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.     

Expression of glutamate receptor subunits in the hypothalamus of the female rat during the afternoon of the proestrous luteinizing hormone surge and effects of antiprogestin treatment and aging

The excitatory transmitter, glutamate has been implicated in the control of reproduction, hormone secretion and neuroendocrine regulation. The present study examined whether the hypothalamic expression of three key ionotropic glutamate receptor subunits (NMDAR1, GluR1 and GluR6) fluctuates significantly on proestrus in the rat, and whether treatment with the antiprogestin, RU486 affected glutamate receptor subunit expression. The studies revealed that NMDAR1, GluR1 and GluR6 mRNA levels in the mediobasal hypothalamus (MBH) and preoptic area (POA) fluctuate little throughout the day of proestrus. However, treatment with the antiprogestin, RU486 induced a significant elevation of GluR6 mRNA levels at 14.00 and 16.00 h on proestrus in the MBH, suggesting that endogenous progesterone (P4) may act to inhibit hypothalamic GluR6 levels. In support of this suggestion, exogenous P4 treatment to estrogen (E2)-primed ovariectomized (ovx) rats significantly suppressed GluR6 mRNA levels in the afternoon (12.00-16.00 h) in the MBH, and at 12.00 h in the POA, which preceded LH surge induction. Likewise, temporal examination of hypothalamic GluR6 protein levels in E2 + P4-treated young and middle-aged ovx rats revealed an early elevation from 12.00 to 14.00 h, which was followed by a fall from 16.00 to 20.00 h. The early elevation of GluR6 protein levels was most pronounced in the POA of the young rat, and this elevation was markedly attenuated in the middle-aged rat. As a whole, the studies suggest that glutamate receptor expression fluctuates little on proestrus in the hypothalamus, but that expression of the kainate GluR6 receptor subunit may be modulated by progesterone and aging.     

FSH- and LH-releasing activity in the hypothalamus of female hamsters during proestrus and estrus: effects of arcuate nucleus lesions

An in vitro bioassay was used to characterize the pattern of follicle-stimulating hormone (FSH)- and luteinizing hormone (LH)-releasing activities in the anterior (AH) and medial basal hypothalamus (MBH) during proestrus (P) and estrus (E) in the hamster. Regularly cycling hamsters were decapitated at 12.00, 16.00, 20.00 and 23.00 h of P and 02.00, 05.00, 08.00, 11.00, 14.00 and 17.00 h of E and sera assayed for FSH and LH. Neutralized extracts of the AH and MBH were incubated with a hemipituitary (HP), which was obtained from estrogen- and progesterone-primed ovariectomized rats. The incubation media were assayed for FSH and LH and the results expressed as nanograms of gonadotropin released per milligram HP. There was a surge of serum FSH and LH at 16.00 h of P and a surge of serum FSH only on E. LH-releasing activity in the AH and MBH declined late on P. This activity in the MBH increased by 08.00 h of E. FSH-releasing activity declined markedly in the MBH early on E when serum levels of FSH, but not LH, were rising. Conversely, low serum levels of FSH and high FSH releasing activity were found in the AH and MBH of animals with a chemically induced lesion of the arcuate nucleus. These data suggest that an FSH-releasing hormone may play an important role in the neuroendocrine control of FSH release.     

Role of proestrous progesterone secretion in suppressing basal pulsatile LH secretion during estrus of the estrous cycle

These studies examined the mechanisms responsible for the paucity of basal LH pulses during estrus. We confirmed our earlier observations that constant infusion of naloxone during estrus results in the immediate appearance of pulsatile LH secretion during estrus, consisting of LH peak heights and LH interpulse intervals that are similar to those observed during other days of the estrous cycle. We then tested whether the proestrous surge of progesterone was responsible for the suppression of pulsatile LH secretion during estrus. Three treatment regimens were used on proestrus to either block progesterone secretion (pentobarbital) or block its action (progesterone antiserum or the progesterone antagonist, RU 486). After treatment at 12.00 h on proestrus, blood samples were collected during estrus every 10 min for 4 h, and the plasma samples were analyzed for the pattern of LH secretion. Treatment with pentobarbital (35 mg/kg at 12:00 h) blocked the proestrous surges of LH and progesterone and resulted in pulsatile LH secretion during estrus. The LH interpulse interval (72 +/- 7 min) was somewhat slower than that observed in the naloxone-infused animals (54 +/- 8 min). Simultaneous treatment with pentobarbital and progesterone at 12:00 h on proestrus completely prevented the appearance of LH pulses during estrus. Treatment with either progesterone antiserum (0.5 ml, i.v.) or RU 486 (1 mg s.c.) resulted in the initiation of pulsatile LH secretion during estrus. In the RU 486-treated animals, LH peak heights and LH interpulse intervals were similar to those observed in naloxone-infused animals and during other days of the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone enhances the surge of luteinizing hormone by increasing the activation of luteinizing hormone-releasing hormone neurons

The ability of progesterone (P) to enhance the surge of LH in the rat is well documented, but whether its primary site of action is on the pituitary or brain is unclear. To determine whether P can alter the activation of LHRH neurons, 1) intact female rats were treated with the P antagonist RU486 (5 mg) at 1230 h on proestrus and killed at specified times during the afternoon and evening for comparison of plasma LH levels and cFos expression in LHRH neurons with untreated proestrous rats. RU486 treatment greatly reduced both the magnitude of the LH surge and the degree of cFos induction (numbers of cells expressing cFos and intensity of cFos staining) in LHRH neurons during proestrus. 2) Ovariectomized (OVX) rats were primed with estradiol benzoate (EB, 1 microgram) and then were treated with EB alone (50 microgram) or EB plus P (5 mg). Treatment with EB without P resulted in significantly lower peak LH levels and a reduced cFos response in LHRH neurons than the EB-P treated rats. These data suggest that the actions of P eventuate in an enhanced activation of LHRH neurons that may be responsible for the increased magnitude of the LH surge.     

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.     

Hypothalamic-pituitary interactions during the periovulatory secretion of follicle-stimulating hormone in the rat

We investigated the importance of anterior afferents to the medial basal hypothalamus (MBH) on the increases in plasma FSH during the periovulatory period in the 4-day cyclic rat. We served the anterior connections to the MBH either at 1200 h on proestrus (before the time of onset of the normal spontaneous LH surge in plasma and the associated first phase of FSH release) or near the end of the LH surge and first phase of FSH release at 2000 h on proestrus (before the onset of the second or selective phase of FSH release). Analyses of FSH and LH in blood collected through indwelling atrial catheters or from the trunk after decapitation showed that anterior deafferentation of the MBH at 1200 h on proestrus blocked the proestrous LH surge, the elevations in plasma FSH during proestrus and estrus, and ovulation. In contrast, when brain surgery was delayed until 2000 h on proestrus, the second phase of FSH release and ovulation occurred. In rats with retrochiasmatic transections made at 1200 h, a constant rate iv infusion of LHRH from 1500-1800 h on proestrus restored the LH surge, both phases of increased plasma FSH, and ovulation. The results suggest that 1) the prevolutory LH surge and the first phase of FSH release are dependent on rostral afferents to the MBH which result in hypothalamic LHRH release and 2) the role of rostral afferents to the MBH in the second phase of FSH release is solely to result in hypothalamic LHRH release during proestrus.     

Vasoactive intestinal peptide inhibits the steroid-induced LH surge in the ovariectomized rat.

The LH surge was induced in ovariectomized rats by sequential treatment with oestradiol benzoate and progesterone. Vasoactive intestinal peptide (VIP) or saline was infused into the third cerebral ventricle from 13.30 to 16.30 h on the afternoon of the anticipated LH surge. Two blood samples were taken by jugular puncture from each animal, one at 12.00 h as a control sample and the other at 16.00, 18.00, 20.00 or 22.00 h. Saline-infused animals showed a normal LH surge, with mean plasma LH concentrations reaching a peak at 18.00 h, declining by 20.00 h and reaching control (12.00 h) levels by 22.00 h. Plasma LH in animals infused with VIP was not significantly higher than control levels at 16.00 or 18.00 h. By 20.00 h, mean LH levels in VIP-infused rats had risen to the levels seen at that time in saline-infused rats, and by 22.00 h LH had returned to control levels in VIP-infused animals. We interpret these findings to mean that VIP inhibits LH secretion during the LH surge. It does not block the surge completely, as pentobarbital during the critical period would have done; nor does VIP appear to affect the timing of the LH surge. Rather, VIP inhibits the increased LH secretion rates of the LH surge only during the period of VIP treatment and for a short time afterward.     

Effects of progesterone on pulsatile luteinizing hormone (LH) secretion and LH subunit messenger ribonucleic acid during lactation in the rat

In ovarian-intact lactating rats, removal of the suckling stimulus leads to restoration of pituitary LH beta mRNA levels and pulsatile LH secretion after 72 h, which correlates with a sharp decrease in plasma progesterone concentrations to basal levels. In contrast, in ovariectomized lactating rats, the increase in pituitary LH function is observed by 24 h after pup removal. To determine if progesterone secretion from the ovary participates in the delayed recovery of LH secretion, we treated lactating rats with the progesterone antagonist RU 486 and determined the effects on the time course of recovery of pulsatile LH secretion and LH subunit mRNA after pup removal and on pituitary responsiveness to GnRH. In ovarian-intact lactating rats treated with RU 486, pulsatile LH secretion was observed in about 40% of the rats within 24 h after pup removal (LH interpulse interval, 43.7 +/- 8.3 min) and in about 90% of the rats within 48 h after pup removal (LH interpulse interval, 46.1 +/- 3.6 min). The mean plasma LH level in the RU 486-treated rats was 10.1 +/- 2.2 ng/ml 24 h after removal of pups (control, less than 5 ng/ml) and had increased to 35.1 +/- 6.4 ng/ml 48 h after pup removal (control, 9.1 +/- 2.5 ng/ml). However, RU 486 treatment had no significant effect on LH mRNA subunit levels. To determine whether progesterone acts at the pituitary to block GnRH stimulation of LH secretion, we tested the effects of RU 486 on LH secretion in response to 2- and 5-ng pulses of GnRH. Pituitary responsiveness was tested 24 h after pup removal. We found that both doses of GnRH were effective in stimulating pulsatile LH secretion, and treatment with RU 486 had no significant effect on this response. We conclude from these studies that progesterone secretion from the ovary contributes to the inhibition of LH secretion that occurs after pup removal, since antagonizing progesterone's action resulted in an earlier restoration of pulsatile LH secretion. The increase in LH secretion occurred in the absence of any significant changes in responsiveness of the pituitary to GnRH stimulation or in LH subunit mRNA levels. Therefore, the primary site of action of progesterone would appear to be at the hypothalamus to suppress pulsatile GnRH secretion.     

Correlation of luteinizing hormone surges with estrogen nuclear and progestin cytosol receptors in the hypothalamus and pituitary gland. I. Estradiol dose response effects

The present studies were designed to answer three questions: (1) how will a progressive increase in serum estradiol (E2) in ovariectomized (OVX) rats affect progesterone (P4)-induced luteinizing hormone (LH) surge concentrations? (2) Can steroid-induced LH surges be correlated with estrogen nuclear receptor (E2Rn) and progestin cytosol receptor (PRc) levels in brain regions known to regulate LH secretion, and (3) do differences in pituitary responsiveness to LHRH in E2- or E2P4-treated OVX rats parallel changes in E2Rn and PRc concentrations in this gland? 1 week after ovariectomy of adult cyclic rats (day 0), Silastic E2 capsules were placed subcutaneously at 09.00 h and produced serum E2 levels of 6-8 (low), 12-19 (medium) and 27-37 (high) pg/ml, respectively. 2 days later (day 2), some rats also received Silastic P4 capsules subcutaneously which elevated serum P4 concentrations to 10-12 ng/ml. In rats with low serum E2, P4 treatment induced peak serum LH levels of 913 ng/ml. When serum E2 was increased to the medium or relatively high physiologic range, P4 treatment resulted in LH surge levels of 4,686 and 5,030 ng/ml. OVX controls and E2-treated OVX rats were sacrificed at 10.00 h on day 2 and E2Rn and PRc were measured concurrently in the preoptic area (POA), mediobasal hypothalamus (MBH), corticomedial amygdala (CMA) and pituitary gland (PIT). Raising serum E2 from OVX levels to the low range significantly increased both E2Rn and PRc in MBH and PIT, but not in the POA or CMA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of estrogen-dependent progesterone receptor in protein kinase C-mediated LH secretion and GnRH self-priming in rat anterior pituitary glands

The aim of the present study was to explore the involvement of pituitary progesterone receptor (PR) in PKC-mediated LH secretion and LHRH self-priming and the role of the estrogen (E) environment. Eight randomly selected hemipituitaries from adult female rats in proestrus or from 2 weeks ovariectomized (OVX) rats were incubated, in the absence of progesterone (P), over 3 h in Dulbecco's modified Eagle's medium (DMEM). In the first experiment, hemipituitaries were incubated continuously with: medium alone, GnRH (10 nM), the PKC stimulator PMA (100 nM), the PKC inhibitor staurosporine (100 nM), the antiprogestin at the receptor RU486 (10 nM), LHRH+staurosporine, GnRH+RU486 or PMA+RU486. In the second experiment, hemipituitaries were incubated, one h apart, with GnRH to determine the GnRH self-priming and this was compared with the priming effect of PMA. Also, the effect of staurosporine and RU486 during the induction period (1st h) on GnRH and PMA priming was evaluated. Medium was aspirated at the end of each h to determine LH accumulation and to evaluate GnRH self-priming. Both GnRH and PMA stimulated LH secretion. Staurosporine and RU486 reduced basal and GnRH-stimulated LH secretion, and RU486 reduced PMA-stimulated LH secretion from proestrus pituitaries. The stimulating effect of GnRH and PMA on LH secretion and the inhibitory action of staurosporine and RU486 on basal or stimulated LH secretion were significantly reduced in OVX-rats. Both GnRH and PMA induced GnRH priming. Staurosporine during the induction h reduced GnRH self-priming while RU486 reduced both GnRH self-potentiation and PMA priming. The magnitude of these inhibitory effects was blunted in OVX-rats. These results showed that PKC signaling pathway in the gonadotrope mediates, at least in part, basal and GnRH-stimulated LH secretion and GnRH self-priming. Also, the results are suggestive of an interaction of PKC signaling pathway with E-dependent PR in a ligand-independent activation manner in the gonadotrope.     

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.     

Immunocytochemical localization of beta endorphin and gonadal steroid regulation of proopiomelanocortin messenger ribonucleic acid in the ewe.

In the ewe, estradiol and progesterone inhibit luteinizing hormone (LH) secretion during the breeding season. Endogenous opioid peptides (EOP) are also inhibitory to LH secretion, and both estrogen and progesterone have been reported to enhance EOP inhibition of LH release. Which EOP are involved in this inhibition is unclear. In this study, we concentrated on beta-endorphin because evidence for its ability to inhibit LH secretion exists in ewes. We first studied the distribution of beta-endorphin-immunoreactive neurons in 4 cycling ewes using immunocytochemistry. Cell bodies were found only within the medial basal hypothalamus (MBH) and were concentrated in arcuate nucleus and mammillary recess of the third ventricle, with a few in the median eminence. Extensive fiber tracts were seen in preoptic area (POA) and median eminence. We next tested the hypothesis that gonadal steroids increase the synthesis of EOP by measuring levels of mRNA for proopiomelanocortin (POMC), the precursor to beta-endorphin. Ovariectomized ewes were treated with no steroids (n = 7) or given subcutaneous Silastic implants containing either estradiol (n = 6) or progesterone (n = 6). After 4 days of treatment, EOP inhibition of LH secretion was measured by determining the LH response to WIN 44,441-3 (WIN), an EOP antagonist. LH pulse frequency and pulse amplitude were determined in blood samples collected at 12-min intervals for 3 h before and after intravenous administration of 12.5 mg WIN. WIN injection increased (p < 0.01) the LH pulse-frequency only in progesterone-treated and pulse amplitude only in estradiol-treated ewes. After blood sampling, the ewes were killed, and POA, MBH, and pituitary gland were removed. Total RNA was extracted from these tissues and dot blotted onto nitrocellulose membranes for hybridization with a DNA probe complementary to the POMC mRNA. The resulting autoradiographs were quantified densitometrically. Levels of POMC mRNA in the MBH were increased (p < 0.01) by both estradiol and progesterone as compared with the no steroid group. There was no detectable POMC mRNA in the POA. These results suggest that estrogen and progesterone enhance EOP inhibition of LH secretion by increasing POMC mRNA levels and thus synthesis of beta-endorphin.     

Temporal changes in the hypothalamic and serum luteinizing hormone-releasing hormone (LH-RH) levels and the circulating ovarian steroids during the rat oestrous cycle.

Cycling female rats were sacrificed at various times during the 4-day oestrous cycle. LH-RH in the medial basal hypothalamus (MBH) and serum LH-RH, LH, oestradiol (Oe2), and progesterone were analyzed by radioimmunoassays. The MBH LH-RH content was lowest at 19.55-20.15 h during pro-oestrus but increased gradually through oestrus and dioestrus I to significantly higher values at noon of dioestrus II, and then decreased precipitously at 18.00 h. Although serum LH levels remained basal from midnight of pro-oestrus through dioestrus II, serum LH-RH levels were significantly elevated at 12.00 and 21.00 h on oestrus (vs. the midnight pro-oestrus levels) and declined between 15.00 and 22.00 h of dioestrus I. In conjunction with high Oe2 titres, LH-RH in the MBH and serum declined initially during pro-oestrus between 14.07-14.45 h and then increased abruptly between 14.55-15.55 h; the pre-ovulatory rise in serum LH was observed from 16.05 h onwards. LH-RH activity in the MBH and serum receded gradually to the early afternoon levels by 19.55-24.00 h while the peak in serum LH was observed at 17.05-18.05 h. These studies suggested that the hypersecretion of the MBH LH-RH (synthesis + release) may be responsible for the pre-ovulatory discharge of LH.     

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.     

Central adrenergic activity in the mediobasal hypothalamus is temporally related to surge outputs of gonadotrophins in oestrogen-stimulated infant female rats

In the mediobasal hypothalamus (MBH) of pro-oestrous rats or acutely ovariectomized oestrogen-treated adults a marked but short-lived increase in adrenergic activity occurs at 16.00 h, 2 h before the oestrogen-dependent surge of gonadotrophins at 18.00 h. In this study oestrogen-stimulated (noon on day 1) 22-day-old female rats were used which are known to produce surge levels of prolactin at 18.00 h on day 2 and surges of both prolactin and LH at 18.00 h on day 3; although similar treatment of 18-day-old animals or oil-treated 22-day-old rats failed to produce these effects. Radioenzymatic assays of adrenaline concentrations and of the activity of its synthesizing enzyme (phenylethanolamine-N-methyl transferase; PNMT, EC 2.1.1.28) in the MBH of oestrogen-treated 22-day-old rats showed significant (P less than 0.05-0.01) increases in both parameters at 16.00 h (i.e. 2 h before surge levels of gonadotrophins) on days 2 and 3 when compared with other times of day. Such effects were not seen in oil-treated 22-day-old animals or in oestrogen-treated 16-day-old rats. Noradrenaline and dopamine concentrations in the MBH of oestrogen-treated 22-day-old rats remained at baseline levels on days 2 and 3 with the exception of noradrenaline at 17.00 h on day 3 when levels appeared higher (P less than 0.05) than at either 15.00 or 16.00 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of gonadectomy and reposition of gonadal steroids on alpha-melanocyte-stimulating hormone concentration in discrete hypothalamic areas during a 24-hour period

Hypothalamic alpha-melanocyte-stimulating hormone (alpha-MSH) was measured by radioimmunoassay in males after orchidectomy and after orchidectomy plus testosterone replacement, and in females after ovariectomy and after ovariectomy plus estradiol or progesterone, or estradiol and progesterone (EP) replacement. Gonadectomy inverted the diurnal rhythm of the alpha-MSH content observed in intact males in the medial basal hypothalamus (MBH) and preoptic hypothalamic area (POA), and produced a notable decrease of alpha-MSH total content in the three regions studied (MBH, POA and dorsolateral hypothalamus, DLH). The addition of testosterone restored the rhythm of the intact males and increased alpha-MSH content in MBH and POA. No diurnal variations in alpha-MSH content were observed in ovariectomized females. A circadian rhythm similar to that of proestrus was observed in MBH after estradiol or EP replacement, and in POA after the addition of any steroid. In DLH the injection of estradiol produced variations through the day, but they are somehow different from those described for proestrus. Treatment with progesterone significantly decreased alpha-MSH content in MBH and DLH, but not in POA. In this region an increase in alpha-MSH content was noticed after EP replacement. We conclude that gonadal steroids can alter the content of hypothalamic alpha-MSH and influence the diurnal variations of the peptide. This may be important in the modulation of several types of behavior or in the neuroendocrine control of gonadotropin release in females, where alpha-MSH seems to modulate the release of luteinizing hormone and prolactin.     

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)     

Changes in alpha-estradiol receptor and progesterone receptor expression in the locus coeruleus and preoptic area throughout the rat estrous cycle

We have previously shown that the locus coeruleus (LC) is essential for triggering surges of LH. Since LC neurons are responsive to estradiol, which induces progesterone receptor (PR) expression, this study aimed to investigate whether LC neurons express the alpha-estradiol receptor (alphaER) and PR as well as comparing such responses to that observed in the preoptic area (POA). Female rats were perfused at 10, 14 and 16 h on each day of the estrous cycle, and a blood sample was collected for estradiol, progesterone and LH measurements. alphaER- and PR immunoreactive (ir) neurons were detected in POA and LC by immunocytochemistry (ICC). Higher plasma estradiol levels were observed on the day of proestrus, when a smaller number of alphaER-ir POA neurons were detected. An increase in the number of alphaER-ir neurons were observed at 16 h of proestrus and estrus. The number of PR-ir neurons increased in POA only at 16 h of proestrus, and remained unchanged during all other days and times. The profile of alphaER-ir and PR-ir neurons in LC changed over the estrous cycle, with a lower expression on metestrus morning and reaching a peak on diestrus afternoon before declining on the day of proestrus. However, on estrus afternoon, alphaER-ir neurons increased, while PR-ir neurons decreased which may be related to the prolactin surge of estrus. These data show that LC neurons express alphaER and PR and seem to be more sensitive to variations in estradiol than POA. Also, the fluctuation in alphaER and PR observed for LC neurons seems to accompany the hormonal events that occur during the estrous cycle. This profile of alphaER and PR expression might be related to the ability of estradiol and progesterone in regulating the activity of LC neurons, which could be associated to the control mechanisms of LH and prolactin release.