Developmental changes in hypothalamic insulin-like growth factor-1: relationship to gonadotropin-releasing hormone neurons

Reproductive development in vertebrates is controlled by changes in hypothalamic GnRH neurons and their inputs from other neurons and glia. One factor involved in the regulation of the GnRH system is the neurotrophic factor, IGF-1. To better understand the regulation of GnRH neurons by hypothalamic IGF-1, we quantified levels of IGF-1 mRNA in hypothalamic and preoptic regions containing GnRH cells, studied the effects of IGF-1 on GnRH gene expression, and examined the neuroanatomical relationship between GnRH neurons and hypothalamic IGF-1 in neonatal, peripubertal, and reproductively mature mice. Our results indicated that IGF-1 mRNA levels in the preoptic area and anterior hypothalamus peaked at postnatal day (P) 5, decreased through P20, and then increased through peripubertal and adult development. Second, IGF-1 had stimulatory effects on GnRH gene expression in explanted preoptic area-anterior hypothalamuses of P5 and peripubertal mice, with results varying by sex and duration of treatment. In contrast, IGF-1 had no effect or even inhibited GnRH gene expression in adult P60 mice. Third, GnRH perikarya coexpressed IGF-1, and this increased throughout sexual maturation. Taken together, the results suggest that IGF-1 can modulate GnRH neurons, that the sensitivity of GnRH neurons to IGF-1 changes developmentally, and that GnRH cells coexpress IGF-1.     

Postnatal development of gonadotropin-releasing hormone and cyclophilin gene expression in the female and male rat brain

The rat preoptic area-anterior hypothalamic continuum (POA-AH) contains about 400-800 neurons that express the decapeptide GnRH and the 56-amino-acid GnRH-associated peptide. Originating from the olfactory placode, these neurons migrate and establish their final distribution and connections in the POA-AH several days before birth. The aim of the present study was to examine whether the biosynthesis of the mRNA encoding the precursor (proGnRH) common to GnRH and GnRH-associated peptide undergoes postnatal changes corresponding to the development of sexual maturation. The POA-AH content of proGnRH messenger RNA (mRNA) was followed from postnatal day 1 to day 90 in female and male Sprague-Dawley rats killed by decapitation between 1000-1200 h. Cytoplasmic RNA fractionated from individual POA-AH homogenates was purified using proteinase K digestion. Cytoplasmic proGnRH mRNA was quantitated simultaneously with cyclophilin mRNA (an internal standard control) using solution hybridization-RNase protection assay, with the protected fragments separated through polyacrylamide gel electrophoresis. In the POA-AH, the concentrations of proGnRH mRNA (femtograms mRNA per microgram total RNA) increased significantly with age in both sexes (P less than 0.001). In males, proGnRH mRNA levels increased by day 30 some 2-fold over the values of days 5 and 10, and the levels established on day 30 were maintained through adulthood. In females, the first rise in proGnRH mRNA levels occurred on day 30, followed by an additional increase on day 45 to levels seen in adulthood. Levels of proGnRH mRNA established in adulthood were significantly higher in females than in males (P less than 0.03). The concentrations of cyclophilin mRNA (picograms mRNA per microgram total RNA) remained essentially unchanged in both sexes during the same period of time when proGnRH mRNA levels were increasing. These results provide evidence for postnatal sex-related increases in the levels of proGnRH mRNA in the rat POA-AH, which are likely to reflect differential regulation by gonadal steroids.     

Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons

The neuropeptide kisspeptin has recently been implicated as having a critical role in the activation of the GnRH neurons to bring about puberty. We examined here the postnatal development of kisspeptin neuronal populations and their projections to GnRH neurons in the mouse. Three populations of kisspeptin neurons located in the 1) anteroventral periventricular nucleus (AVPV) and the preoptic periventricular nucleus (PeN), 2) dorsomedial hypothalamus, and 3) arcuate nucleus were identified using an antisera raised against mouse kisspeptin-10. A marked 10-fold (P<0.01), female-dominant sex difference in the numbers of kisspeptin neurons existed in the AVPV/PeN but not elsewhere. Kisspeptin neurons in the AVPV/PeN of both sexes displayed a similar pattern of postnatal development with no cells detected at postnatal day (P) 10, followed by increases from P25 to reach adult levels by puberty onset (P<0.01; P31 females and P45 males). This pattern was not found in the dorsomedial hypothalamus or arcuate nucleus. Dual immunofluorescence experiments demonstrated close appositions between kisspeptin fibers and GnRH neuron cell bodies that were first apparent at P25 and increased across postnatal development in both sexes. These studies demonstrate kisspeptin peptide expression in the mouse hypothalamus and reveal the postnatal development of a sexually dimorphic continuum of kisspeptin neurons within the AVPV and PeN. This periventricular population of kisspeptin neurons reaches adult-like proportions at the time of puberty onset and is the likely source of the kisspeptin inputs to GnRH neurons.     

Gonadal regulation of gonadotropin subunit gene expression: evidence for regulation of follicle-stimulating hormone-beta messenger ribonucleic acid by nonsteroidal hormones in female rats

Gonadectomy results in a rise in gonadotropin secretion and subunit gene expression, although the relative contributions of declining gonadal hormones or increasing hypothalamic GnRH secretion are uncertain. To further delineate the roles of the hypothalamus and gonads in regulation of gonadotropin gene expression, male and female rats were castrated and gonadotropin subunit messenger RNA (mRNA) concentrations measured 2, 7, 14, or 21 days (d) later. In males, FSH beta mRNA was maximal (2-fold increase) by 7 d while peak levels of alpha (3-fold) and LH beta (3-fold) were seen by 14 d. Testosterone (T) replacement restored all three subunit mRNA concentrations to intact values. In females, FSH beta mRNA also reached plateau levels (8-fold increase) earlier than alpha (3-fold) or LH beta (11-fold). When female rats ovariectomized 7 days earlier were given estradiol (E2) and progesterone (P) implants for up to 14 d, suppression of alpha and LH beta to intact levels was observed. However, FSH beta mRNA concentrations only decreased to 67% of castrate values, and remained 2- to 3-fold higher than levels in intact female rats. Female rats were also given E2 replacement at the time of ovariectomy. LH beta mRNA was maintained at intact levels for 14 days while alpha and FSH beta showed partial castration responses (2-fold and 3-fold, respectively). Finally, to determine whether E2 and P regulate gonadotropin subunit expression directly or by reducing GnRH secretion, female rats were ovariectomized and immediately replaced with E2, P, or E2 + P in the presence or absence of a GnRH antagonist (A) for 2 d. alpha mRNA was increased (2-fold) by E2 but not by E2 + A suggesting that E2 requires the presence of GnRH to increase alpha mRNA. P alone was ineffective, but both E2 and A prevented the LH beta mRNA response to ovariectomy. The effects of E2 and A were not additive, suggesting that E suppresses LH beta mRNA by inhibiting the increase in GnRH secretion. In contrast, the FSH beta mRNA response to ovariectomy was only partially suppressed by E2, E2 + P, or E2 + P + A. These data indicate that in castrate males, replacement of T suppresses all three subunit mRNAs to intact levels. However, replacement of E2 to ovariectomized females did not prevent the increase in alpha and FSH beta mRNAs. In female rats, LH beta mRNA is predominantly regulated by GnRH. alpha mRNA expression is also mainly regulated by GnRH, and E2 appears to augment GnRH action on alpha mRNA expression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sexually dimorphic and estrogen-dependent expression of estrogen receptor beta in the ventromedial hypothalamus during rat postnatal development

The ventromedial hypothalamus (VMH) is a sexually dimorphic region of the brain related to female reproductive behavior. The effect of estrogen in the adult rat VMH is thought to be mediated predominantly via estrogen receptor (ER)alpha, because this receptor is expressed at considerably higher levels than ER beta. The present study revealed, using in situ hybridization and immunohistochemistry, that both ER beta mRNA and protein were expressed in the ventrolateral portion of the caudal VMH, at remarkably higher levels during early postnatal development than in adulthood. In addition, the expression was sexually dimorphic, with females having significantly more ER beta-immunoreactive (-ir) cells than males, between postnatal d 5 (P5) and P14, although the sex difference was not significant by P21. Double-label immunofluorescence revealed that 66% of ER beta-ir cells coexpressed ER alpha in the caudal VMH of the P5 female rat. Furthermore, neonatal treatment with E2 benzoate down-regulated ER beta mRNA in the female rat VMH at P5 and decreased VMH ER beta-ir cells during the period between P5 and P14. In contrast to females, no differences in expression of ER beta mRNA or protein were detected between control and E2 benzoate-treated males. These results suggest that estrogen is involved in regulating the sexually dimorphic expression of ER beta in the VMH during early postnatal development of the rat.     

Effects of ovariectomy on GnRH mRNA, proGnRH and GnRH levels in the preoptic hypothalamus of the female rat

Experiments were carried out to investigate the effects of ovariectomy on gonadotropin-releasing hormone (GnRH) messenger RNA (mRNA), proGnRH and GnRH peptide levels in the hypothalamus of female rats. Intact proestrous female rats and female rats, which had been ovariectomized for 2 weeks, were sacrificed at 9.00 h and the preoptic area (POA) and basal hypothalamus (BH) were dissected out and frozen on dry ice. One group of tissues from proestrous control and ovariectomized females were extracted in acetic acid, centrifuged at 13,000 g and the supernatant purified on a C18 column. The purified extract was then radioimmunoassayed for proGnRH, using a specific antiserum to rat proGnRH (ARK-2), and for GnRH using the E1-14 antiserum. Total cellular RNA was isolated from another group of tissues and prepared as Northern blots. Hybridization with 32P-labeled GnRH cRNA was used to detect GnRH mRNA. A third group of proestrous and ovariectomized female rats were perfused, and 50 microns vibratome sections were cut. These were immunostained with proGnRH or GnRH antiserum, followed by in situ hybridization with 35S-labeled GnRH cRNA to detect GnRH mRNA. Based on the histochemical staining, mRNA was colocalized to the cell soma of neurons containing proGnRH and GnRH throughout the POA and BH. Based on the radioimmunoassay, proGnRH levels were 2 times higher in the POA versus the BH, but GnRH levels were 6-7 times higher in the BH. Ovariectomy significantly decreased proGnRH levels in both the POA and BH, while GnRH decreased in the BH. In contrast, quantitative Northern blot analysis demonstrated that ovariectomy had no effect on mRNA levels in the POA and BH. These data indicate that the effects of ovariectomy on proGnRH and GnRH levels are a result of altered translation, posttranslational processing and/or secretion of GnRH.     

Neuroendocrine aging in the female rat: the changing relationship of hypothalamic gonadotropin-releasing hormone neurons and N-methyl-D-aspartate receptors

The reproductive axis undergoes alterations during aging, resulting in acyclicity and the loss of reproductive function. In the hypothalamus, changes intrinsic to GnRH neurons may play a critical role in this process, as may changes in inputs to GnRH neurons from neurotransmitters such as glutamate. We investigated the effects of age and reproductive status on neuroendocrine glutamatergic NMDA receptors (NRs), their regulation of GnRH neurons, and their expression on GnRH neurons, in female rats. First, we quantified NR subunit messenger RNAs (mRNAs) in preoptic area-anterior hypothalamus (POA-AH) and medial basal hypothalamus (MBH), the sites of GnRH perikarya and neuroterminals, respectively. In POA-AH, NR1 mRNA levels varied little with age or reproductive status. NR2a and NR2b mRNA levels decreased significantly between cycling and acyclic rats. In MBH, NR mRNAs all increased with aging, particularly in acyclic animals. Second, we tested the effects of N-methyl-D,L-aspartate (NMA) on GnRH mRNA levels in POA-AH of aging rats. NMA elevated GnRH mRNA levels in young rats, but decreased them in middle-aged rats. Third, we quantified expression of the NR1 subunit on GnRH perikarya in aging rats using double label immunocytochemistry. NR1 expression on GnRH cell bodies varied with age and reproductive status, with 30%, 19%, and 46% of GnRH somata double labeled with NR1 in young proestrous, middle-aged proestrous, and middle-aged persistent estrous rats, respectively. Thus, 1) the expression of hypothalamic NR subunit mRNAs correlates with reproductive status; 2) changes in NR subunit mRNA levels, if reflected by changes in protein levels, may result in alterations in the stoichiometry of the NR during aging, with possible physiological consequences; 3) the effects of NR activation on GnRH mRNA switches from stimulatory to inhibitory during reproductive aging; and 4) expression of the NR1 subunit on GnRH perikarya changes with reproductive status. These molecular, physiological, and cellular neuroendocrine changes are proposed to be involved in the transition to acyclicity in aging female rats.     

Neuropeptide Y (NPY): a possible role in the initiation of puberty

NPY modulates the rat hypothalamic-pituitary-gonadal axis at both the adenohypophysial and central levels. Previously published studies have consistently shown elevations of GnRH content in the preoptic area (POA) and hypothalamus starting with the appearance of GnRH immunoreactivity around fetal day 12-14 until stabilization around the time of puberty. In the present studies, irNPY content of male and female rat hypothalami and POA was examined during days 0 to 36 of postnatal development. Hypothalamic irNPY content rose steadily from 4.54 +/- 0.19 ng/fragment (males) and 2.72 +/- 0.55 ng/fragment (females) at birth to 34.14 +/- 3.94 ng/fragment (males) and 46.79 +/- 6.16 ng/fragment (females) at day 36, corresponding approximately to the time of vaginal opening. A similar elevation of irNPY content was observed in the POA. At day 0, POA content was 1.91 +/- 0.18 ng/fragment (males) and 2.02 +/- 0.25 ng/fragment (females) and progressively increased to 42.26 +/- 3.94 ng/fragment and 41.33 +/- 3.72 ng/fragment by postnatal day 36. In subsequent investigations, hypophysial-portal and peripheral plasma irNPY was determined around the time of vaginal opening, revealing a surge in portal levels of irNPY which preceded the prepubertal LH surge. The progressive postnatal increase in hypothalamic and POA irNPY content culminating in a prepubertal surge of irNPY secretion into the hypophysial-portal circulation suggests involvement of this neuropeptide in reproductive development and the onset of puberty.     

Implication of dopaminergic systems on GnRH and GnRHR genes expression in the hypothalamus and GnRH-R gene expression in the anterior pituitary gland of anestrous ewes.

We examined by Real-time PCR how prolonged inhibition of dopaminergic D-2 receptors (DA-2) in the hypothalamus of anestrous ewes by infusion of sulpiride into the third cerebral ventricle affected GnRH and GnRH-R gene expression in discrete parts of this structure and GnRH-R gene expression in the anterior pituitary. Blockaded DA-2 receptors significantly decreased GnRH mRNA levels in the ventromedial hypothalamus but did not evidently affect GnRH mRNA in the preoptic/ anteriorhypothalamicarea. Blockaded DA-2 receptors led to different responses in GnRH-R mRNA in various parts of the hypothalamus; increased GnRH-R mRNA levels in the preoptic/anterior hypothalamic area, and decreased GnRH-R mRNA amounts in the ventromedial hypothalamus stalk/median eminence. An infusion of sulpiride into the III-rd ventricle increased GnRH mRNA levels in the anterior pituitary gland and LH secretion. It is suggested that the increase of GnRH gene expression in the anterior pituitary gland and LH secretion in sulpiride-treated ewes are related with an increase of biosynthesis GnRH with concomitant decreased biosynthesis of GnRH-R protein in the ventromedial hypothalamus/stalk median eminence allowing to an increase of GnRH release.     

3H-estradiol distribution in female, androgenized female, and male rats at 100 and 200 days of age.

Tissue distribution of radioactivity was studied 48 h after gonadectomy and 1 h after iv injection of 3H-estradiol (1 mug/kg body wt) in 100- and 200-day-old normal female rats, female rats androgenized with 30, 100 or 1250 mug testosterone propionate (TP) at 5 days of age, and male rats. Receptor-mediated uptake of estradiol, as shown by diethylstilbestrol (DES) competition, was highest in preoptic area-anterior hypothalamus (POA-AH) and median eminence-basal hypothalamus (ME-BH), but also occurred in dorsal hypothalamus, pre-hypothalamic area, amygdala and septum in all groups. At 100 days of age there were no differences in brain radioactivity levels between females and androgenized females or males. At 200 days of age radioactivity levels in POA-AH and ME-BH tended to be lower in androgenized female and male rats than in normal females. Also, radioactivity levels in the amygdala were lower in the 1250 mug TP-treated females than in normal females. When expressed per unit fresh weight, uptake in the anterior pituitary tended to be lower in androgenized rats at 100 days of age and was higher in males at 200 days of age than in normal females, but did not differ among any of the groups when expressed as uptake per organ. Thus, the well-known differences among these groups in neural regulation of gonadotropin secretion and sex behavior were not correlated with consistent differences in specific estradiol binding by hypothalamic or other brain areas or pituitary. The uterus took up less estradiol in androgenized females than in control females at both ages. Estradiol receptor activity was demonstrated in kidney of all animals and in seminal vesicles. At both ages radioactivity levels in the toluene and ethanol extracts of lever and kidney were strikingly higher in males than in the female groups. Reviewing the data, it appears that despite some evidence of differences between groups, males, females and androgenized females all have relatively similar limited-capacity, estradiol-uptake systems in brain and pituitary, as measured under the conditions of the present study.     

Progesterone facilitation of sexual receptivity in rats with neural implantation of estrogen.

The neural substrate for estrogen (E) AND E plus progesterone (P)-induced sexual receptivity was examined in 3 classes of adult rats: (1) adult females ovariectomized for 11 days, and (2) females and (3) males gonadectomized on the day of birth. E responses were found only after implantation in the preoptic or medial hypothalamus; adult females had the largest fraction of responders. The addition of P significantly increased the fraction of adult and neonatally ovariectomized females responding when E implantation was made throughout the hypothalamus from preoptic to mammillary bodies. No E-P synergistic effects were seen in the day-1 gonadectomized males. E-P synergistic effects were found in the amygdala of the adult ovariectomized females. However, midbrain implants of E were ineffective for facilitating E-P-induced receptivity in the 3 classes of animals studied.     

Autoregulation of the gonadotropin-releasing hormone (GnRH) system during puberty: effects of antagonistic versus agonistic GnRH analogs in a female rat model

To address whether gonadotropin-releasing hormone (GnRH) regulates its own expression and the expression of its receptor in the hypothalamus and ovary, we treated five groups of prepubertal/peripubertal female rats from postnatal days 25-36 with either the GnRH agonist triptorelin (TRIP) or the GnRH antagonist cetrorelix (CET), each 10 or 100 microgram/day, or a placebo. We compared their effects regarding pubertal development, serum gonadotropins and the expression of GnRH and GnRH-receptor in the hypothalamus, pituitary, ovary and uterus. Onset of puberty was determined by vaginal opening, and expression levels of GnRH and GnRH-receptor were determined using either quantitative real-time PCR or competitive RT-PCR. Onset of puberty was retarded by both analogs but CET (100 microgram/day) inhibited while TRIP (10 and 100 microgram/day) stimulated serum gonadotropins (P<0.05). The expression of GnRH in the preoptic area did not show significant differences among the treatment groups but ovarian GnRH mRNA levels were significantly stimulated by CET (100 microgram/day). GnRH mRNA could not be detected in the uterus by either real-time PCR or competetive RT-PCR. The GnRH-receptor expression in the hypothalamus (preoptic area and mediobasal hypothalamus) did not vary among any of the groups, whereas in the pituitary GnRH-receptor mRNA levels were stimulated by TRIP (10 microgram/day) but inhibited by CET (100 microgram/day). In contrast, in the ovary GnRH-receptor mRNA levels were inhibited by both TRIP (100 microgram/day) and CET (100 microgram/day). Interestingly, the GnRH-receptor was even expressed in the uterus where it was strongly stimulated by both CET and TRIP in a dose-related manner. This shows that in addition to their different pituitary effects, the GnRH analogs cetrorelix and triptorelin exert different actions at the hypothalamic, ovarian and uterine level. This study also demonstrates an organ-specific regulation of GnRH and GnRH-receptor gene expression which is likely part of a local autoregulatory system. We conclude that the ovarian and uterine effects of GnRH analogs must be considered in addition to their known pituitary effects when deciding which GnRH analog is most suitable for treating precocious puberty.     

A daily neural signal for luteinizing hormone release in the untreated ovariectomized rat: changes in gonadotropin-releasing hormone content of the preoptic area and hypothalamus throughout the day.

Gonadotropin-releasing hormone (GnRH) content of preoptic areas (POA) and hypothalami, and serum gonadotropins of rats ovariectomized six weeks earlier were measured throughout the day in two experiments. In the first, rats were decapitated at 2 h intervals between 0800 and 1800 h. The entire preoptic-hypothalamic region was removed and extracted for radioimmunoassay (RIA) of GnRH. Serum gonadotropins measured by RIA were highly variable but mean concentrations were not significantly different throughout the day. However, preoptic hypothalamic content of GnRH declined markedly between 1000 and 1200 h. In the second experiment, 75 rats were divided into three groups and were untreated or were implanted sc with empty Silastic capsules or capsules containing estradiol-17beta (E2). Two days later, groups of five rats from each of the three treatment groups were decapitated at 0800, 1100, 1400, 1700 and 2000 h. The preoptic area was separated from the hypothalamus by a transverse cut at the caudal aspect of the optic chiasm. POA and hypothalamic content of GnRH correlated well (r=0.74, P less than 0.001, n=75). Two-way analysis of variance failed to reveal any effect of treatment on the GnRH content in either the POA or hypothalamus. GnRH content of both regions decreased significantly between 1100 and 1700h regardless of whether E2 was administered. In striking contrast, gonadotropin surges occurred in the late afternoon only in the E2-treated rats. Serum GnRH was undetectable (less than 5 pg/ml) in all groups of animals. These experiments demonstrate that in the untreated ovariectomized rat GnRH content of the POA and hypothalamus decreases during the early afternoon. This study supports the concent of a daily neural signal for LH release and that E2 is necessary for expression of the daily LH surge in the ovariectomized rat.     

Blockage of N-methyl-D-aspartate receptors decreases testosterone levels and enhances postnatal neuronal apoptosis in the preoptic area of male rats

Sexual dimorphism has been found in the preoptic area of the hypothalamus (POA), a major site of glutamate actions via N-methyl-D-aspartate (NMDA) receptors. The sexually dimorphic nucleus of the preoptic area (SDN-POA) of male rats exhibits about seven-fold greater nuclear volume than that of females. A naturally occurring neonatal neuronal apoptosis, that can be prevented by testosterone, may contribute to this sexual difference in SDN-POA nuclear volume. Since activation of NMDA receptors in the POA induces GnRH secretion, it may be involved in both elevation of serum testosterone and prevention of neuronal death in the SDN-POA. In the present study, protein expression of NMDA receptors in the POA of male and female fetuses was quantified on the day preceding the fetal testosterone peak (embryonic day 16; ED 16). Rats were then distributed in four groups: (1) untreated males, (2) untreated females, (3) males pretreated with MK-801 (a noncompetitive NMDA receptor antagonist), and (4) females pretreated with MK-801. Serum levels of testosterone were estimated on the afternoon of ED 18. Expression of Bcl-2 and Bax, as well as neuronal apoptosis in SDN-POA, were observed on postnatal day 8. The results showed that (1) expression of NMDA receptors in the POA of male fetuses was higher than that of females on ED 16; (2) levels of testosterone were lower in MK-801 pretreated male fetuses than in intact males on ED 18; (3) expression of Bcl-2 in the POA of MK-801 pretreated male rats was significantly less than that of control males; (4) the apoptotic incidence in the SDN-POA of MK-801 pretreated male rats was significantly greater than in control males, while there was no significant difference in apoptotic incidence in the SDN-POA between MK-801 pretreated and intact females. These results suggest that the NMDA receptor is highly expressed in prenatal male fetuses, and that it might play an important role in the elevation of testosterone levels. Moreover, activation of NMDA receptors may protect SDN-POA neurons from naturally occurring neuronal death, by modulating testosterone and/or Bcl-2 expression.     

The central effect of beta-endorphin and naloxone on the expression of GnRH Gene and GnRH receptor (GnRH-R) gene in the hypothalamus, and on GnRH-R gene in the anterior pituitary gland in follicular phase ewes.

The effect of prolonged intermittent infusion of beta-endorphin or naloxone into the third cerebral ventricle in ewes during the follicular phase of the estrous cycle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined by Real time-PCR. Activation of micro opioid receptors decreased GnRH mRNA levels in the hypothalamus and led to complex changes in GnRH-R mRNA: an increase of GnRH-R mRNA in the preoptic area, no change in the anterior hypothalamus and decrease in the ventromedial hypothalamus and stalk/median eminence. In beta-endorphin treated ewes the levels of GnRH-R mRNA in the anterior pituitary gland also decreased significantly. These complex changes in the levels of GnRH mRNA and GnRH-R mRNA were reflected in the decrease of LH secretion. Blockade of micro opioid receptors affected neither GnRH mRNA and GnRH-R mRNA nor LH levels secretion. These results indicate that beta-endorphin displays a suppressive effect on the expression of the GnRH gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland, but affects GnRH-R gene expression in a specific manner in the various parts of hypothalamus; altogether these events lead to the decrease in GnRH/LH secretion.     

Ovarian steroids stimulate adenosine triphosphate-sensitive potassium (KATP) channel subunit gene expression and confer responsiveness of the gonadotropin-releasing hormone pulse generator to KATP channel modulation

The ATP-sensitive potassium (K(ATP)) channels couple intracellular metabolism to membrane potential. They are composed of Kir6.x and sulfonylurea receptor (SUR) subunits and are expressed in hypothalamic neurons that project to GnRH neurons. However, their roles in regulating GnRH secretion have not been determined. The present study first tested whether K(ATP) channels regulate pulsatile GnRH secretion, as indirectly reflected by pulsatile LH secretion. Ovariectomized rats received sc capsules containing oil, 17beta-estradiol (E(2)), progesterone (P), or E(2)+P at 24 h before blood sampling. Infusion of the K(ATP) channel blocker tolbutamide into the third ventricle resulted in increased LH pulse frequency in animals treated with E(2)+P but was without effect in all other groups. Coinfusion of tulbutamide and the K(ATP) channel opener diazoxide blocked this effect, whereas diazoxide alone suppressed LH. Effects of steroids on Kir6.2 and SUR1 mRNA expression were then evaluated. After 24hr treatment, E(2)+P produced a modest but significant increase in Kir6.2 expression in the preoptic area (POA), which was reversed by P receptor antagonism with RU486. Neither SUR1 in the POA nor both subunits in the mediobasal hypothalamus were altered by any steroid treatment. After 8 d treatment, Kir6.2 mRNA levels were again enhanced by E(2)+P but to a greater extent in the POA. Our findings demonstrate that 1) blockade of preoptic/hypothalamic K(ATP) channels produces an acceleration of the GnRH pulse generator in a steroid-dependent manner and 2) E(2)+P stimulate Kir6.2 gene expression in the POA. These observations are consistent with the hypothesis that the negative feedback actions of ovarian steroids on the GnRH pulse generator are mediated, in part, by their ability to up-regulate K(ATP) channel subunit expression in the POA.