Role of the progesterone receptor in restrained glutamic acid decarboxylase gene expression in the hypothalamus during the preovulatory luteinizing hormone surge

Previous work by our laboratory demonstrated that activation of the progesterone receptor through exogenous administration of progesterone suppressed glutamic acid decarboxylase-67 (GAD(67)) mRNA in the hypothalamus of the estrogen-primed ovariectomized rat. Since GAD(67) is the major synthetic enzyme for the inhibitory transmitter, gamma-aminobutyric acid, the finding raised the possibility that the endogenous activation of the progesterone receptor may act to restrain GAD(67) expression during the natural preovulatory gonadotropin surge during proestrus in the rat, thereby allowing GnRH secretion and the resultant LH surge. To test this hypothesis, the progesterone receptor antagonist, RU486, was administered to regularly cycling proestrous rats and the effect on GAD(67) and GAD(65) mRNA levels in the preoptic area (POA) and medial basal hypothalamus (MBH) was examined. Serum luteinizing hormone (LH) levels were also examined in order to identify correlations between changes in POA and MBH GAD levels and production of the LH surge. GAD(67) mRNA levels in the POA were increased in the cycling rat during proestrus at 18.00 h at the peak and just preceding the termination of the LH surge. There was no change in GAD(67) mRNA levels in the MBH, and GAD(65) expression was also unchanged during proestrus in the POA and MBH. Treatment with the antiprogestin RU486 resulted in an increase in GAD(67) mRNA levels at 12.00 and 14.00 h in the POA, and in the MBH at 14.00, 16.00, and 18.00 h during proestrus, effects which preceded and correlated with the attenuated LH surge in RU486-treated rats at 18.00 h. GAD(65) mRNA levels were also elevated by RU486 at 14.00 and 16.00 h in the POA, and at 14.00 h in the MBH during proestrus. These findings suggest that the progesterone receptor plays a role in restraining GAD expression in the hypothalamus during proestrus, and that this effect may be important for the production of the GnRH and LH surge.     

Evidence that neuronal nitric oxide synthase but not heme oxygenase increases in the hypothalamus on proestrus afternoon

Nitric oxide (NO) has been implicated in the control of the proestrus luteinizing hormone (LH) surge in the rat but to date no studies have attempted to measure neuronal nitric oxide synthase (nNOS) or NO production on proestrus in the hypothalamus in order to determine if endogenous NO is increased on proestrus afternoon to activate gonadotropin-releasing hormone (GnRH) neurons. To address this deficit in our knowledge, we measured nNOS mRNA and protein levels as well as NOS activity levels in rat preoptic area (POA) and medial basal hypothalamus (MBH) fragments at 12.00, 14.00, 16.00, and 18.00 h of proestrus. Serum LH levels were also assessed to determine whether NOS changes correlate to the LH surge. To determine the specificity of observed changes we also measured mRNA levels for the enzyme heme oxygenase-2, which is responsible for production of another putative gaseous transmitter, carbon monoxide. In all studies a metestrus 12.00 h control group was included since steroid and LH levels would be basal at this time as compared to proestrus. The results revealed that nNOS mRNA and protein levels, as well as NOS activity did not change significantly in the MBH on proestrus. In contrast, nNOS mRNA levels were significantly elevated in the POA at proestrus 12.00 and 14.00 h, as compared to metestrus 12.00 h. Likewise, at the protein and activity level, nNOS protein levels in the POA were significantly elevated on proestrus at 14.00 and 16.00 h, with NOS activity significantly increased at 16.00 h on proestrus. The elevation of nNOS protein and activity levels in the POA occurred at the time of initiation of the LH surge. The elevation of nNOS was specific as mRNA levels for the CO-synthetic enzyme heme oxygenase-2 did not change significantly on proestrus in the POA or MBH. As a whole, the current studies provide new evidence that nNOS is elevated in the POA on proestrus, and thus could play a role in the activation of GnRH neurons to produce the preovulatory LH surge.     

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.     

Selective regulation of glutamic decarboxylase isoform 65, but not isoform 67, in the bed nucleus of the stria terminalis and the preoptic area of the ewe brain across the estrous cycle.

gamma-Aminobutyric acid neurons in the preoptic area (POA) of the brain may regulate GnRH neurons. The level of expression of two isoforms (65 and 67) of glutamic acid decarboxylase (GAD) in the ewe brain was determined across the estrous cycle by in situ hybridization. GAD mRNA expression (cell number and silver grains/cell) was examined in the subdivisions of the bed nucleus of stria terminalis (BnST), in the diagonal band of Broca, and the POA. The number of cells expressing GAD(65) and GAD(67) mRNA did not change across the cycle. Within the rostro-dorsal BnST, the number of silver grains/cell for GAD(65) mRNA was lower in the follicular phase than the luteal phase or at estrus. In the rostro-lateral division, expression was lower in the follicular phase. In the POA, the number of silver grains/cell for GAD(65) mRNA was lower at estrus than during the luteal phase. The number of silver grains/cell for GAD(67) mRNA did not change across the estrous cycle. GAD(65) is thought to be the active enzyme during periods of high demand of GABA and our results are consistent with the GABA neurons of BnST being most active during the luteal phase of the estrous cycle.     

Effects of age on GABA turnover rates in specific hypothalamic areas in female rats.

During aging in female rats the estrous cycle ceases and the animals develop phases of constant estrous (CE) or constant diestrous (CD) prior to the irreversible transition into anestrous. In young rats, gamma-aminobutyric acid (GABA) is of pivotal importance for the release of GnRH. In the medial-preoptic area (MPO) where the majority of the GnRH perikarya are located in the rat, GABA release decreases at the time of the preovulatory LH surge. The suprachiasmatic nucleus (SCN) contains numerous GABA neurons. Neurochemical signals from this hypothalamic nucleus provide temporal information to GnRH neurons and thereby influence the preovulatory LH surge and the length of estrous cycles. To investigate aging-related changes of the activity of hypothalamic GABAergic neurons, we determined GABA turnover rates in various hypothalamic nuclei of CE and CD rats and compared them to those determined in young estrous (E) or diestrous rats (D1). In old female rats, GABA activity in the MPO was significantly decreased compared to E and D1 rats. A selective increase of GABA turnover rates was observed in the SCN of CE animals. No age-related changes were observed in the other examined brain areas. These data provide the first evidence for alterations in GABAergic activity in specific hypothalamic areas that depend on age and reproductive status. These may cause changes in ability to induce preovulatory LH surges and to maintain regular estrous cyclicity.     

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.     

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.     

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.     

A subset of gonadotropin-releasing hormone neurons in the ovine medial basal hypothalamus is activated during increased pulsatile luteinizing hormone secretion

GnRH neurons active in the preovulatory LH surge have been identified in several species using the early intermediate gene product, Fos, but the GnRH neurons active during episodic LH secretion remain unknown. In this study, we have used Fos and Fos-related antigens (FRA) to determine whether a subset of GnRH neurons is active when pulsatile LH secretion is acutely stimulated in sheep. In experiment 1, episodic LH secretion was stimulated in five of six ewes by injection of an opioid antagonist to luteal phase ewes. These five ewes had a 6-fold increase in the percentage of GnRH neurons in the medial basal hypothalamus (MBH) expressing Fos/FRA, compared with control ewes that had no LH pulses before death. Fos/FRA expression was not increased in GnRH neurons found in any other area. In experiment 2, episodic LH secretion was induced in rams by introduction of estrous ewes. This treatment increased Fos/FRA expression in MBH GnRH neurons approximately 10-fold compared with control rams. Again, this increase in Fos/FRA expression in GnRH neurons was limited to the MBH. This selective activation of MBH GnRH neurons could reflect the preferential inhibition of these perikarya by endogenous opioid peptides. It also raises the possibility that a subset of GnRH neurons in the MBH may be responsible for episodic GnRH secretion in sheep.     

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.     

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.     

In vitro gonadotropin-releasing hormone release from hypothalamic tissues of ovariectomized estrogen-treated cynomolgus macaques

The effects of in vivo 17 beta-estradiol (E2) treatment on in vitro GnRH release and serum LH levels were studied to determine the loci of E2 feedback actions and to examine the hypothalamic mechanisms by which this steroid may regulate LH secretion in monkeys. Ovariectomized cynomolgus macaques received sc Silastic capsule implants containing E2 and were killed 12, 36, 42, or 48 h later. At least one control (CTL) animal received a blank implant and was killed concurrently with each E2-treated monkey. Three untreated animals were used in validation experiments. Before death, each animal was anesthetized with ketamine (15 mg/kg, im), and blood samples were drawn for subsequent LH analysis by Leydig cell bioassay. A diencephalic tissue block was obtained at autopsy and immediately immersed in Krebs-Ringer-phosphate medium (KRP). Mediobasal hypothalamic (MBH) and anterior hypothalamic/preoptic (AH/POA) fragments were quickly dissected from the block and placed in separate superfusion chambers maintained at 37 C. Tissues were superfused at 50 microliter/min with KRP, and 10-min fractions were collected, acidified, and stored at -20 C for subsequent GnRH RIA. Basal immunoreactive GnRH (IR-GnRH) release was measurable from MBH (0.367 +/- 0.063 pg/min) and AH/POA (0.176 +/- 0.065 pg/min) fragments from CTL monkeys. In validation experiments, IR-GnRH release was increased 3- to 7-fold by superfusion with 60 mM K+-KRP only in the presence of Ca+2. Superfusate IR-GnRH coeluted with synthetic GnRH from a Sephadex G-25 chromatographic column, and superfusate and tissue extract GnRH showed appropriate LH-releasing capacities, as determined by rat pituitary cell culture assay. IR-GnRH release rates from MBH or AH/POA tissues varied as a function of in vivo estrogen treatment. GnRH release from both tissues was increased in the E2-treated group killed at 12 h when LH levels were suppressed. Thirty-six hours after E2 treatment, in vitro GnRH release was not significantly different from CTL values. GnRH release rates from MBH and AH/POA tissues obtained 42 h after E2 treatment were significantly greater than CTL release rates (P less than 0.01). This increased in vitro GnRH release at 42 h occurred during the apparent rising phase of the LH surge. Elevated GnRH release was not sustained at 48 h, when surge levels of LH were apparent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of hypothalamic gonadotropin-releasing hormone and neuropeptide Y concentrations by progesterone and corticosteroids in immature rats: correlation with luteinizing hormone and follicle-stimulating hormone release.

In a previous study, we demonstrated that progesterone (P4) and the synthetic glucocorticoid triamcinolone acetonide (TA), but not cortisol, could induce LH and FSH release in estrogen-primed ovariectomized immature rats. Therefore, the purpose of this study was to determine if the stimulatory effect of P4 and TA on LH and FSH release were associated with changes in GnRH or NPY concentrations in the medial basal hypothalamus (MBH) or preoptic area (POA). Ovariectomized immature rats primed with estradiol at 27 and 28 days received either vehicle, P4, TA or cortisol (1 mg/kg BW) at 9.00 h on day 29. Animals were killed at 9.30, 10.00, 12.00 and 13.00 h on day 29 for serum LH and FSH measurements, and the MBH and POA were dissected and analyzed for GnRH and NPY concentrations via RIAs. P4- and TA-treated animals showed significantly elevated serum LH and FSH levels from 13.00 h to 15.00 h. Cortisol was without effect. P4 significantly increased MBH GnRH and NPY concentrations at 12.00 h followed by a significant fall at 13.00 h. P4 modulated POA GnRH and NPY concentrations in a fashion similar to that seen in the MBH, except POA NPY concentrations did not fall at 13.00 h after the elevation at 12.00 h. TA had no significant effect on MBH GnRH and NPY levels at 12.00 h compared to the values at 9.30 h and 10.00 h but, as with P4, there was a significant fall in MBH GnRH and NPY levels at 13.00 h. TA had no significant effect on POA GnRH and NPY concentrations at any time point studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential gene expression response to gonadal hormones by preoptic regulatory factors-1 and -2 in the female rat brain

Steroids and neuropeptides interact in the central nervous system (CNS) to regulate reproductive function and behavior. The preoptic regulatory factors, PORF-1 and PORF-2, are unique neuropeptides for which roles in gender-related brain development and function have been proposed. PORF-1 and PORF-2 expression in rat brain are age, region and gender dependent, and castration or hypophysectomy alter the metabolism of the PORF-1 and PORF-2 mRNAs in male rat brain and testes. If these two peptides have a role in gender-dependent brain function, then gonadal steroids might well affect their expression. The present study was designed to investigate the response of the PORF-1 and PORF-2 mRNAs to sex steroids in the female rat brain and to compare this response to that of two peptides whose roles in the neuroendocrinology of reproduction are well established, gonadotropin-releasing hormone (GnRH) and neuropeptide Y (NPY). Rats were ovariectomized and treated with placebo, estradiol (E2), progesterone (P4) or a combination of the two (E2/P4) and NPY, PORF-2, GnRH and PORF-1 mRNAs were quantified by nuclease protection assays. PORF-1, PORF-2 and GnRH mRNAs were also measured in intact rats during estrus and proestrus. Responses were compared in the preoptic anterior hypothalamus (POA), medial basal hypothalamus (MBH), cerebral cortex (CC) and hippocampus (HIPP). Expression of PORF-1 and PORF-2 was also confirmed in the female rat hypothalamus by in situ hybridization analysis. PORF-1 and PORF-2 mRNAs were detected in the adult female rat brain by both in situ hybridization and ribonuclease protection analyses. In situ hybridization analysis demonstrated that PORF-1 and PORF-2 mRNAs are expressed in hypothalamic neurons. RNase protection analysis showed that PORF-1, PORF-2 and NPY mRNAs were present in all four brain regions examined while GnRH expression was detected only in the MBH and POA. Estradiol alone upregulated expression of the PORF-1 and PORF-2 mRNAs in the ovariectomized rat in the POA and HIPP, and of NPY mRNA in the MBH and HIPP. Progesterone alone had a stimulatory effect on NPY mRNA in the MBH and HIPP. Treatment with a combination of E2/P4 downregulated PORF-2 mRNA in the POA as well as PORF-1, PORF-2 and NPY mRNAs in the CC. In contrast, E2/P4 upregulated the PORF-2 and NPY mRNAs in the HIPP and NPY mRNA in the MBH. In the cycling rat, PORF-1 mRNA levels were higher during proestrus than estrus in both the MBH and POA, while PORF-2 mRNA levels did not change. In contrast GnRH mRNA was lower in the POA and higher in the MBH during proestrus compared with estrus. Thus, intrinsic factors, most likely both ovarian and neuroendocrine, regulate PORF-1 and GnRH expression in the intact cycling rat CNS in a region-dependent manner. In the ovariectomized rat, PORF-1, PORF-2, NPY and GnRH mRNAs all respond in a region-specific manner to sex steroid treatment. These data support the role of PORF-1 and PORF-2 in gender-dependent brain function in the adult female rat.     

Changes in the pulsatile pattern of luteinizing hormone secretion during the rat estrous cycle

To ascertain whether changes in the pattern of GnRH release from the hypothalmus occur during the 4-day rat estrous cycle, the pattern of LH release was characterized on each day of the estrous cycle, and the results were interpreted in light of the changes in pituitary responsiveness to GnRH previously described by this laboratory to occur during this time. Blood samples were taken from intact, freely moving rats via venous catheters at 6- to 10-min intervals for 3-4 h. LH pulse height and LH interpulse interval were quantified on each day of the cycle, and the transition on the afternoon of proestrus from tonic LH release to the preovulatory LH surge was detailed. The effects on the pattern of LH release during estrus of small doses of GnRH (0.4 ng) and the continuous infusion of the opioid antagonist naloxone were also examined. Plasma LH concentrations (NIAMDD rat LH-RP-1) were determined with a highly sensitive LH RIA. LH pulses were identified using the PULSAR algorithim. The LH interpulse intervals of 46 +/- 2 min on diestrous-1 day, 49 +/- 4 min on diestrous day 2, and 60 +/- 8 min on proestrus immediately before the LH surge were not significantly different. There were no changes immediately preceding the preovulatory LH surge on the afternoon of proestrus in either the LH interpulse interval or the LH pulse height. Instead, the transition from tonic LH secretion to the preovulatory LH surge was found to occur abruptly. These data suggest that an abrupt increase in GnRH secretion during the afternoon of proestrus initiates the dramatic rise in LH concentrations. The pattern of LH secretion during the day of estrus differed significantly from that on the other days of the cycle in that no LH pulses were observed. However, the administration of small pulses of GnRH elicited physiological elevations in LH release. Furthermore, the continuous infusion of naloxone to estrous rats immediately stimulated a pulsatile pattern of LH secretion, with a LH interpulse of 56 +/- 4 min. These data indicate that the absence of LH pulses during estrus may result from a deficit in GnRH release. Similar modifications in GnRH release during the other days of the cycle were inferred from the observed changes in LH pulse heights. The LH pulse height of 21 +/- 3 ng/ml on diestrous day 2 was significantly less than the LH pulse height of 41 +/- 4 ng/ml on diestrous day 1 or 35 +/- 4 ng/ml on proestrus before the surge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decreases in mediobasal hypothalamic and preoptic area opioid ([3H]naloxone) binding are associated with the progesterone-induced luteinizing hormone surge

In view of evidence implicating hypothalamic opioid systems in the control of LH release, we have examined the binding of [3H]naloxone (NAL) to slices of mediobasal hypothalamus (MBH) and preoptic area (POA) during the induction of an afternoon LH surge (1630-1700 h) in estradiol benzoate (EB)-primed ovariectomized (OVX; day 0) rats by treatment with progesterone (P; day 2). Such a surge was invariably accompanied by a decrease from early morning (1000 h) values in the number of NAL-binding sites detectable in the MBH, while the affinity of the binding site was not affected over the course of the day. Time-course studies indicated that P injection at 1000 h on day 2 was followed by a transient midday elevation in the amount of NAL bound to slices of MBH; the binding decreased significantly before the onset of and during the LH surge. A similar diurnal change was not observed in MBH slices of either oil-treated OVX rats (controls) or EB-treated OVX rats, which displayed only a 2-fold increase in LH release in the afternoon. Further studies indicated a similar change in NAL binding to slices of the POA of EBP-treated rats. Since hypothalamic opioid systems inhibit LH release, the decrease in opioid binding to MBH as well as POA slices suggests that P may curtail the existing opioid inhibitory influence in these areas before and during the course of the afternoon LH surge.     

Proenkephalin and opioid mu-receptor mRNA expression in ovine hypothalamus across the estrous cycle

The neural mechanism underlying the preovulatory surge of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) is thought to include reduced opioid inhibition of GnRH secretion (disinhibition). Possible mechanisms for disinhibition include reduced endogenous opioid peptide or receptor mRNA expression. Proenkephalin and opioid mu-receptor mRNA expression were measured by in situ hybridization using 35S-labeled cRNA probes and computer-assisted grain counting in hypothalamic nuclei of ovary-intact ewes (n = 4) killed on day 10 of the luteal phase or 24 or 48 h into the follicular phase. In a second experiment, proenkephalin and mu-receptor mRNA expression were compared in ewes killed on day 10 of the luteal phase or during the preovulatory LH surge. Cells expressing proenkephalin mRNA were more widely distributed in ovine hypothalamus than previously described. In the periventricular nucleus, there was a significant reduction in the grain count per cell and the number of labeled cells during the follicular phase and during the LH surge, as compared to the luteal phase. In the ventromedial hypothalamus, there was a significant reduction in the grain count per cell during the follicular phase and LH surge as compared to the luteal phase, but no change in the number of labeled cells. No differences in proenkephalin mRNA expression were detected in the medial septum, diagonal band of Broca, preoptic area, anterior hypothalamic area or paraventricular nucleus across the estrous cycle. Cells expressing opioid mu-receptor mRNA were also widely distributed. No difference in mu-receptor mRNA expression was detected in the medial septum, diagonal band, medial preoptic area, anterior hypothalamus or bed nucleus of the stria terminalis across the cycle. We conclude that in sheep, proenkephalin gene expression in the periventricular nucleus and ventromedial hypothalamus is reduced during the follicular phase and at the time of the LH surge. This may be part of the neural mechanism underlying the GnRH/LH surge in this species.     

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.     

Correlative changes of the gonadotropin-releasing hormone and gonadotropin-releasing-hormone-associated peptide immunoreactivities in the pituitary portal plasma in female rats

The precursor protein of gonadotropin-releasing hormone (GnRH) contains a 56-amino acid peptide, known as GnRH-associated peptide (GAP), and GnRH. Both of these peptides are localized in the same neurons and coprocessed under varieties of physiological conditions. In the present study, we evaluated whether these two peptides are cosecreted into the pituitary portal blood in female rats under the conditions in which the secretion of hypothalamic GnRH and pituitary luteinizing hormone (LH) are known to be altered. The immunoreactivities of GAP-like peptide (IR-GAP-LI) and GnRH (IR-GnRH) in the portal plasma were 2- to 15-fold higher than those observed in peripheral plasma of female rats. In the pubertal females, the preovulatory LH surge which occurred in the afternoon of the day before vaginal opening (puberty) was found to coincide with surges of IR-GAP-LI and IR-GnRH in the pituitary portal plasma. The surges of IR-GAP-LI and IR-GnRH in portal plasma corresponded with a fall in the preoptic and hypothalamic contents of these peptides. In the adult rats, the levels of IR-GAP-LI and IR-GnRH in portal plasma and LH in peripheral plasma were significantly higher during the afternoon of proestrus than those in the afternoon of diestrus. Ovariectomy increased the portal plasma levels of IR-GAP-LI and GnRH and peripheral plasma levels of LH as compared to the level of these hormones in diestrous females. These results indicate that both GnRH and GAP-LI are cosecreted into pituitary portal blood and that changes in the endocrine environment similarly affect both GnRH and GAP secretion.