Neuroendocrine consequences of prenatal androgen exposure in the female rat: absence of luteinizing hormone surges, suppression of progesterone receptor gene expression, and acceleration of the gonadotropin-releasing hormone pulse generator

Preovulatory GnRH and LH surges depend on activation of estrogen (E2)-inducible progesterone receptors (PGRs) in the preoptic area (POA). Surges do not occur in males, or in perinatally androgenized females. We sought to determine whether prenatal androgen exposure suppresses basal or E2-induced Pgr mRNA expression or E2-induced LH surges (or both) in adulthood, and whether any such effects may be mediated by androgen receptor activation. We also assessed whether prenatal androgens alter subsequent GnRH pulsatility. Pregnant rats received testosterone or vehicle daily on Embryonic Days 16-19. POA-hypothalamic tissues were obtained in adulthood for PgrA and PgrB (PgrA+B) mRNA analysis. Females that had prenatal exposure to testosterone (pT) displayed reduced PgrA+B mRNA levels (P < 0.01) compared with those that had prenatal exposure to vehicle (pV). Additional pregnant animals were treated with vehicle or testosterone, or with 5alpha-dihydrotestosterone (DHT). In adult ovariectomized offspring, estradiol benzoate produced a 2-fold increase (P < 0.05) in PgrA+B expression in the POA of pV females, but not in pT females or those that had prenatal exposure to DHT (pDHT). Prenatal testosterone and DHT exposure also prevented estradiol benzoate-induced LH surges observed in pV rats. Blood sampling of ovariectomized rats revealed increased LH pulse frequency in pDHT versus pV females (P < 0.05). Our findings support the hypothesis that prenatal androgen receptor activation can contribute to the permanent defeminization of the GnRH neurosecretory system, rendering it incapable of initiating GnRH surges, while accelerating basal GnRH pulse generator activity in adulthood. We propose that the effects of prenatal androgen receptor activation on GnRH neurosecretion are mediated in part via permanent impairment of E2-induced PgrA+B gene expression in the POA.     

Repair of reproductive deficits by neural transplantation

Transplantation of brain tissue has been used to ameliorate the genetic lesion of the hypogonadal mutant mouse. This animal does not synthesize gonadotropin-releasing hormone (GnRH) and so has an infantile reproductive system. Implantation of normal fetal or neonatal preoptic area containing GnRH neurons reverses many aspects of the reproductive deficiency. Pituitary and plasma levels of gonadotropins rise, followed by growth of the gonads and sexual organs. Pituitary release of gonadotropins is episodic, suggesting that the grafted tissue is integrated into the "pulse generator." The vast majority of grafted animals do not show castration-induced elevations of luteinizing hormone (LH) nor respond to exogenous steroids with a depression in circulating LH. Negative feedback of gonadal steroids seems to be inoperative. In contrast, some females can show ovulatory surges of LH in response to mating (reflex ovulation), after administration of exogenous steroid (progesterone), and, on rare occasion, ovulation cycles occur spontaneously. Anatomical studies demonstrate that reproductive recovery is dependent on the outgrowth of GnRH axons to the host median eminence. Some but not all of the GnRH neurons within the grafts contribute to this innervation. GnRH axons exit into the host along well-defined pathways, recapitulating in part the paths taken by normal axons. How the graft and host are integrated to produce the panoply of reproductive responses is the subject of current study.     

Loss of luteinizing hormone surges induced by chronic estradiol is associated with decreased activation of gonadotropin-releasing hormone neurons

Chronic exposure of young ovariectomized rats to elevated circulating estradiol causes loss of steroid-induced LH surges. Such LH surges are associated with cFos-induced activation of GnRH neurons; therefore, we hypothesized that chronic estradiol treatment abolishes LH surges by decreasing activation of GnRH neurons. Regularly cycling rats were ovariectomized and immediately received an estradiol implant or remained untreated. Three days or 2 or 4 wk later, the estradiol-treated rats received vehicle or progesterone at 1200 h, and 7 hourly blood samples were collected for RIA of LH. Thereafter, all rats were perfused, and the brains were examined for immunocytochemical localization of cFos and GnRH. The GnRH neurons from untreated ovariectomized rats rarely expressed cFos. As reported, LH surges induced by 3 days of estradiol treatment were associated with a 30% increase in cFos-containing GnRH neurons, and progesterone enhanced both the amplitude of LH surges and the proportion of cFos-immunopositive GnRH neurons. As hypothesized, the abolition of LH surges caused by 2 or more weeks of estradiol was paralleled by a reduction in the percentage of cFos-containing GnRH neurons, and this effect was delayed by progesterone. These results suggest that chronic estradiol abolishes steroid-induced LH surges in part by inactivating GnRH neurons.     

Increased milk yield in transgenic mice expressing insulin-like growth factor 1

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 was reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in mammary gland, transgenic mice were produced by pronuclear microinjection of a construct containing a bovine alpha-lactalbumin (alpha-LA) promoter linked to an ovine IGF-1 cNDA. This alpha-LA promoter has previously been shown to direct expression of a human factor VIII gene specifically to the mammary gland of transgenic mice. Three transgenic mouse lines were established as a result of microinjection of 398 embryos. Transgene expression was found in mammary gland at day 1 of lactation from these three lines. Progeny test were carried out by mating two transgenic males/one transgenic female to two nontransgenic females/one nontransgenic male. Mice from one line (line 1225) were all nonexpressors and the other (line 1372) failed to produce offspring. Milk yield was analyzed in the line 1137 that produced 10 mice, of which three were transgenic females and three nontransgenic females. All of the three transgenic females showed integration of the transgene and expressed transgene IGF-1 mRNA in the mammary gland. Milk yields from days 5, 10, and 15 of lactation were significant greater in transgenic expressors than in their nontransgenic littermates. Specifically, there is 17.9% increase in total milk yield from these three days for transgenics compared with nontransgenics. These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable to stimulating milk yield during the first lactation.     

Increased Milk Yield in Transgenic Mice Expressing Insulin-like Growth Factor 1

Abstract

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 was reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in mammary gland, transgenic mice were produced by pronuclear microinjection of a construct containing a bovine α-lactalbumin (α-LA) promoter linked to an ovine IGF-1 cNDA. This α-LA promoter has previously been shown to direct expression of a human factor VIII gene specifically to the mammary gland of transgenic mice. Three transgenic mouse lines were established as a result of microinjection of 398 embryos. Transgene expression was found in mammary gland at day 1 of lactation from these three lines. Progeny test were carried out by mating two transgenic males/one transgenic female to two nontransgenic females/one nontransgenic male. Mice from one line (line 1225) were all nonexpressors and the other (line 1372) failed to produce offspring. Milk yield was analyzed in the line 1137 that produced 10 mice, of which three were transgenic females and three nontransgenic females. All of the three transgenic females showed integration of the transgene and expressed transgene IGF-1 mRNA in the mammary gland. Milk yields from days 5, 10, and 15 of lactation were significant greater in transgenic expressors than in their nontransgenic littermates. Specifically, there is 17.9% increase in total milk yield from these three days for transgenics compared with nontransgenics. These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable to stimulating milk yield during the first lactation.     

Detailed examination of the mechanism and site of action of progesterone and corticosteroids in the regulation of gonadotropin secretion: hypothalamic gonadotropin-releasing hormone and catecholamine involvement.

Progesterone and certain corticosteroids, such as deoxycorticosterone (DOC) and triamcinolone acetonide (TA), can stimulate gonadotropin surges in rats. The mechanism of these steroids could involve a pituitary or hypothalamic site of action, or both. Progesterone and TA did not alter the ability of GnRH to release LH or FSH either before, during, or after the gonadotropin surge induced by these steroids in estrogen-primed ovariectomized female rats. Furthermore, progesterone, TA and DOC were unable to induce a gonadotropin surge in short-term estrogen-primed castrated male rats. These results suggested a hypothalamic rather than a pituitary site of action of progesterone and corticosteroids in the release of gonadotropins. Since progestin and corticosteroid receptors are present in catecholamine neurons, a role for catecholamine neurotransmission in progesterone and corticosteroid-induced surges of LH and FSH in estrogen-primed ovariectomized rats was examined. Catecholamine synthesis inhibitors and specific alpha 1 (prazosin), alpha 2 (yohimbine), and beta (propranolol) receptor antagonists were used to determine the role of catecholamine neurotransmission in the steroid-induced surges of LH and FSH. Both of the catecholamine synthesis inhibitors, alpha-methyl-p-tyrosine HCl (alpha-MPT), a tyrosine hydroxylase inhibitor, and sodium diethyldithiocarbamate (DDC), an inhibitor of dopamine-beta-hydroxylase, attenuated the ability of progesterone, TA, and DOC to induce LH surges when administered 3 h and 1 h, respectively, before the steroid. DDC also suppressed the ability of progesterone, TA, and DOC to induce FSH surges. Rats treated with alpha-MPT had lower mean FSH values than did steroid controls, but the effect was not significant. Both the alpha 1 and alpha 2 adrenergic antagonists, prazosin and yohimbine, significantly suppressed the ability of progesterone, TA, and DOC to induce LH and FSH surges. In contrast, the beta adrenergic receptor blocker, propranolol, had no effect upon the ability of progesterone, TA, or DOC to facilitate LH and FSH secretion. Finally, the stimulatory effect of progesterone and TA upon LH and FSH release was found to be blocked by prior treatment with a GnRH antagonist, further suggesting hypothalamic involvement. In conclusion, this study provides evidence that the stimulation of gonadotropin release by progesterone and corticosteroids is mediated through a common mechanism, and that this mechanism involves the release of GnRH, most likely through catecholaminergic stimulation. Furthermore, catecholamine neurotransmission, through alpha 1 and alpha 2 but not beta receptor sites, is required for the expression of progesterone and corticosteroid-induced surges of LH and FSH in estrogen-primed ovariectomized rats.     

Effects of applying gamma-aminobutyric acid(B) drugs into the medial basal hypothalamus on basal luteinizing hormone concentrations and on luteinizing hormone surges in the female sheep

Prior investigations have shown that localized infusion by microdialysis of gamma-aminobutyric acid(B) (GABA(B)) agonists into the medial basal hypothalamus of male sheep rapidly increases GnRH and LH pulse amplitude. The objectives of these studies were to determine if infusion of GABA(B) agonists SKF 97541 or baclofen into the medial basal hypothalamus of female sheep would affect basal LH secretion and if infusion of a potent antagonist would alter expression of LH surges induced by injection of estrogen. Infusion of either SKF 97541 (10 or 40 microM) or baclofen (1 mM) into estrogen-treated ovariectomized ewes did not alter basal LH secretory patterns, whereas both drugs significantly elevated mean LH and LH pulse amplitude in ovariectomized ewes during the nonbreeding season. Infusion of the antagonist CGP 52432 (250 or 500 microM) did not affect expression of estrogen-induced LH surges in ovariectomized ewes. These observations support the concept that GABA(B) receptors in the medial basal hypothalamus regulate basal LH secretion but do not regulate the surge mode of LH secretion in the female sheep.     

FOS expression in grafted gonadotropin-releasing hormone neurons in hypogonadal mouse: Mating and steroid induction

We used FOS expression, widely accepted as a marker for neuronal activation, to evaluate physiologically induced activation of gonadotropin-releasing hormone (GnRH) neurons within intraventricular preoptic area grafts in hypogonadal (hpg) female mice. Hpg mice lack endogenous GnRH due to a mutated gene, but can respond to grafted GnRH neurons with reproductive development. The purpose of this study was to determine the degree to which the host brain regulates grafted GnRH neurons. FOS expression in grafted GnRH neurons was induced in progesterone-primed female mice paired with sexually active males. The degree of sexual activity did not affect the outcome, with 40.9 ± 12.2% of the grafted GnRH cells expressing FOS when male partners performed intromissions, and 47.5 ± 10.2% when they also ejaculated. There was little or no FOS expression in the grafts of unprimed hpg mice paired with sexually active males, in unpaired mice primed with progesterone or sequential estradiol benzoate and progesterone, or in controls. The pattern of FOS expression in the brains of the female hpg mice engaged in mating behavior was similar to that reported in other species, with moderate to high expression in the medial preoptic area, ventromedial nucleus, and medial amygdala in females paired with males that ejaculated. The present results support the hypothesis that host-derived activation of grafted GnRH neurons underlies aspects of reproductive responses seen in hpg mice with grafts, and further, that at least a portion of the host-graft connectivity is steroid sensitive. © 1996 John Wiley & Sons, Inc.     

A Population of Kisspeptin/Neurokinin B Neurons in the Arcuate Nucleus May Be the Central Target of the Male Effect Phenomenon in Goats

Exposure of females to a male pheromone accelerates pulsatile gonadotropin-releasing hormone (GnRH) secretion in goats. Recent evidence has suggested that neurons in the arcuate nucleus (ARC) containing kisspeptin and neurokinin B (NKB) play a pivotal role in the control of GnRH secretion. Therefore, we hypothesized that these neurons may be the central target of the male pheromone. To test this hypothesis, we examined whether NKB signaling is involved in the pheromone action, and whether ARC kisspeptin/NKB neurons receive input from the medial nucleus of the amygdala (MeA)—the nucleus suggested to relay pheromone signals. Ovariectomized goats were implanted with a recording electrode aimed at a population of ARC kisspeptin/NKB neurons, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA) volleys, was measured. Pheromone exposure induced an MUA volley and luteinizing hormone (LH) pulse in control animals, whereas the MUA and LH responses to the pheromone were completely suppressed by the treatment with an NKB receptor antagonist. These results indicate that NKB signaling is a prerequisite for pheromone action. In ovariectomized goats, an anterograde tracer was injected into the MeA, and possible connections between the MeA and ARC kisspeptin/NKB neurons were examined. Histochemical observations demonstrated that a subset of ARC kisspeptin/NKB neurons receive efferent projections from the MeA. These results suggest that the male pheromone signal is conveyed via the MeA to ARC kisspeptin neurons, wherein the signal stimulates GnRH pulse generator activity through an NKB signaling-mediated mechanism in goats.     

Does the seasonal increase in estradiol negative feedback prevent luteinizing hormone surges in anestrous ewes by suppressing hypothalamic gonadotropin-releasing hormone pulse frequency?

To test the hypothesis that the anestrous increase in estradiol negative feedback prevents estrous cycles by suppressing hypothalamic gonadotropin-releasing hormone (GnRH) pulse frequency, a variety of regimens of increasing GnRH pulse frequency were administered to anestrous ewes for 3 days. A luteinizing hormone (LH) surge was induced in 45 of 46 ewes regardless of amplitude or frequency of GnRH pulses, but only 19 had luteal phases. Estradiol administration induced LH surges in 6 of 6 ewes, only 3 having luteal phases. Anestrous luteal phase progesterone profiles were similar in incidence, time course, and amplitude to those of the first luteal phases of the breeding season, which in turn had lower progesterone maxima than late breeding season luteal phases. In the remaining ewes, progesterone increased briefly or not at all, the increases being similar to the transient rises in progesterone occurring in most ewes at the onset of the breeding season. These results demonstrate that increasing GnRH pulse frequency induces LH surges in anestrus and that the subsequent events are similar to those at the beginning of the breeding season. Finally, they support the hypothesis that the negative feedback action of estradiol prevents cycles in anestrus by suppressing the frequency of the hypothalamic pulse generator.     

Evidence for a pituitary site of gonadal steroid stimulation of GnRH receptors in female mice

Treatment of GnRH-deficient (hpg) female mice with oestradiol-17 beta (E2) for 7 days increased GnRH receptors from 4.1 +/- 0.4 fmol/pituitary (control) to 7.2 +/- 0.7 fmol/pituitary (GnRH-treated), and consistently increased pituitary FSH content. Treatment of hpg female mice with E2 plus progesterone (P) for 14 days stimulated GnRH receptors more than did E2 alone, although values still remained lower than those of normal intact female mice. In contrast, GnRH treatment of intact hpg female mice alone, or combined with E2 + P, increased GnRH receptors to values similar to those of intact normal female mice. In contrast, the receptor rise after GnRH treatment alone of ovariectomized hpg mice was significantly less than in intact hpg mice similarly treated. However, the combination of GnRH + E2 + P treatment of ovariectomized hpg mice increased GnRH receptors to normal intact female values, indicating the synergistic actions of these hormones on GnRH receptor up-regulation at the pituitary. Oestradiol treatment of ovariectomized normal female mice prevented the receptor fall after ovariectomy, and when combined with exogenous GnRH further increased receptors to values identical to those of intact female mice receiving GnRH alone. Ovariectomy of hpg mice had no effect on GnRH receptor, serum or pituitary LH and FSH values. There was no change in serum LH concentration after GnRH treatment of hpg female mice, but serum FSH increased and this was accentuated by ovariectomy, indicating that in intact mice an ovarian factor(s) normally inhibits GnRH-stimulated FSH release. This factor did not appear to be an ovarian steroid since serum FSH was not suppressed in intact or ovariectomized GnRH-treated hpg mice concurrently receiving E2 + P treatment. These results suggest that: (1) gonadal steroids alone have a major direct stimulatory action on the pituitary to increase GnRH receptors; (2) the oestrogen-induced increase in GnRH receptors is enhanced in the presence of GnRH; (3) steroids exert inhibitory feedback on gonadotrophin secretion that is mediated at some cellular regulatory locus other than the GnRH-receptor complex.     

Role of GnRH in the regulation of pituitary GnRH receptors in female mice

The fall in pituitary GnRH receptors in female mice after ovariectomy (Ovx) was further decreased (greater than 50%), rather than prevented, by treatment with a GnRH antiserum, despite suppression of the post-gonadectomy increase in serum gonadotrophins, suggesting that increased endogenous GnRH secretion is not the mediator of GnRH receptor fall after ovariectomy in mice. Furthermore, GnRH antiserum reduced GnRH receptors by 30-50% in intact normal females, without altering receptor affinity, and rendered serum LH and FSH undetectable but did not reduce receptors in GnRH-deficient, hpg mice. When GnRH was administered to ovariectomized mice this failed to restore receptor values (fmol/pituitary) (intact = 55.3 +/- 2.4; Ovx = 30.1 +/- 2; Ovx + GnRH = 31.6 +/- 2.8), but serum LH was reduced from high post-ovariectomy values (231 +/- 42 ng/ml) to values normal for intact females (24 +/- 2 ng/ml). In contrast, multiple GnRH injections to intact female mice increased GnRH receptor by 35%, while serum LH was reduced to just detectable levels. A marked dissociation between GnRH receptor and serum gonadotrophin concentrations was observed. Administration of oestrogen (E2) plus progesterone (P) to ovariectomized mice in which endogenous GnRH had been immunoneutralized reversed the inhibitory effect of GnRH antiserum on GnRH receptors and increased values above those of ovariectomized controls, although no increase in serum or pituitary gonadotrophin levels was seen in ovariectomized mice treated with E2 + P + GnRH antiserum. Treatment with E2 and P of intact females receiving GnRH antiserum did not prevent the inhibitory effect of antiserum on receptors, while E2 + P treatment alone of intact female mice reduced GnRH receptors by 30%. These data suggest that the gonadal steroids reduce GnRH receptors in intact female mice by inhibiting hypothalamic GnRH secretion, and that a certain degree of pituitary exposure to GnRH is required for maintenance of a normal receptor complement. These results suggest that (1) the fall in GnRH receptors after ovariectomy is primarily attributable to removal of gonadal factors. The fall is not a reflection of alteration in endogenous GnRH interaction with the gonadotroph; (2) homologous ligand 'up-regulation' of GnRH receptors in female mice depends upon the presence of the ovaries; (3) endogenous GnRH is also required for GnRH receptor maintenance in intact female mice; and (4) GnRH receptor and serum gonadotrophin responses to hormonal changes can be dissociated and their relationship is complex.     

GABAergic integration of progesterone and androgen feedback to gonadotropin-releasing hormone neurons

Steroid feedback regulates GnRH secretion and previous work has implicated gamma-aminobutyric acid (GABA)ergic neurons as a mediator of these effects. We examined GABAergic postsynaptic currents (PSCs) in green fluorescent protein-identified GnRH neurons from mice exposed to different steroid milieus in vivo. Adult mice were ovariectomized and treated with estradiol (OVX+E, controls) or E plus progesterone (P, OVX+E+P). P decreased PSC frequency, a presynaptic effect, and PSC size, which could be via pre- and/or postsynaptic mechanisms. In contrast, dihydrotestosterone (DHT, OVX+E+DHT) increased both GABAergic PSC frequency and size in GnRH neurons. Tetrodotoxin (TTX), which eliminates action-potential-dependent presynaptic effects, did not alter frequency, suggesting DHT may have increased PSC frequency by increasing connectivity between GABAergic and GnRH neurons. TTX reduced PSC size below control values, indicating DHT may augment presynaptic GABA release but inhibits the postsynaptic GnRH neuron response. In mice treated with both P and DHT (OVX+E+P+DHT), PSC frequency and size were similar to controls, suggesting these steroids counteract one another. These results demonstrate GABAergic neurons participate in integrating and conveying steroid feedback to GnRH neurons, defining a potential central mechanism for steroid regulation of GnRH neurons during the reproductive cycle, and providing one possible mechanism for increased activity of these cells in hyperandrogenic females.     

Compensation by pulsatile GnRH infusions for incompetence for oestradiol-induced LH surges in long-term ovariectomized gilts and castrated male pigs.

The aim of this study was to investigate incompetence for oestradiol-induced LH surges in long-term ovariectomized gilts and male pigs. Gilts (250 days old; n = 36), which had been ovariectomized 30 (OVX 30) or 100 days (OVX 100) before the start of treatment, were challenged i.m. with oestradiol benzoate and were either given no further treatment, fed methallibure to inhibit endogenous GnRH release or fed methallibure and given i.v. pulses of 100 or 200 ng GnRH agonist at 1 h intervals during the LH surge (48-96 h after oestradiol benzoate). The same treatments were applied to long-term orchidectomized male pigs (ORC, n = 23). In addition, one ORC group was not injected with oestradiol benzoate but was fed methallibure and given pulses of 200 ng GnRH agonist. Oestradiol benzoate alone induced an LH surge in the OVX 30 group only (5/6 gilts), methallibure suppressed (P < 0.05) oestradiol benzoate-induced LH secretion, while pulses of 100 ng GnRH agonist in animals fed methallibure produced LH surges in four of six OVX 30 and four of six OVX 100 gilts. The induced LH surges were similar to those produced by oestradiol benzoate alone in OVX 30 gilts. Pulses of 200 ng GnRH agonist produced LH surges in OVX 30 (6/6) and OVX 100 (6/6) gilts and increased the magnitude of the induced LH surge in OVX 100 gilts (P < 0.05 compared with 100 ng GnRH agonist or OVX 30 control). Pulses of 200 ng GnRH agonist also induced LH surge release in ORC male pigs (5/6), but were unable to increase LH concentrations in a surge-like manner in ORC animals that had not been given oestradiol benzoate, indicating that oestradiol increases pituitary responsiveness to GnRH. These results support the hypothesis that oestradiol must inhibit secretion of LH before an LH surge can occur. It is concluded that incompetence for oestradiol-induced LH surges in long-term ovarian secretion-deprived gilts and in male pigs is due to the failure of oestradiol to promote a sufficient increase in the release of GnRH.     

Fos-induction in gonadotropin-releasing hormone neurons receiving vasoactive intestinal polypeptide innervation is reduced in middle-aged female rats

A hallmark of reproductive aging in rats is a delay in the initiation and peak, and a decrease in the amplitude, of both proestrous and steroid-induced surges of LH and a decrease in the number of GnRH neurons that express Fos during the surge. The altered timing of the LH surge and the decline in Fos expression in GnRH neurons may be due to changes in the rhythmic expression of vasoactive intestinal polypeptide (VIP), a neuropeptide that carries time-of-day information from the circadian pacemaker, located in the suprachiasmatic nuclei (SCN), to GnRH neurons. The goals of our study were to determine if aging alters 1) the innervation of GnRH neurons by VIP and 2) the ability of VIP to activate GnRH neurons by examining the effects of aging on the number of GnRH neurons apposed by VIP fibers and the number of GnRH neurons that receive VIP input that express Fos. Immunocytochemistry for GnRH and VIP; or GnRH, VIP, and Fos was performed on tissue sections collected from young (2-4 mo), regularly cycling females and middle-aged (10-12 mo) females in constant estrus. The number of GnRH neurons, GnRH neurons apposed by VIP fibers, and GnRH neurons that express Fos and apposed by VIP fibers were counted in both age groups. Our results clearly demonstrate that aging does not alter the number of GnRH neurons that receive VIP innervation. However, the number of GnRH neurons that receive VIP innervation and coexpress Fos decreases significantly. We conclude that the age-related delay in the timing of the LH surge is not due to a change in VIP innervation of GnRH neurons, but instead may result from a decreased sensitivity of GnRH neurons to VIP input.     

Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons

Expression of GFP in GnRH neurons has allowed for studies of individual GnRH neurons. We have demonstrated previously the preservation of physiological function in male GnRH-GFP mice. In the present study, we confirm using biocytin-filled GFP-positive neurons in the hypothalamic slice preparation that GFP-expressing somata, axons, and dendrites in hypothalamic slices from GnRH-GFP rats are GnRH1 peptide positive. Second, we used repetitive sampling to study hormone secretion from GnRH-GFP transgenic rats in the homozygous, heterozygous, and wild-type state and between transgenic and Wistar males after ~4 yr of backcrossing. Parameters of hormone secretion were not different between the three genetic groups or between transgenic males and Wistar controls. Finally, we performed long-term recording in as many GFP-identified GnRH neurons as possible in hypothalamic slices to determine their patterns of discharge. In some cases, we obtained GnRH neuronal recordings from individual males in which blood samples had been collected the previous day. Activity in individual GnRH neurons was expressed as total quiescence, a continuous pattern of firing of either low or relatively high frequencies or an intermittent pattern of firing. In males with both intensive blood sampling (at 6-min intervals) and recordings from their GnRH neurons, we analyzed the activity of GnRH neurons with intermittent activity above 2 Hz using cluster analysis on both data sets. The average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h. Therefore, the GnRH pulse generator may be maintained in the sagittal hypothalamic slice preparation.     

Separate neural systems mediate the steroid-dependent and steroid-independent suppression of tonic luteinizing hormone secretion in the anestrous ewe

In the ewe, two types of seasonal fluctuations in secretion of tonic luteinizing hormone (LH) have been described: a steroid-dependent change whereby estradiol gains the capacity to suppress LH pulse frequency in anestrus, and a steroid-independent decrease in pulse frequency in ovariectomized animals during anestrus. We have proposed that the former reflects activation, in anestrus, of estradiol-sensitive catecholaminergic neurons that inhibit gonadotropin-releasing hormone (GnRH). Three results reported here support this hypothesis: dopaminergic (pimozide) and alpha-adrenergic (phenoxybenzamine) antagonists increased LH in intact anestrous ewes without altering pituitary responses to GnRH; other dopaminergic (fluphenazine) and alpha-adrenergic (dibenamine) antagonists also increased LH in anestrus; agonists for dopaminergic (apomorphine) and alpha-adrenergic (clonidine) receptors suppressed LH secretion in both seasons, suggesting that the appropriate receptors are present in breeding-season ewes. In contrast, catecholamines do not appear to mediate the steroid-independent suppression of pulse frequency; neither pimozide nor phenoxybenzamine increased LH pulse frequency in ovariectomized ewes during anestrus. When antagonists for 6 other neurotransmitter receptors (muscarinic and nicotinic cholinergic, GABAnergic, serotonergic, opioid, and beta-adrenergic) were tested in anestrus, only cyproheptadine, the serotonergic antagonist, increased pulse frequency in ovariectomized ewes. Cyproheptadine had no effect on frequency during the breeding season. On the basis of these results, we propose that the steroid-dependent and -independent actions of anestrous photoperiod occur via catecholaminergic and serotonergic neurons, respectively.     

Exposure to ram wool stimulates gonadotropin-releasing hormone pulse generator activity in the female goat

In the sheep and goat, exposure of anestrous females to a conspecific male odor enhances reproductive activity. Interestingly, a previous report indicated that male goat hair stimulated pulsatile luteinizing hormone (LH) secretion in the ewe. In the present study, we addressed whether ram wool affects the gonadotropin-releasing hormone (GnRH) pulse generator activity in the female goat. Five ovariectomized (OVX) goats were chronically implanted with recording electrodes in the mediobasal hypothalamus, and manifestations of the GnRH pulse generator were monitored as characteristic increases in multiple-unit activity (MUA volleys). Wool or hair samples were collected from a mature ram, ewe and male goat, and their effects on the MUA volley were examined. The exposure to ram wool induced an MUA volley within 1 min in all five OVX goats, as did the exposure to male goat hair. The ewe wool had no effect on the timing of an MUA volley occurrence. An invariable association of MUA volleys with LH pulses in the peripheral circulation was also confirmed in two OVX goats exposed to ram wool. The present results clearly indicate that exposure to ram wool stimulates pulsatile GnRH/LH release in the female goat. Since exposure to male goat hair enhances pulsatile LH secretion in the ewe, it is likely that very similar, if not identical, molecules are contained in the male-effect pheromone in the sheep and goat.     

Internal and external determinants of the timing of puberty in the female

A working hypothesis is proposed to account for the timing of puberty in female sheep. In the immature female, the frequency of LH pulses is low, and ovarian follicles do not develop to an advanced stage. During the pubertal transition, the frequency of LH pulses increases to drive follicular development and the production of oestradiol which evokes the gonadotrophin surge and ovulation. Central to the hypothesis is the hypothalamic pulse generator for GnRH that directs the pattern and level of LH secretion. Growth-related cues are monitored to regulate the activity of the GnRH pulse generator. When a sufficient body size is attained, the frequency of LH pulses increases both because the sensitivity to oestradiol inhibitory feedback decreases and because the GnRH pulse generator can be accelerated by the steroid. This increase in LH pulse frequency occurs provided the female has experienced the requisite exposure to photoperiod, i.e. the long days of summer followed by the short days of autumn. These photoperiodic cues are transduced by the pineal gland into a humoral signal which is an increased nocturnal production of melatonin. Failure to grow to the appropriate body size or to experience the appropriate exposure to photoperiod leads to a maintenance of the prepubertal anovulatory condition because the GnRH pulse generator operates at low frequency.