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.     

Inhibition of metastin (kisspeptin-54)-GPR54 signaling in the arcuate nucleus-median eminence region during lactation in rats

Follicular development and ovulation are suppressed during lactation in various mammalian species, mainly due to the suppression of pulsatile GnRH/LH secretion. Metastin (kisspeptin-54), a KiSS-1 gene product, is an endogenous ligand for GPR54, a G-protein-coupled receptor, and suggested to play a critical role in regulating the gonadal axis. The present study therefore aims to determine whether metastin (kisspeptin-54)-GPR54 signaling in discrete brain areas is inhibited by the suckling stimulus that causes suppression of LH secretion in lactating rats. Quantitative RT-PCR revealed that the KiSS-1 mRNA level was significantly lower in the arcuate nucleus (ARC)-median eminence region in lactating ovariectomized (OVX) and estrogen-treated OVX rats than in nonlactating controls. KiSS-1 mRNA in the anteroventral periventricular nucleus was kept at a low level in both lactating and nonlactating rats despite estrogen treatment. GPR54 mRNA levels were significantly lower in lactating than nonlactating rats in the anteroventral periventricular nucleus, but the levels in lactating mothers of the preoptic area and ARC-median eminence were comparable with nonlactating controls. Although KiSS-1 mRNA-expressing cells or metastin (kisspeptin-54) immunoreactivities were densely located in the ARC of nonlactating controls, few were found in the ARC of lactating OVX animals. Various doses of metastin (kisspeptin-54) (0.02, 0.2, and 2 nmol) injected into the third ventricle caused a significant increase in LH secretion in both lactating and nonlactating OVX rats, suggesting that lactating rats are responsive to metastin (kisspeptin-54) stimulus. Thus, the present study demonstrated that KiSS-1 mRNA/metastin (kisspeptin-54) expression is inhibited in the ARC by the suckling stimulus, suggesting that the inhibition is most probably involved in suppressing LH secretion in lactating rats.     

Immunocytochemical localization of kisspeptin neurons in the rat forebrain with special reference to sexual dimorphism and interaction with GnRH neurons

Kisspeptin/metastin has been implicated as a critical regulator in luteinizing hormone (LH) secretion and the reproductive system mediating the effect of estrogen on GnRH neurons. In the present study we examined the sex differences in the effects of estrogen on Kiss1/kisspeptin expression in the forebrain by using gonadectomized rats to assess the interaction of kisspeptin and GnRH neurons. Kiss1/kisspeptin cell bodies were abundant in the rostral periventricular area of the third ventricle (RV3P) and the arcuate nucleus (ARC). A few cell bodies were also observed in other portions of the forebrain, i.e. the bed nucleus of the stria terminalis (BST), the paraventricular hypothalamic nucleus (PaAP), the ventromedial hypothalamic nucleus (VMH), and the medial amygdaloid nucleus (MeA). Kisspeptin-immunoreactive fibers were found mainly in the median eminence (ME), the ARC, and the RV3P, but were scarce in the preoptic area (POA), where GnRH neurons are localized. We also found that estrogen triggers expression of the Kiss1 gene and peptide within all the regions except the ARC, and that the effects in the RV3P, BST, PaAP, and VMH are greater in estrogen treated ovariectomized female rat. It is noteworthy that kisspeptin and GnRH neurons were densely associated in the ME but were rarely in contact in the POA. Thus, our results suggest that kisspeptin-positive neurons, except for the ones in the ARC, are related not only to estrogen-positive feedback, but also sex dimorphism, and that kisspeptin regulates GnRH release in the ME rather than the POA.     

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.     

Evidence that the arcuate nucleus is an important site of progesterone negative feedback in the ewe

There is now considerable evidence that dynorphin neurons mediate the negative feedback actions of progesterone to inhibit GnRH and LH pulse frequency, but the specific neurons have yet to be identified. In ewes, dynorphin neurons in the arcuate nucleus (ARC) and preoptic area (POA) are likely candidates based on colocalization with progesterone receptors. These studies tested the hypothesis that progesterone negative feedback occurs in either the ARC or POA by determining whether microimplants of progesterone into either site would inhibit LH pulse frequency (study 1) and whether microimplants of the progesterone receptor antagonist, RU486, would disrupt the inhibitory effects of peripheral progesterone (study 2). Both studies were done in ovariectomized (OVX) and estradiol-treated OVX ewes. In study 1, no inhibitory effects of progesterone were observed during treatment in either area. In study 2, microimplants of RU486 into the ARC disrupted the negative-feedback actions of peripheral progesterone treatments on LH pulse frequency in both OVX and OVX+estradiol ewes. In contrast, microimplants of RU486 into the POA had no effect on the ability of systemic progesterone to inhibit LH pulse frequency. We thus conclude that the ARC is one important site of progesterone-negative feedback in the ewe. These data, which are the first evidence on the neural sites in which progesterone inhibits GnRH pulse frequency in any species, are consistent with the hypothesis that ARC dynorphin neurons mediate this action of progesterone.     

Role of Na+ and Ca2+ channels in the preoptic LH surge generating mechanism in proestrous rats

We studied whether Na+ and Ca2+ channels are involved in the neural mechanism responsible for the surge of gonadotropin-releasing hormone (GnRH) in proestrous rats. In experiment 1, female rats in proestrus were i.p. injected at 1345 h with pentobarbital sodium (35 mg/kg) to block spontaneous surge of LH and electrical stimulation was applied between 1400 and 1600 h to the preoptic area (POA) together with POA injection of 0.5 microl saline containing the Na+ channel blocker tetrodotoxin (TTX) at a concentration of 1 microM, 2 microM, or 5 microM. Since 5 microM TTX completely blocked the increase in serum LH concentrations evoked by the POA stimulation, we used this concentration in experiment 2 to observe the TTX effect on the spontaneous LH surge. In experiment 2, bilateral injections of 1.5 microl of 5 microM TTX at 1430 h in the POA in proestrous rats postponed the peak time and reduced the peak level of the LH surge. In experiment 3, bilateral injections of 1.5 microl of 5 microM L-type Ca2+ channel blocker nifedipine at 1430 h in the POA completely blocked the LH surge. Since the cell bodies of GnRH neurons are primarily concentrated in the POA in rats, these results suggest that both voltage-sensitive Na+ channels and Ca2+ channels contribute to the generation of action potentials at GnRH cell bodies for the surge release of GnRH.     

Expression of Fos-like proteins in gonadotropin-releasing hormone neurons of Syrian hamsters: effects of estrous cycles and metabolic fuels.

In female mammals, reproduction is sensitive to the availability of metabolic fuels, and food deprivation has been shown to suppress pulsatile LH secretion, attenuate the preovulatory LH surge, and prevent ovulation. It has been suggested that food deprivation impairs fertility by reducing the secretion of GnRH by GnRH-producing neurons in the forebrain. A series of experiments tested this hypothesis by examining the effects of estrous cycles and manipulations of metabolic fuel availability on the expression of Fos-like proteins (Fos-IR) in GnRH-immunoreactive (GnRH-IR) neurons in the forebrain of Syrian hamsters. GnRH-IR neurons were detected in several areas, including the diagonal band of Broca (DBB), medial septum (MS), rostral medial preoptic area (mPOA), and caudal POA. In the more rostral regions (DBB and MS/mPOA), GnRH-IR neurons expressed Fos-IR almost exclusively on day 4 of the cycle, just after the preovulatory LH surge. However, in the caudal POA, GnRH-IR neurons expressed Fos-IR across the entire cycle, including days 1-3, when LH secretion is pulsatile. Food deprivation on days 1 and 2 of the cycle, which attenuates the LH surge and blocks ovulation in hamsters, significantly reduced the proportion of GnRH-IR neurons that expressed Fos-IR on days 2 and 4 (caudal POA) or only on day 4 (DBB and MS/mPOA). Suppression of fuel availability with insulin or 2-deoxy-D-glucose on day 1 of the cycle mimicked the effects of food deprivation and reduced the proportion of caudal POA GnRH-IR neurons that expressed Fos-IR. The results of these experiments suggest that in Syrian hamsters, there are separate populations of GnRH-IR neurons associated with pulsatile and surge modes of LH secretion. In addition, the fact that manipulations of metabolic fuel availability cause changes in the expression of Fos-IR in both populations of GnRH-IR neurons provides strong support for the hypothesis that nutritional infertility is due in part to decreased GnRH secretion.     

Morphological evidence for direct interaction between kisspeptin and gonadotropin-releasing hormone neurons at the median eminence of the male goat: an immunoelectron microscopic study

Kisspeptin has been thought to play pivotal roles in the control of both pulse and surge modes of gonadotropin-releasing hormone (GnRH) secretion. To clarify loci of kisspeptin action on GnRH neurons, the present study examined the morphology of the kisspeptin system and the associations between kisspeptin and GnRH systems in gonadally intact and castrated male goats. Kisspeptin-immunoreactive (ir) and Kiss1-positive neurons were found in the medial preoptic area of intact but not castrated goats. Kisspeptin-ir cell bodies and fibers in the arcuate nucleus (ARC) and median eminence (ME) were fewer in intact male goats compared with castrated animals. Apposition of kisspeptin-ir fibers on GnRH-ir cell bodies was very rare in both intact and castrated goats, whereas the intimate association of kisspeptin-ir fibers with GnRH-ir nerve terminals was observed in the ME of castrated animals. Neurokinin B immunoreactivity colocalized not only in kisspeptin-ir cell bodies in the ARC but also in kisspeptin-ir fibers in the ME, suggesting that a majority of kisspeptin-ir fibers projecting to the ME originates from the ARC. A dual immunoelectron microscopic examination revealed that nerve terminals containing kisspeptin-ir vesicles made direct contact with GnRH-ir nerve terminals at the ME of castrated goats. There was no evidence for the existence of the typical synaptic structure between kisspeptin- and GnRH-ir fibers. The present results suggest that the ARC kisspeptin neurons act on GnRH neurons at the ME to control (possibly the pulse mode of) GnRH secretion in males.     

Kisspeptin expression in the brain: Catalyst for the initiation of puberty

In 2003, two independent groups of researchers discovered almost simultaneously that inactivating mutations of the G protein coupled receptor, GPR54, cause hypogonadotropic hypogonadism in mice and men. Since this discovery, kisspeptins, the natural ligands for GPR54, have been thrust into the reproductive neuroendocrine spotlight, as major regulators of GnRH function. Kisspeptins are the peptide products of the KiSS-1 gene, and potently stimulate gonadotrophin secretion when administered either centrally or peripherally. Expression of KiSS-1 has been localised to specific regions of the hypothalamus in many species and is regulated by gonadal steroids and across the estrous cycle. It appears that kisspeptin transmits steroid feedback signals to GnRH cells, especially the positive feedback effect of estrogen that causes the preovulatory GnRH/LH surge. Importantly, kisspeptin function appears to be fundamental to the initiation of puberty.     

Morphological evidence for direct interaction between arcuate nucleus neuropeptide Y (NPY) neurons and gonadotropin-releasing hormone neurons and the possible involvement of NPY Y1 receptors.

Neuropeptide Y (NPY) neurons in the arcuate nucleus of the hypothalamus (ARH) have been shown to play an important role in modulating LH secretion. One mechanism by which the ARH NPY system may regulate LH secretion is by modulating GnRH neuronal function. Thus, the present study examined whether the ARH NPY system provided direct input to GnRH cell bodies in the preoptic area (POA), as well as to their nerve terminals in the median eminence (ME). The possible involvement of the NPY Y1 receptor subtype in mediating the effects of NPY was also investigated. Lactating rats were used in these studies because they have increased hypothalamic NPY content, especially in the ARH/ME areas, making it easier to detect NPY fibers and terminals. The anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), was iontophoresed into the ARH of lactating rats; and triple-label immunofluorescence was performed, with the aid of confocal microscopy, to visualize NPY, PHA-L, and GnRH. GnRH cell bodies were found scattered throughout the organum vasculosum laminae terminalis (OVLT)/POA region, and NPY/ PHA-L double-labeled fibers were found in very close proximity to numerous GnRH perikarya. In the ME, double-labeled NPY/PHA-L fibers were found in the inner and external zones, and they were found in close proximity to GnRH neuronal fibers. Using a NPY Y1 specific antibody, double-label immunofluorescence was performed to examine whether the Y1 receptor subtype was expressed in GnRH neurons. No convincing Y1-positive staining was found in GnRH cell bodies in the OVLT/POA region. However, abundant Y1-positive fiber and cell staining were observed throughout the region, and Y1-positive fibers were found in close apposition to GnRH cell bodies. In contrast, numerous GnRH nerve fibers and terminals in both the OVLT and ME were colocalized with Y1-positive staining. The results of this study suggest that ARH NPY neurons come in close contact with GnRH neurons and may provide direct input to both GnRH cell bodies in the POA region and to their nerve terminals in the ME. The Y1 receptor subtype may be directly involved in NPY modulation of GnRH secretion from its nerve terminals.     

The morphometry of astrocytes in the rostral preoptic area exhibits a diurnal rhythm on proestrus: relationship to the luteinizing hormone surge and effects of age

The morphometry of astrocytes in the arcuate nucleus exhibits cyclic changes during the estrous cycle leading to dynamic changes in the communication between neurotransmitters and neuropeptides that regulate pituitary hormone secretion. Data suggest that remodeling of direct and/or indirect inputs into GnRH neurons may influence the timing and/or amplitude of the preovulatory LH surge in young rats. We have previously found that aging alters the timing and amplitude of the LH surge. Therefore, the purpose of this study was to focus on the rostral preoptic area where GnRH cell bodies reside. We assessed the possibility that the morphometry of astrocytes in the rostral preoptic area displays time-related and age-dependent changes on proestrus. Our results demonstrate that, in young rats, astrocyte cell surface area decreases between 0800 h and 1200 h, before the initiation of the LH surge. Changes in surface area over the cycle were specific to astrocytes in close apposition to GnRH neurons. In contrast, in middle-aged rats astrocyte surface area was significantly less than in young rats and did not change during the day. These findings suggest that a loss of astrocyte plasticity could lead to the delayed and attenuated LH surge that has been previously observed in middle-aged rats.     

Regulation of NKB pathways and their roles in the control of Kiss1 neurons in the arcuate nucleus of the male mouse

Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17β-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.     

Evidence that dynorphin plays a major role in mediating progesterone negative feedback on gonadotropin-releasing hormone neurons in sheep

Endogenous opioid peptides (EOP) mediate progesterone-negative feedback in many species, but the specific EOP systems involved remain unresolved. We first addressed this question in sheep by determining the role of different EOP receptor subtypes in the medial basal hypothalamus (MBH) and preoptic area (POA). Local administration of EOP receptor antagonists to luteal phase ewes indicated that kappa-, but not micro- or delta-, receptors mediate the inhibition of LH secretion in the MBH. In contrast, both kappa- and micro-, but not delta-receptor, antagonists increased LH pulse frequency when placed in the POA. We next examined close appositions between dynorphin (kappa ligand) and beta-endorphin (micro ligand) containing varicosities and GnRH perikarya in luteal phase ewes using dual immunocytochemistry and light microscopy. Approximately 90% of MBH GnRH neurons had close associations by dynorphin-containing varicosities, but only 40-50% of GnRH perikarya elsewhere had such close associations. In contrast, the percentage of beta-endorphinergic varicosities close to GnRH neurons was similar among all regions. Electron microscopic analysis demonstrated both dynorphinergic synapses and beta-endorphinergic synapses onto GnRH perikarya. These and other data lead to the hypothesis that dynorphin neurons play a major role in progesterone-negative feedback in the ewe and that this inhibition may be exerted directly on GnRH perikarya within the MBH, whereas dynorphin and beta-endorphin input to GnRH neurons in the POA provide redundancy to this system or are involved in other actions of progesterone or estradiol in the control of the GnRH surge.     

Colocalization of progesterone receptors in parvicellular dynorphin neurons of the ovine preoptic area and hypothalamus

Recent evidence suggests that the dynorphin-kappa receptor opioid system acts to mediate the inhibitory effect of progesterone (P) on GnRH pulse frequency during the luteal phase of the ovine estrous cycle. It is known that progesterone receptors (PRs) are required for the actions of P on GnRH secretion. Therefore, if P acts directly on dynorphin (DYN) neurons, then these neurons should contain PRs. To test this hypothesis, we used a dual-label immunoperoxidase procedure to visualize PRs and DYN in the preoptic area (POA) and hypothalamus of ovary-intact ewes killed during the luteal phase of the estrous cycle. The PR was colocalized in more than 90% of parvicellular DYN neurons in the POA, anterior hypothalamus (AHA), and arcuate nucleus (ARC). By contrast, none of magnocellular DYN cells of the paraventricular and supraoptic nuclei coexpressed immunoreactive PRs. The high percentage of colocalization of PRs in parvicellular DYN cells of the POA, AHA, and ARC suggests that these cells are prime targets of P. In addition, DYN cells in the ARC, but not the POA or AHA, were found to receive synaptic inputs from DYN-positive axon terminals. This observation raises the possibility that an ultrashort feedback loop controls the release of DYN from ARC neurons.     

Progesterone increases dynorphin a concentrations in cerebrospinal fluid and preprodynorphin messenger ribonucleic Acid levels in a subset of dynorphin neurons in the sheep

Recent studies suggest that the endogenous opioid peptide, dynorphin, is an important mediator of progesterone negative feedback on GnRH pulse frequency in the ewe. These experiments tested this hypothesis by examining the effects of progesterone on dynorphin A concentrations in cerebrospinal fluid (CSF) collected from the third ventricle and expression of preprodynorphin (PPD) mRNA in hypothalamic nuclei. CSF was collected every 10 min for 5 h in three groups of ewes: 1) ovary-intact ewes during the luteal phase (d 6-7 of estrous cycle); 2) ewes 6-7 d after ovariectomy (OVX); and 3) OVX ewes treated for 6-7 d with implants that produced luteal-phase progesterone levels. Diencephalic tissue from these ewes was then collected and processed for in situ hybridization using an ovine cDNA probe against PPD. Progesterone treatment increased dynorphin A concentrations in CSF over that observed in untreated OVX ewes; CSF dynorphin A concentrations in ovary-intact ewes were midway between the other groups. OVX significantly decreased the number of PPD mRNA-expressing cells in the preoptic area (POA), anterior hypothalamic area (AHA), and arcuate nucleus (ARC), with no change seen in any other PPD-expressing nuclei. Progesterone treatment of OVX ewes restored PPD expression in the POA and AHA to levels seen in luteal-phase animals but had no effect on PPD expression in the ARC. These results are consistent with the hypothesis that progesterone acts via dynorphin neurons to inhibit pulsatile GnRH secretion and point to dynorphin neurons in the POA, AHA, and ARC as potential mediators of this action during the luteal phase.     

Neurotensin gene expression increases during proestrus in the rostral medial preoptic nucleus: potential for direct communication with gonadotropin-releasing hormone neurons.

Neurotensin (NT)-containing neurons in the rostral portion of the medial preoptic nucleus (rMPN) of the brain may play a key role in regulating the pattern of secretion of GnRH, thereby influencing the reproductive cycle in females. The major goals of this study were to determine whether NT messenger RNA (mRNA) levels in the rMPN exhibit a unique pattern of expression in temporal association with the preovulatory LH surge and to assess whether NT neurons may communicate directly with GnRH neurons. We analyzed NT gene expression in rats using in situ hybridization over the day of proestrus and compared this with diestrous day 1. We also determined whether the high-affinity NT receptor (NT1) is expressed in GnRH neurons using dual-label in situ hybridization and whether this expression varies over the estrous cycle. We found that NT mRNA levels in the rMPN increase significantly on the day of proestrus, rising before the LH surge. No such change was detected on diestrous day 1, when the LH surge does not occur. Furthermore, we observed that a significant number of GnRH neurons coexpress NT1 mRNA and that the number of GnRH neurons expressing NT1 mRNA peaks on proestrus. Together with previous findings, our results suggest that increased expression of NT in the rMPN may directly stimulate GnRH neurons on proestrus, contributing to the LH surge. In addition, our results suggest that responsiveness of GnRH neurons to NT stimulation is enhanced on proestrus due to increased expression of NT receptors within GnRH neurons.     

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.     

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.