Circadian control of kisspeptin and a gated GnRH response mediate the preovulatory luteinizing hormone surge

In spontaneously ovulating rodents, the preovulatory LH surge is initiated on the day of proestrus by a timed, stimulatory signal originating from the circadian clock in the suprachiasmatic nucleus (SCN). The present studies explored whether kisspeptin is part of the essential neural circuit linking the SCN to the GnRH system to stimulate ovulation in Syrian hamsters (Mesocricetus auratus). Kisspeptin neurons exhibit an estrogen-dependent, daily pattern of cellular activity consistent with a role in the circadian control of the LH surge. The SCN targets kisspeptin neurons via vasopressinergic (AVP), but not vasoactive intestinal polypeptide-ergic, projections. Because AVP administration can only stimulate the LH surge during a restricted time of day, we examined the possibility that the response to AVP is gated at the level of kisspeptin and/or GnRH neurons. Kisspeptin and GnRH activation were assessed after the administration of AVP during the morning (when AVP is incapable of initiating the LH surge) and the afternoon (when AVP injections stimulate the LH surge). Kisspeptin, but not GnRH, cellular activity was up-regulated after morning injections of AVP, suggesting that time-dependent sensitivity to SCN signaling is gated within GnRH but not kisspeptin neurons. In support of this possibility, we found that the GnRH system exhibits pronounced daily changes in sensitivity to kisspeptin stimulation, with maximal sensitivity in the afternoon. Together these studies reveal a novel mechanism of ovulatory control with interactions among the circadian system, kisspeptin signaling, and a GnRH gating mechanism of control.     

Increased gonadotropin-releasing hormone pulse frequency associated with estrogen-induced luteinizing hormone surges in ovariectomized ewes

Hypophyseal portal blood samples were taken from ovariectomized (OVX) ewes given 50 micrograms estradiol benzoate. This estrogen treatment elicited a biphasic alteration (decrease then increase) in LH secretion. During the negative feedback phase, pulsatile GnRH secretion continued; at this time the interpulse interval for the GnRH pulses (49.5 +/- 5.7 min, mean +/- SE, n = 6) was similar to that in 7 control OVX ewes (53.4 +/- 8.7 min). During the positive feedback phase the GnRH interpulse interval (26.8 +/- 9.8 min; n = 6) was significantly (P less than 0.05) less than in the controls. In 3/7 cases the GnRH pulse frequency in OVX controls was within the range observed for estrogen-treated sheep during the positive feedback phase. These data suggest that, in most cases, the LH surge that can be induced by estrogen in OVX ewes, is associated with an increased GnRH pulse frequency. In some animals the inherent GnRH pulse frequency may already be at a rate that is high enough to permit an LH surge by action of estrogen on the pituitary. In general, the mean concentrations of GnRH in portal blood during the LH surge were higher than those in untreated animals, suggesting an overall increase in GnRH output during the LH surge. Pulsatile GnRH secretion continues throughout the early negative feedback phase, suggesting that the predominant effect of estrogen at this time is at the pituitary level.     

The dorsomedial suprachiasmatic nucleus times circadian expression of Kiss1 and the luteinizing hormone surge

Ovulation in mammals is gated by a master circadian clock in the suprachiasmatic nucleus (SCN). GnRH neurons represent the converging pathway through which the brain triggers ovulation, but precisely how the SCN times GnRH neurons is unknown. We tested the hypothesis that neurons expressing kisspeptin, a neuropeptide coded by the Kiss1 gene and necessary for the activation of GnRH cells during ovulation, represent a relay station for circadian information that times ovulation. We first show that the circadian increase of Kiss1 expression, as well as the activation of GnRH cells, relies on intact ipsilateral neural input from the SCN. Second, by desynchronizing the dorsomedial (dm) and ventrolateral (vl) subregions of the SCN, we show that a clock residing in the dmSCN acts independently of the light-dark cycle, and the vlSCN, to time Kiss1 expression in the anteroventral periventricular nucleus of the hypothalamus and that this rhythm is always in phase with the LH surge. In addition, we show that although the timing of the LH surge is governed by the dmSCN, its amplitude likely depends on the phase coherence between the vlSCN and dmSCN. Our results suggest that whereas dmSCN neuronal oscillators are sufficient to time the LH surge through input to kisspeptin cells in the anteroventral periventricular nucleus of the hypothalamus, the phase coherence among dmSCN, vlSCN, and extra-SCN oscillators is critical for shaping it. They also suggest that female reproductive disorders associated with nocturnal shift work could emerge from the desynchronization between subregional oscillators within the master circadian clock.     

Pro-gonadotropin-releasing hormone (ProGnRH) and GnRH content in the preoptic area and the basal hypothalamus of anterior medial preoptic nucleus/suprachiasmatic nucleus-lesioned persistent estrous rats

The content of GnRH and its precursor peptide were quantified in female rats bearing lesions in the anterior medial preoptic nucleus (AMPO) and the suprachiasmatic nucleus (SCN), and the effects of the lesions on the synthetic activity of the GnRH neurons were evaluated. Electrolytic lesions which induced persistent estrous (PE), or irregular estrous cycles, were produced by passing 5-10 microA of direct current into the AMPO or the SCN of female rats which exhibited regular 4 days estrous cycles before the lesions. Approximately 5 weeks after lesion placement, blood samples were withdrawn from catheterized, freely moving animals and plasma LH, PRL, estrogen, and progesterone were determined by RIA. The preovulatory surges of LH and PRL were eliminated in AMPO- or SCN-lesioned PE rats. Moreover, the LH surge was eliminated and the PRL surge significantly attenuated after estrogen and progesterone treatment of rats bearing complete lesions, irrespective of the presence of ovaries. Irregular cycling animals with incomplete AMPO or SCN lesions, exhibited attenuated LH surge and PRL surge similar to proestrous controls. In one incidence this occurred spontaneously, and could also be induced by sequential estrogen and progesterone injections. After ovariectomy, plasma LH levels were significantly lower in the lesioned animals as compared to sham operated rats (P less than 0.05). Similar secretory patterns of LH and PRL were obtained from a second series of sham-operated rats during the different stages of the estrous cycle or from AMPO- or SCN-lesioned rats during persistent estrus. After 2 months the animals were killed between 0830 and 0930 h, and the preoptic area and the basal hypothalamus were microdissected from the brain sections. After extraction and purification, proGnRH and GnRH levels were measured by RIA. ProGnRH levels in the preoptic area were significantly reduced in AMPO- or in SCN-lesioned rats, compared to proestrous controls (P less than 0.01). In contrast, GnRH levels in either area did not differ in AMPO- or in SCN-lesioned animals compared to sham-operated, proestrous controls. Therefore, lesions of the AMPO or the SCN produce PE and reduce proGnRH without reducing GnRH levels. These data would suggest that the AMPO and the SCN participate in the control of the estrous cycle and are necessary for preovulatory surges of PRL and LH to occur and that the AMPO and the SCN form part of the neural circuit that regulates GnRH synthesis and/or release.     

Role of endogenous opioid peptides in mediating progesterone-induced disruption of the activation and transmission stages of the GnRH surge induction process.

How progesterone blocks the E2-induced GnRH surge in females is not known. In this study we assessed whether the endogenous opioid peptides (EOPs) that mediate progesterone negative feedback on pulsatile GnRH secretion also mediate the blockade of the GnRH surge. We treated ovariectomized ewes with physiological levels of E2 and progesterone to stimulate and block the GnRH surge, respectively, using LH secretion as an index of GnRH release. A pilot study confirmed that blocking opioidergic neurotransmission with the opioid receptor antagonist, naloxone (NAL; 1 mg/kg.h, i.v.), could prevent the suppression of pulsatile LH secretion by progesterone in our model. By contrast, antagonizing EOP receptors with NAL did not restore LH surges in ewes in which the E2-induced GnRH surge was blocked by progesterone treatment during the E2-dependent activation stage (Exp 1) of the GnRH surge induction process. However, in ewes treated with progesterone during the E2-independent transmission stage (Exp 2), NAL partially restored blocked LH surges, as indicated by increased fluctuations in LH that, in some cases, resembled LH surges. We conclude, therefore, that the EOPs that mediate progesterone negative feedback on pulsatile GnRH secretion are not involved in blockade of activation of the E2-induced GnRH surge by progesterone, but do appear to be part of the mechanism by which progesterone disrupts the transmission stage.     

Effects of sex steroids on the positive estrogen feedback mechanism in intact women and castrate men

We studied the effects of prolonged testosterone treatment on ovulatory function and positive estrogen feedback in women and of prolonged estrogen priming on gonadotropin feedback in castrate men. An estrogen provocation test was carried out in 4 groups of transsexual subjects: 12 female transsexuals in their early follicular phase (days 3-5; group 1A), 8 females who had been treated with Depo-testosterone (T) for 3-6 months (group 1B), 11 men who had been castrated 3 months previously (group 2A), and 4 male castrates treated with oral estrogen for 3 months starting 3 months after castration (group 2B). The estrogen provocation test consisted of 3 GnRH tests (100 micrograms) carried out immediately before (0 h) and 44 and 92 h after an im injection of estradiol valerate (10 mg). Responses to the estrogen provocation test in women with normal menstrual cycles (group 1A) were typically female. After initial suppression at 44 h, a LH surge (positive feedback) occurred at 92 h. Pituitary responsiveness, however, was amplified both at 44 and 92 h. Prolonged T priming of women in group 1B did not inhibit the estrogen-induced LH surge, nor was the amplitude of the surge blunted. Removal of androgens and other testicular factors (group 2A) did not result in the appearance of an estrogen-induced LH surge. On the other hand, prolonged estrogen priming in male castrates (group 2B) resulted in activation of the positive feedback mechanism; a LH surge in response to the estrogen provocation occurred. The results of the present study imply that 1) contrary to an earlier suggestion, testosterone does not block or blunt the LH surge, indicating that it is probably not responsible for suppressing the LH surge in normal men; 2) testosterone can cause ovulatory failure without suppressing the LH surge in women; and 3) prolonged estrogen priming may be involved in activation of the positive feedback mechanism in humans.     

Classical estrogen receptor alpha signaling mediates negative and positive feedback on gonadotropin-releasing hormone neuron firing

During the female reproductive cycle, the neuroendocrine action of estradiol switches from negative feedback to positive feedback to initiate the preovulatory GnRH and subsequent LH surges. Estrogen receptor-alpha (ERalpha) is required for both estradiol negative and positive feedback regulation of LH. ERalpha may signal through estrogen response elements (EREs) in DNA and/or via ERE-independent pathways. Previously, a knock-in mutant allele (ERalpha-/AA) that selectively restores ERE-independent signaling onto the ERalpha-/- background was shown to confer partial negative but not positive estradiol feedback on serum LH. The current study investigated the roles of the ERE-dependent and ERE-independent ERalpha pathways for estradiol feedback at the level of GnRH neuron firing activity. The above ERalpha genetic models were crossed with GnRH-green fluorescent protein mice to enable identification of GnRH neurons in brain slices. Targeted extracellular recordings were used to monitor GnRH neuron firing activity using an ovariectomized, estradiol-treated mouse model that exhibits diurnal switches between negative and positive feedback. In wild-type mice, GnRH neuron firing decreased in response to estradiol during negative feedback and increased during positive feedback. In contrast, both positive and negative responses to estradiol were absent in GnRH neurons from ERalpha-/- and ERalpha-/AA mice. ERE-dependent signaling is thus required to increase GnRH neuron firing to generate a GnRH/LH surge. Furthermore, ERE-dependent and -independent ERalpha signaling pathways both appear necessary to mediate estradiol negative feedback on serum LH levels, suggesting central and pituitary estradiol feedback may use different combinations of ERalpha signaling pathways.     

Diurnal and estradiol-dependent changes in gonadotropin-releasing hormone neuron firing activity

A robust gonadotropin-releasing hormone (GnRH) surge is a prerequisite signal for the luteinizing hormone (LH) surge that triggers ovulation. In rodents, the GnRH surge is initiated by elevated estradiol and a diurnal switch in estrogen action from negative to positive feedback. The ability of constant estradiol treatment to induce daily LH surges was tested in adult mice that were ovariectomized (OVX) or OVX and treated with estradiol implants (OVX+E). LH in OVX mice showed no time-of-day difference. In contrast, OVX+E mice showed a large LH surge (8- to 124-fold relative to the a.m.) in p.m. samples on d 2-5 post-OVX+E. Targeted extracellular recordings were used to examine changes in firing activity of GnRH neurons in brain slices. There was no time-of-day difference in cells from OVX mice. In contrast, OVX+E cells recorded in the p.m. showed an increased mean firing rate and instantaneous firing frequency, which could increase GnRH release, and decreased duration of quiescence between bouts of firing, possibly reflecting increased pulse frequency, compared with cells recorded in the a.m. In the a.m., OVX+E cells showed changes in GnRH neuron firing reflecting negative feedback compared with OVX cells, whereas in the p.m., OVX+E cells exhibited changes suggesting positive feedback. These data indicate that differences in pattern and level of individual GnRH neuron firing may reflect the switch in estradiol action and underlie GnRH surge generation. The persistence of altered GnRH neuron activity in slices indicates that this approach can be used to study the neurobiological mechanisms of surge generation.     

Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats

Ovulation is caused by a sequence of neuroendocrine events: GnRH and LH surges that are induced by positive feedback action of estrogen secreted by the mature ovarian follicles. The central mechanism of positive feedback action of estrogen on GnRH/LH secretion, however, is not fully understood yet. The present study examined whether metastin, the product of metastasis suppressor gene KiSS-1, is a central neuropeptide regulating GnRH/LH surge and then estrous cyclicity in the female rat. Metastin had a profound stimulation on LH secretion by acting on the preoptic area (POA), where most GnRH neurons projecting to the median eminence are located, because injection of metastin into the third ventricle or POA increased plasma LH concentrations in estrogen-primed ovariectomized rats. Metastin neurons were immunohistochemically found in the arcuate nucleus (ARC) to be colocalized with estrogen receptors with some fibers in the preoptic area (POA) in close apposition with GnRH neuronal cell bodies or fibers. Quantitative RT-PCR has revealed that KiSS-1 and GPR54 mRNAs were expressed in the ARC and POA, respectively. The blockade of local metastin action in the POA with a specific monoclonal antibody to rat metastin completely abolished proestrous LH surge and inhibited estrous cyclicity. Metastin-immunoreactive cell bodies in the ARC showed a marked increase and c-Fos expression in the early proestrus afternoon compared with the day of diestrus. Thus, metastin released in the POA is involved in inducing the preovulatory LH surge and regulating estrous cyclicity.     

Vasoactive intestinal polypeptide can excite gonadotropin-releasing hormone neurons in a manner dependent on estradiol and gated by time of day

A surge of GnRH release signals the LH surge that triggers ovulation. The GnRH surge is dependent on a switch in estradiol feedback from negative to positive and, in rodents, a daily neural signal, likely from the suprachiasmatic nuclei. Vasoactive intestinal polypeptide (VIP) may be involved in suprachiasmatic nuclei-GnRH neuron communication. Here we assessed the effects of acute VIP (5 min treatment) on GnRH neuron function using targeted extracellular recordings of firing activity of GnRH neurons in brain slices. We examined the effect of VIP on firing rate at different times of day using an established ovariectomized, estradiol-treated (OVX+E) mouse model that exhibits daily LH surges timed to the late afternoon. Cells from OVX animals (no estradiol) did not respond to VIP, regardless of time of day. With estradiol, the effect of VIP on GnRH neurons was dependent on the time of recording. During negative feedback, OVX+E cells did not respond. VIP increased firing in cells recorded during surge onset, but this excitatory response was reduced at surge peak. Acute treatment of OVX+E cells during surge peak with a VIP receptor antagonist decreased GnRH neuron firing. This suggests endogenous VIP may both increase GnRH neuron firing during the surge and occlude response to exogenous VIP. These data provide functional evidence for VIP effects on GnRH neurons and indicate that both estradiol and time of day gate the GnRH neuron response to this peptide. VIP may provide an excitatory signal from the circadian clock that helps time the GnRH surge.     

Differential effects of hypothalamic IGF-I on gonadotropin releasing hormone neuronal activation during steroid-induced LH surges in young and middle-aged female rats

Interactions between brain IGF-I receptors and estrogen receptors regulate female reproductive physiology and behavior. The present study investigated potential mechanisms by which IGF-I receptors in the neuroendocrine hypothalamus regulate GnRH neuronal activation and LH release in young and middle-aged female rats under estradiol (E2) positive feedback conditions. We infused vehicle, IGF-I, or JB-1, a selective antagonist of IGF-I receptors, into the third ventricle of ovariectomized female rats primed with E2 and progesterone or vehicle. In young females, blockade of IGF-I receptors attenuated the steroid hormone-induced LH surge, reduced the percent of GnRH neurons expressing c-fos on the day of the LH surge, and decreased the total number of neurons expressing c-fos in the preoptic area. Middle-aged females had fewer GnRH neurons expressing c-fos during the LH surge than young females, and the LH surge amplitude was attenuated. Infusion of an IGF-I dose previously shown to increase LH surge amplitude did not increase the percent of GnRH neurons expressing c-fos in middle-aged females. Brain IGF-I receptor blockade did not modify E2 induction of progestin receptor-immunoreactive neurons in the preoptic area, arcuate, or ventromedial hypothalamus of young rats. These findings indicate that brain IGF-I receptors are required for E2 activation of GnRH neurons in young rats and for robust GnRH release from axon terminals in middle-aged females. IGF-I likely exerts its effects by actions on E2-sensitive neurons that are upstream of GnRH neurons and terminals.     

The preovulatory LH surge A case of a neuroendocrine switch.

The preovulatory surge in LH is a unique endocrine event that involves a switch from the negative feedback effect of estrogen to a positive feedback effect. This occurs at both the level of the brain and that of the pituitary gland. Within the brain the mechanism appears to involve disinhibition of negative inputs to GnRH neurons, as well as stimulation of positive inputs. The positive feedback effects on the brain and the pituitary appear to be coordinated so that the effect of estrogen involves a number of time-delayed mechanisms.     

Metastin/Kisspeptin and control of estrous cycle in rats

Estrous cyclicity is controlled by a cascade of neuroendocrine events, involving the activation of the hypothalamo-pituitary-gonadal axis. Two modes of gonadotropin-releasing hormone (GnRH) are well established to regulate the estrous cycle: one is a tonic or pulse mode of secretion which is responsible for the stimulation of follicular development and steroidogenesis; the other is a surge mode, which is solely responsible for the induction of luteinizing hormone (LH) surges, eventually leading to ovulation. Metastin/kisspeptin-GPR54 signaling has been suggested to control ovarian cyclicity through regulating the two modes of GnRH release. A population of metastin/kisspeptin neurons located in the anteroventral periventricular nucleus (AVPV) is considered to trigger GnRH surge and thus to mediate the estrogen positive feedback action on GnRH release. The other hypothalamic population of metastin/kisspeptin neurons is located in the arcuate nucleus (ARC) and could be involved in generating GnRH pulses and mediating negative feedback action of estrogen on GnRH release. GnRH neurons express mRNA for GPR54, a metastin/kisspeptin receptor, and have a close association with metastin/kisspeptin neurons at the cell body and terminal level, but the precise mechanism by which this peptide regulates the two modes of GnRH release needs to be determined. Metastin/kisspeptin, therefore, is a key hypothalamic neuropeptide, which is placed immediately upstream of GnRH neurons and relays the peripheral steroidal information to GnRH neurons to control estrous cyclicity.     

Estradiol down-regulates RF-amide-related peptide (RFRP) expression in the mouse hypothalamus

In most mammals, RF-amide-related peptides are synthesized in the dorsomedial hypothalamic nucleus and regulate reproduction via inhibiting GnRH neurons and, possibly, adenohypophyseal gonadotrophs. In the present study, we investigated the possibility that RFRP-synthesizing neurons are involved in estrogen feedback signaling to the reproductive axis in mice. First, we used quantitative in situ hybridization and compared the expression of prepro-RFRP mRNA of ovariectomized mice, with and without 17β-estradiol (E2) replacement. Subcutaneous administration of E2 via silastic capsules for 4 d significantly down-regulated prepro-RFRP mRNA expression. The underlying receptor mechanism was investigated with immunohistochemistry. In ovariectomized mice, low levels of nuclear estrogen receptor (ER)-α immunoreactivity were detectable in 18.7 ± 3.8% of RFRP neurons. The majority of RFRP neurons showed no ER-α signal, and RFRP neurons did not exhibit ER-β immunoreactivity. Results of these studies indicate that RFRP is a negatively estradiol-regulated neurotransmitter/neuromodulator in mice. The estrogenic down-regulation of RFRP expression may contribute to estrogen feedback to the reproductive axis. The issue of whether E2 regulates RFRP neurons directly or indirectly remains open given that ER-α immunoreactivity is present only at low levels in a subset of these cells.     

Absence of gonadotropin surges and gonadotropin-releasing hormone self-priming in ovariectomized (OVX), estrogen (E2)-treated, progesterone receptor knockout (PRKO) mice.

It is well known that estrogen (E2) stimulates expression of progesterone receptors (PRs), thereby inducing responsiveness of several tissues to the actions of progesterone (P). Recent studies have also suggested, however, that biological actions previously ascribed to E2 alone may also be mediated by activation of E2-induced PRs, even independently of signal changes in P concentrations. In the present experiments, the progesterone receptor knockout (PRKO) mice were used to assess the role of PR activation in the positive feedback actions of E2 on gonadotropin release. Ovariectomized (OVX) PRKO mice were tested for their capacity to mount primary gonadotropin surges in response to exogenous E2, and to exhibit a GnRH self-priming effect in response to sequential injections of the decapeptide. Wild-type (WT) and PRKO mice were OVX, treated with both 17beta-estradiol and estradiol benzoate (EB), and then killed at 1900 h on day 7 postOVX. Plasma LH RIA revealed that WT mice exhibited surges in response to the E2 treatment; the PRKO mice, however, showed no elevation in plasma LH above untreated controls. Instead, plasma LH levels in E2-treated, OVX PRKO mice decreased significantly in comparison to untreated OVX PRKO mice, suggesting that E2 can exert a negative feedback influence on LH release in PRKO mice, despite the absence of positive feedback effects. A slight but significant rise in plasma FSH was observed in E2-treated OVX WT mice in comparison to untreated controls: an effect not seen in E2-treated OVX PRKO mice, reinforcing the observation that estrogen's positive feedback effects are compromised in PRKO mice. In a second experiment, E2-treated OVX WT and PRKO mice were given either one or two pulses of GnRH 60 min apart, and killed 10 min later. The WT mice were found to exhibit a robust GnRH self-priming effect, as WT mice receiving two GnRH pulses displayed LH responses approximately 2-fold greater than those receiving only one pulse. By contrast, PRKO mice receiving two GnRH pulses exhibited no additional increase in plasma LH levels. We conclude that PR activation is obligatory for expression of the GnRH self-priming effect as well as for generation of E2-induced LH and FSH surges. The extent to which failure of LH surge secretion in PRKO mice is due to the absence of GnRH self-priming, lack of hypothalamic GnRH surges, and/or defects in other processes remains to be determined. These observations clearly demonstrate, however, that the presence of PR is an absolute requirement for the transmission of E2-induced signals leading to gonadotropin surges.     

Orphanin FQ: evidence for a role in the control of the reproductive neuroendocrine system

Orphanin FQ (OFQ), also known as nociceptin, is a member of the endogenous opioid peptide family that has been functionally implicated in the control of pain, anxiety, circadian rhythms, and neuroendocrine function. In the reproductive system, endogenous opioid peptides are involved in the steroid feedback control of GnRH pulses and the induction of the GnRH surge. The distribution of OFQ in the preoptic area and hypothalamus overlaps with GnRH, and in vitro evidence suggests that OFQ can inhibit GnRH secretion from hypothalamic fragments. Using the sheep as a model, we examined the potential anatomical colocalization between OFQ and GnRH using dual-label immunocytochemistry. Confocal microscopy revealed that approximately 93% of GnRH neurons, evenly distributed across brain regions, were also immunoreactive for OFQ. In addition, almost all GnRH fibers and terminals in the external zone of the median eminence, the site of neurosecretory release of GnRH, also colocalized OFQ. This high degree of colocalization suggested that OFQ might be functionally important in controlling reproductive endocrine events. We tested this possibility by examining the effects of intracerebroventricular administration of [Arg(14), Lys(15)] OFQ, an agonist to the OFQ receptor, on pulsatile LH secretion. The agonist inhibited LH pulse frequency in both luteal phase and ovariectomized ewes and suppressed pulse amplitude in the latter. The results provide in vivo evidence supporting a role for OFQ in the control of GnRH secretion and raise the possibility that it acts as part of an ultrashort, autocrine feedback loop controlling GnRH pulses.     

Kisspeptin is essential for the full preovulatory LH surge and stimulates GnRH release from the isolated ovine median eminence

Kisspeptins are the product of the Kiss1 gene and potently stimulate GnRH secretion. In sheep, Kiss1 mRNA-expressing cells are found in the arcuate nucleus (ARC) and dorsal-lateral preoptic area and both appear to mediate the positive feedback effect of estradiol to generate the preovulatory GnRH/LH surge. To determine the role of kisspeptin in transmitting estrogen-positive feedback in the hypothalamus, we administered the kisspeptin antagonist p-271 to ewes subjected to an estradiol benzoate-induced LH surge. Kisspeptin antagonist treatment significantly attenuated these LH surges. We further examined the response to kisspeptin treatment prior to the LH surge. Kisspeptin significantly stimulated GnRH secretion into the hypophysial portal system, but the response to kisspeptin was similar in luteal and late-follicular phase ewes. Kiss1r mRNA expression in GnRH neurons was also similar across the estrous cycle. To examine alternative pathways for kisspeptin stimulation of GnRH neurons, we examined the origin of kisspeptin neuronal fibers in the external zone of the median eminence (ME) using neuronal tracing and immunohistochemical techniques. ARC populations of kisspeptin neurons project fibers to the ME. Finally, we showed kisspeptin stimulates GnRH release from ovine ME-cultured explants. This suggests direct kisspeptin to GnRH terminal-to-terminal communication within the ME. Overall, these data indicate an essential role for kisspeptin in receiving stimulatory estrogen signals and generating the full positive feedback GnRH/LH surge. Kisspeptin neurons of the ARC project to the external zone of the ME and kisspeptin acts upon the GnRH fibers at this level.     

Separate negative feedback effects of estrogen on the pituitary and the central nervous system in the ovariectomized rhesus monkey

The site(s) of the negative feedback action of estrogen on gonadotropin secretion were studied in the ovariectomized rhesus monkey by observing the serum LH and FSH responses to intravenous GnRH injections at various times after implantation of Silastic capsules filled with estrogen. Circulating estrogen concentrations produced by the capsules were within the normal midcycle range for this species. Four h after estrogen implantation, no LH or FSH response was seen to the GnRH injection, indicating a suppressive effect of the steroid directly on the pituitary. Twelve and 22 h after estrogen implantation, however, the LH and FSH responses were equal to or larger than control responses. Since preinjection LH and FSH levels were below control values at these times and the pituitary responded to exogenous GnRH, it appears that endogenous GnRH secretion was affected, indicating an inhibitory action of estrogen on the central nervous system. Thus these experiments suggest 2 separate negative feedback actions of estrogen: a transient one directly on the pituitary and a longer lasting effect on the central nervous system.