In the second experiment, we tested the hypothesis that endotoxin inhibits pituitary responsiveness to exogenous GnRH pulses. Hourly pulses of GnRH were delivered to ovariectomized ewes in which endogenous GnRH secretion was blocked. Endotoxin suppressed the amplitude of GnRH-induced LH pulses. Together, these observations support the conclusion that endotoxin inhibits pituitary responsiveness to GnRH.

Endotoxin inhibits pituitary responsiveness to gonadotropin-releasing hormone.

Immune/inflammatory challenges powerfully suppress reproductive neuroendocrine activity. This inhibition is generally considered to be centrally mediated via mechanisms that regulate GnRH secretion. The present study provides two lines of evidence that bacterial endotoxin, a commonly used model of immune/inflammatory challenge, also acts to inhibit pituitary responsiveness to GNRH: In the first experiment, pulsatile secretion of GnRH into pituitary portal blood and LH into peripheral blood were monitored in ovariectomized ewes treated with a low dose of endotoxin. Although this treatment only marginally suppressed GnRH pulsatile secretion, it markedly disrupted LH pulsatility. In extreme cases, the low dose of endotoxin blocked LH pulses without inhibiting endogenous GnRH pulses, thereby uncoupling GnRH and LH pulsatile suppression. In the second experiment, we tested the hypothesis that endotoxin inhibits pituitary responsiveness to exogenous GnRH pulses. Hourly pulses of GnRH were delivered to ovariectomized ewes in which endogenous GnRH secretion was blocked. Endotoxin suppressed the amplitude of GnRH-induced LH pulses. Together, these observations support the conclusion that endotoxin inhibits pituitary responsiveness to GNRH:

Phytoestrogens and gonadotropin-releasing hormone pulse generator activity and pituitary luteinizing hormone release in the rat.

Phytoestrogens can produce inhibitory effects on gonadotropin secretion in both animals and humans. The aims of this study were 2-fold: 1) to determine in vivo whether genistein and coumestrol act on the GnRH pulse generator to suppress hypothalamic multiunit electrical activity volleys and associated LH pulses and/or on the pituitary to suppress the LH response to GnRH; and 2) to examine the effect of these phytoestrogens on GnRH-induced pituitary LH release in vitro and to determine whether estrogen receptors are involved. Wistar rats were ovariectomized and chronically implanted with recording electrodes and/or indwelling cardiac catheters, and blood samples were taken every 5 min for 7--11 h. Intravenous infusion of coumestrol (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) resulted in a profound inhibition of pulsatile LH secretion, a 50% reduction in the frequency of hypothalamic multiunit electrical activity volleys, and a complete suppression of the LH response to exogenous GnRH. In contrast, both genistein (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) and vehicle were without effect on pulsatile LH secretion. Coumestrol (10(-5) M; over 2 or 4 h) suppressed GnRH-induced pituitary LH release in vitro, an effect blocked by the antiestrogen ICI 182,780. It is concluded that coumestrol acts centrally to reduce the frequency of the hypothalamic GnRH pulse generator. In addition, the inhibitory effects of coumestrol on LH pulses occur at the level of the pituitary by reducing responsiveness to GnRH via an estrogen receptor-mediated process.

The importance of autosomal genes in Kallmann syndrome: genotype-phenotype correlations and neuroendocrine characteristics.

Kallmann syndrome (KS) consists of congenital, isolated, idiopathic hypogonadotropic hypogonadism (IHH) and anosmia. The gene responsible for the X-linked form of KS, KAL, encodes a protein, anosmin, that plays a key role in the migration of GnRH neurons and olfactory nerves to the hypothalamus. In addition to X-linked pedigrees, autosomal dominant and recessive kindreds with KS have been reported. The relative importance of these autosomal vs. X-linked genes in producing KS, and the frequency of KAL mutations, are currently unknown because these are rare disorders and large series are unusual. We examined 101 individuals with IHH (+/- anosmia) and their families to determine their modes of inheritance, incidence of mutations in the coding sequence of KAL, genotype-phenotype correlations, and [in a subset (n = 38)] their neuroendocrine phenotype. Of the 101 patients, 59 had true KS (IHH + anosmia/hyposmia); whereas, in the remaining 42, no anosmia was evident in the patients or their families. Of the 59 KS patients, 21 were familial, whereas 38 were sporadic cases. Mutations in the coding sequence of KAL were identified in only 3 of 21 familial cases (14%) and 4 of 38 (11%) of the sporadic cases. Of the X-linked cases confirmed by mutational analysis, only 1 of 3 pedigrees appeared X-linked by inspection whereas the other 2 contained only affected brothers. Female members of known KAL mutation families (n = 3) exhibited no reproductive phenotype and were not anosmic, whereas families with anosmic women (n = 3) were not found to carry mutations in KAL. Mutations were uniformly absent in nonanosmic IHH probands (n = 42), as well as in families with both anosmic and nonanosmic members (n = 2). Overall, 4 novel mutations were identified (C172R, R191x, R457x, and delC@L600). With respect to neuroendocrine phenotype, KS men with documented KAL mutations (n = 8) had completely apulsatile LH secretion, whereas those with autosomal modes of inheritance demonstrated a more variable spectrum with evidence of enfeebled (but present) GnRH-induced LH pulses. Our conclusions are: 1) Confirmed mutations in the coding sequence of the KAL gene occur in the minority of KS cases, i.e. only 14% of familial and 11% of sporadic cases; 2) The majority of familial (and presumably sporadic) cases of KS are caused by defects in at least two autosomal genes that are currently unknown; 3) Obligate female carriers in families with KAL mutations have no discernible phenotype; 4) KAL mutations are uniformly absent in patients with either normosmic IHH or in families with both anosmic and nonanosmic individuals; and 5) Patients with KAL mutations have apulsatile LH secretion consistent with a complete absence of GnRH migration of GnRH cells into the hypothalamus, whereas evidence of present (but enfeebled) GnRH-induced LH pulses may be present in autosomal KS cases. Taken together, these findings suggest that autosomal genes account for the majority of familial cases of KS, and that unique neuroendocrine phenotypes consistent with some GnRH neuronal migration may exist in these patients.

Interactions of insulin-like growth factor-I, insulin and estradiol with GnRH-stimulated luteinizing hormone release from female rat gonadotrophs.

It is well established that ovarian steroids modulate gonadotropin secretion from anterior pituitary cells. It has been speculated that insulin and IGF-I might influence gonadotropin secretion. To investigate the effects of IGF-I and estradiol alone, or combinations of IGF-I with insulin and estradiol on GnRH-stimulated LH release from female rat pituitary cells in serum-supplemented and serum-free culture conditions. Pituitary cells were incubated for 24 h or 48 h with a series of increasing concentrations of IGF-I or estradiol and stimulated with 1 nmol/l GnRH for 3 h. To determine the interaction of IGF-I and estradiol on GnRH-stimulated LH secretion, cells were exposed to increasing concentrations of IGF-I and 100 pmol/l estradiol for 24 h. We also investigated the effects of combined treatment with IGF-I and insulin on GnRH-stimulated LH secretion. Our findings indicate that long-term IGF-I treatment (24 h) alone has a significant augmenting effect on GnRH-stimulated LH release in serum-free medium only, with a maximum at low concentrations (10 and 100 pmol/l). Estradiol significantly increased GnRH-induced LH release in a dose-dependent manner. The extent of GnRH-stimulated LH secretion by long-term estradiol treatment (24 h) was significantly greater in serum-supplemented (+42%) medium than in serum-free medium. Estradiol facilitated IGF-I-primed LH responses to GnRH in serum-free medium. In contrast, in serum-supplemented medium, the facilitating potential of estradiol was lower. We also found that, in GnRH-stimulated cells, LH release was augmented by insulin treatment, in contrast to quiescent cells that had been pretreated with 100 pmol/l IGF-I alone and 1 nmol/l insulin alone. IGF-I and to a lesser extent insulin stimulate GnRH-induced LH secretion from pituitary gonadotrophs. This action is enhanced by estradiol treatment of the cells. However, the well known stimulatory action of estradiol on LH secretion is dependent on the presence of growth factors.

Different effects of melatonin pretreatment on cAMP and LH responses of the neonatal rat pituitary cells.

The rhythm of the pineal hormone melatonin transduces the effect of photoperiod on seasonal functions. Duration of the melatonin pulse provides information about season and the long melatonin pulse induces reproductive involution in the long day breeders such as photoperiodic rodents. The length of melatonin pulse thus carries photoperiodic information, which regulates the function of target cells. Therefore, we have studied the effects of melatonin pretreatment of various lengths on responsiveness of the neonatal rat pituitary cells cultured in vitro to GnRH or forskolin. In these cells, melatonin treatment inhibits the GnRH-induced LH release as well as the forskolin-induced cAMP accumulation. However, long preincubation with melatonin has a paradoxical stimulatory effect on the cellular responsiveness. When the cells are pretreated with melatonin for 16 hr or more, then rinsed thoroughly and treated with forskolin for 30 min, the increase of cAMP is potentiated. Moreover, in the melatonin-pretreated cells. the subsequent melatonin treatment inhibits the forskolin-induced cAMP accumulation relatively more than in the non-pretreated cells. Although melatonin pretreatment does not potentiate the GnRH-induced LH release, it protects the gonadotrophs against the GnRH-induced desensitization: pretreatment with GnRH for 12 hr or more renders the cells insensitive to subsequent GnRH stimulation, while after pretreatment with GnRH and melatonin, the subsequent GnRH treatment induces significant increase of LH release. These observations indicate that long pretreatment with melatonin improves responsiveness of the pituitary cells to the subsequent stimulation, but its effects on cAMP accumulation and LH release are different.

Ovarian regulators of gonadotropin secretion.

Great progress in the ovarian mechanisms that control gonadotrophin secretion in women has recently been achieved. Estradiol (E2) is the main component of the ovarian negative effect on basal gonadotropin secretion during the normal menstrual cycle. However, nonsteroidal substances such as inhibins and activins that can affect follicle-stimulating hormone (FSH) secretion in vitro may also participate in the control of FSH secretion in vivo. Recent evidence has shown that the ovaries also produce another nonsteroidal substance, named gonadotropin surge-attenuating factor (GnSAF), that specifically attenuates GnRH-induced LH secretion and the endogenous LH surge in superovulated women. It is possible that during the normal menstrual cycle GnSAF controls the amplitude of the midcycle LH surge by antagonizing the stimulating effect of E2 on the pituitary.

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory lh surge

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n=417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n=219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P<0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 ± 6.1 (SEM; n=10 heifers) vs 26.4 ± 1.0% (n=2 replicates), and 39.1 ± 5.1% vs 20.6 ± 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.

Effect of progesterone on the GnRH-induced secretion of oestradiol and androstenedione from the autotransplanted ovary of the anoestrous ewe.

Two experiments were conducted during the anoestrous period in Border Leicester x Merino ewes with ovarian autotransplants to study the effects of a single injection of 20 mg progesterone on follicular steroid secretion. The aim of these experiments was to determine whether pretreatment with a 20 mg intramuscular injection of progesterone could reduce GnRH-induced ovarian steroid secretion in anoestrous ewes. In both experiments, an injection of 150 ng GnRH induced an LH pulse in all ewes with a maximum concentration 10 min (the first post-injection sample) after injection. Oestradiol and androstenedione secretion increased progressively after the GnRH-induced LH pulse and reached maximum rates of secretion between 60 and 90 min before decreasing slowly to pre-injection rates at 150 min. There were no differences in the pattern of secretion of oestradiol (measured in both experiments) or androstenedione (measured only in Expt 2). In Expt 1, the injection of progesterone 72 h before the challenge with GnRH had no effect on the maximum rate of oestradiol secretion from the autotransplanted ovary. However, in Expt 2, when progesterone was given either 36 or 60 h before GnRH, there was a significant suppression in the maximum rate of secretion of both oestradiol and androstenedione between 60 and 90 min after GnRH injection. These data show that pretreatment of anoestrous sheep with progesterone can suppress LH-stimulated steroid secretion from the ovary and indicate that progesterone may have a direct effect on oestrogenic follicles in sheep.

Inhibitory effect of melatonin on GnRH-induced LH release

Inhibitory effect of melatonin on GnRH-induced LH release

Inhibitory effect of melatonin on GnRH-induced LH release.

Melatonin inhibits GnRH-induced release of LH and FSH from the neonatal, but not the adult, rat anterior pituitary gland. This action of melatonin is mediated by the specific high-affinity membrane-bound receptors that are absent in adult rats. The intracellular mechanism of melatonin action involves a decrease in intracellular calcium [Ca2+]i in the gonadotrophs; melatonin inhibits GnRH-induced Ca2+ release from endoplasmic reticulum as well as Ca2+ influx through voltage-sensitive channels. Melatonin also inhibits GnRH-induced accumulation of cAMP, which may result in the decreased influx of Ca2+, because cAMP, acting through protein kinase A, stimulates Ca2+ influx into the gonadotrophs. This age-dependent effect of melatonin on gonadotrophin release from the pituitary may be involved in the timing of puberty.

Non-steroidal factors in bovine follicular fluid inhibit or facilitate the action of pulsatile administration of GnRH on LH release in the female rat.

Using steroid-free bovine follicular fluid (bFF), we studied the action of gonadotrophin surge-inhibiting factor/attenuating factor (GnSIF/AF) on GnRH-induced self-priming in phenobarbital-blocked female rats. For the experiments we used intact rats, short-term (4 h) ovariectomized (OVX) rats and long-term (14 days) OVX rats. In the latter case the rats were injected with 17beta-oestradiol benzoate (OB, 40 micrograms) or vehicle only, 2 or 48 h before the experiment. GnRH (10-50 pmol/kg body weight) was injected intra-arterially in 5 or 15 pulses, respectively 60 or 20 min apart, starting 1 or 4 h after injection of bFF (0.5 or 1.0 ml). In response to 25 pmol/kg GnRH pulses (1/h), we observed no effect in the long-term OVX rats, a minor effect in the intact rats and an enhanced self-priming effect in the short-term OVX rats. Administration of bFF attenuated or even completely inhibited the self-priming process. However, in the case of long-term OVX rats LH release was inhibited only after long-term OB priming. Furthermore, 4 h after administration of bFF, LH release in response to 25 pmol/kg GnRH pulses (3/h) was inhibited transiently in intact rats and long-term OVX rats. The results support the hypothesis of a functional antagonistic action between GnRH and GnSIF/AF. However, when injected 1 h before, bFF facilitated the initial release of the surge-like LH pattern in intact rats in response to 3 pulses/h of GnRH. These results are consistent with an important role of GnSIF/AF and other non-steroidal ovarian factors in the control of both low LH concentrations and the generation of the LH surge. Some genomic action of oestradiol might be a prerequisite for the inhibitory effect of GnSIF/AF on GnRH-induced LH release.

The effect of season and melatonin on GnRH-induced LH secretion in oestradiol-treated orchidectomized sheep.

The biphasic effect of oestradiol (E2) on gonadotrope responsiveness is clearly evident in orchidectomized sheep (wethers) receiving E2 and hourly pulses of GnRH. We hypothesized that the duration of E2-induced reduction in gonadotrope responsiveness differed between the breeding (November) and anoestrous (May) seasons in sheep. To test this hypothesis wethers (n = 6/group) were infused (i.v.) with E2 (2 micrograms/50 kg per h) and received hourly pulses of GnRH (200 ng/50 kg per pulse) or saline in May or November. The pattern of LH secretion during the 72 h infusion period was determined. Serum concentrations of LH did not differ with season in control wethers receiving vehicle alone. Similarly, continuous infusion of E2 resulted in a 3-fold reduction in serum LH, irrespective of season. This E2-induced suppression of serum LH was reversed by concurrent episodic delivery of GnRH. The interval between initiation of infusion and return of pretreatment concentrations of LH was taken as a measure of the duration of E2-induced suppression of gonadotrope responsiveness. The duration of this E2-dependent response varied with season, with suppression of gonadotrope responsiveness more prolonged (P < 0.05) in May (36.7 +/- 2.9 h) than in November (14.3 +/- 1.1 h). In a companion study we examined the effect of melatonin on the duration of E2-induced suppression of gonadotrope responsiveness. Wethers received blank or melatonin-containing implants in March. Sixty days after implant insertion (mid-May) wethers received E2 (2 micrograms/50 kg per h) and hourly pulses of GnRH (200 ng/50 kg per pulse) or saline for 72 h. Continuous delivery of E2 alone resulted in a 3-fold decrease in serum concentrations of LH in both control and melatonin-treated wethers. The duration of E2-induced suppression of gonadotrope responsiveness in animals receiving E2 and GnRH was extended (P < 0.05) in wethers with blank implants (48.0 +/- 0.7 h), relative to the duration of suppression in melatonin-treated wethers (14.5 +/- 1.0 h). Taken together these data indicate that E2-induced suppression of gonadotrope responsiveness is more extended during the anoestrous season. However, this seasonal effect can be reversed by continuous administration of melatonin.

Effects of thyroidectomy and thyroxine replacement on seasonal reproduction in the red deer hind.

Two experiments were conducted to test whether thyroid hormones are required for the cessation of the breeding season in female red deer. In Expt 1, 16 mature hinds were allocated early in the breeding season (May) to the following groups: thyroidectomized (THX), thyroidectomized and treated with subcutaneous thyroxine implants which increased plasma tri-iodothyronine to physiological concentrations (THX + T4), or euthyroid controls. Plasma progesterone concentrations consistent with oestrous cyclicity were evident in all hinds during the breeding season (May-September) but after 23 September plasma progesterone concentrations became generally low (< 2.0 nmol l-1) in control and THX + T4 hinds. In contrast, thyroidectomized hinds not receiving thyroxine continued to exhibit circulating progesterone comparable with breeding season concentrations until the end of the experiment (late December). Nine hinds were ovariectomized or ovariectomized and thyroidectomized in May and treated with subcutaneous oestradiol-impregnated capsules, which were removed for periods of about 1 months during August, November and January, to test whether thyroid hormones are involved in the seasonal decline of LH secretion (Expt 2). In the presence of oestradiol, basal LH concentrations and episodic LH secretion were low during the non-breeding season (September-March) (P < 0.05). During this season, basal and GnRH-induced LH concentrations and LH pulse frequency remained similar for both groups except in the absence of oestradiol, when basal LH (3.1 +/- 0.8 versus 1.3 +/- 0.5 ng ml-1, P < 0.05 and GnRH-induced LH (25.9 +/- 3.6 versus 4.7 +/- 0.4 ng ml-1, P < 0.001) concentrations were higher in thyroidectomized hinds than in euthyroid hinds, respectively. Twenty-five hinds were ovariectomized and thyroidectomized during the breeding season and treated with subcutaneous thyroxine implants at different times to identify when thyroid hormones are required to be present to suppress LH concentrations during the non-breeding season in the absence of oestradiol. Thyroxine treatment at the beginning of or during the non-breeding season was effective in suppressing plasma LH concentration, but this action of thyroid hormones did not occur during the breeding season. These results indicate that, in red deer hinds thyroxine is required for termination of the breeding season and that thyroid gland secretions specifically block steroid-independent inhibition of reproductive activity during the non-breeding season. This inhibitory mechanism requires thyroid hormones to be present only from around the time of the end of the breeding season for their normal expression, and they remain responsive to thyroid hormones after this period.

Ovarian modulation of the oestradiol-induced LH surge in prepubertal and sexually mature gilts.

The role of ovarian secretions in modulating positive oestrogen feedback on LH release in late prepubertal and sexually mature gilts was studied. Gilts were either ovariectomized (OVX) at 230 days of age (sexually mature, OVX 230, control), ovariectomized at 160 days of age (pre-pubertal, OVX 160) or ovariectomized at 160 days of age and given either low, high or very high oestradiol substitution therapy (two or three Silastic implants, 5 or 8 cm in length) from 160 days to 230 days (OVX 160 + 2E5, OVX 160 + 2E8 and OVX 160 + 3E8, respectively). The LH surge responses to three i.m. injections of oestradiol benzoate given at intervals of 12 h (in total 10 micrograms oestradiol benzoate kg-1 bodyweight) were compared at 260 days. An additional group of sexually mature gilts was ovariectomized at 260 days (OVX 260) and challenged with oestradiol benzoate at 360 days. LH surges with peak LH concentrations below pretreatment values (resembling LH responses to oestradiol benzoate in immature gilts) were classified as immature, and those with peak LH concentrations above pretreatment values as mature. LH concentrations before treatment with oestradiol benzoate were reduced (P < 0.05) in OVX 160 + 2E8 and OVX 160 + 3E8 gilts compared with OVX 230 (control). When compared with the control group, the time to LH surge peak was longer in OVX 260 gilts, LH peak amplitude was reduced in the OVX 160 group and the area under the curve of the LH surge (ng LH ml-1 plasma (48 h)-1) was less in all other groups (P < 0.05). Classification of LH surges by mature:immature criteria indicated a high mature:immature ratio in OVX 230 gilts (6/1) and a low ratio in OVX 160 (1/7) and OVX 260 (1/6) gilts. The long-term effect of ovariectomy was partially overcome in the OVX 160 + 2E5 group (mature:immature = 3/3). However, previous exposure to supraphysiological concentrations of oestradiol (as in groups OVX 160 + 2E8 and OVX 160 + 3E8) resulted in a high percentage of animals (66% and 100%, respectively) that did not respond to the oestradiol benzoate challenge with an LH surge. Immediately before oestradiol benzoate administration (30 days after removal of implants) plasma oestradiol concentrations in these two groups were still high (P < 0.05) compared with all other groups. GnRH-induced (0.2 microgram kg-1 bodyweight) LH secretion, evaluated 10 days after treatment with oestradiol benzoate, was depressed (P < 0.05) in OVX 160 + 3E8 gilts, but not affected by age at ovariectomy. We suggest that continuous ovarian secretion is necessary for the final maturation of the LH surge mechanism in late prepubertal gilts and also for maintaining the full functionality of this mechanism in sexually mature gilts. The range over which ovarian oestrogens tune the final maturation of this process appears to be rather narrow; however, other ovarian factors may contribute to the final maturation. Furthermore, continued exposure to supraphysiological concentrations of oestradiol was found to be detrimental to the development of the LH surge mechanism.

The in vitro effect of substance P on the GnRH-induced LH release depends on the steroidal environment and is reverted by a NK1 receptor antagonist (RP 67580) in the cycling female rat.

A previously study reported that administration of substance P on the morning of the proestrous day induces an inhibition of afternoon gonadotropin preovulatory surges in the female rat. It has also been shown, with a non-peptide specific antagonist of the neurokinin 1 (NK1) receptor (RP 67580), that this effect is mediated by NK1 receptors. The present study used perifused anterior pituitaries from proestrous morning female rats and showed that the SP modulation of the GnRH-induced LH release is markedly dependent on the steroidal environment. In the absence of steroids or in the presence of 17beta estradiol, or a combination of 17beta estradiol and progesterone, SP inhibited the GnRH-induced LH release. In contrast, SP stimulated the GnRH-induced LH secretion in the presence of progesterone alone. However, the inhibitory or stimulatory effect of SP was antagonized by the specific NK1 receptor antagonist RP 67580.