Estrogen-primed Ovariectomized Rats Research Articles

Prostaglandin E2 modulates presynaptic regulation of GnRH neurons via EP4 receptors in accordance with estrogen milieu

Prostaglandin E2 (PGE2) promotes gonadotropin secretion by regulating the activity of neurons that release gonadotropin-releasing hormone (GnRH) in the hypothalamus. However, the mechanisms of action of PGE2 at these neurons have yet to be fully explored. We examined the effects of PGE2 on the generation of miniature excitatory postsynaptic currents (mEPSCs) at GnRH neurons as measured by whole-cell, patch-clamp recordings. GnRH neurons were identified in slices prepared from the preoptic areas of female GnRH-EGFP rats. Exposure to PGE2 significantly increased the frequency, but not the amplitude, of the mEPSCs generated on the day of proestrus, but neither frequency nor amplitude was altered on day 1 of diestrus. These data suggest that the action of PGE2 on mEPSC frequency varies depending on the stage of estrous. An estrogen-dependence of PGE2’s action was further supported by the increased frequency, but not amplitude, of mEPSCs generated at GnRH neurons prepared from estrogen-primed ovariectomized rats. Conversely, PGE2 had no effect on mEPSC frequency or amplitude at GnRH neurons in cholesterol-treated rats. Subsequent experiments to identify candidate receptors for PG2E’s action revealed that exposure to a PGE2 receptor 4 (EP4) agonist, but not EP1 or EP2 agonists, mimicked the effects achieved by PGE2 exposure. These effects of mEPSCs could be reversed using an EP4 antagonist, illustrating the specificity of the effect. Collectively, these data demonstrate that PGE2 can alter excitatory synaptic neurotransmission at GnRH neurons via EP4 signaling at presynaptic site(s) in an estrogen-dependent fashion during proestrus.

Involvement of dopamine D2 receptor in the diurnal changes of tuberoinfundibular dopaminergic neuron activity and prolactin secretion in female rats.

BackgroundAn endogenous dopaminergic (DA) tone acting on D3 receptors has been shown to inhibit tuberoinfundibular (TI) DA neuron activity and stimulate prolactin (PRL) surge in the afternoon of estrogen-primed ovariectomized (OVX+E2) rats. Whether D2 receptor (D2R) is also involved in the regulation of TIDA and PRL rhythms was determined in this study.ResultsIntracerebroventricular (icv) injection of PHNO, a D2R agonist, in the morning inhibited TIDA and midbrain DA neurons’ activities, and stimulated PRL secretion. The effects of PHNO were significantly reversed by co-administration of raclopride, a D2R antagonist. A single injection of raclopride at 1200 h significantly reversed the lowered TIDA neuron activity and the increased serum PRL level at 1500 h. Dopamine D2R mRNA expression in medial basal hypothalamus (MBH) exhibited a diurnal rhythm, i.e., low in the morning and high in the afternoon, which was opposite to that of TIDA neuron activity. The D2R rhythm was abolished in OVX+E2 rats kept under constant lighting but not in OVX rats with regular lighting exposures. Pretreatment with an antisense oligodeoxynucleotides (AODN, 10 μg/3 μl/day, icv) against D2R mRNA for 2 days significantly reduced D2R mRNAs in central DA neurons, and reversed both lowered TIDA neuron activity and increased serum PRL level in the afternoon on day 3. A diurnal rhythm of D2R mRNA expression was also observed in midbrain DA neurons and the rhythm was significantly knocked down by the AODN pretreatment.ConclusionsWe conclude that a diurnal change of D2R mRNA expression in MBH may underlie the diurnal rhythms of TIDA neuron activity and PRL secretion in OVX+E2 rats.

Testosterone exposure during the critical period decreases corticotropin-releasing hormone-immunoreactive neurons in the bed nucleus of the stria terminalis of female rats

We previously described sex differences in the number of corticotropin-releasing hormone-immunoreactive (CRH-ir) neurons in the dorsolateral division of the bed nucleus of the stria terminalis (BSTLD). Female rats were found to have more CRH neurons than male rats. We hypothesized that testosterone exposure during the critical period of sexual differentiation of the brain decreased the number of CRH-ir neurons in the hypothalamus, including the BSTLD and preoptic area. In the present study we confirm that testosterone exposure during the neonatal period results in changes to a variety of typical aspects of the female reproductive system, including estrous cyclicity as shown by virginal smear, the positive feedback effects of estrogen alone or combined with progesterone, luteinizing hormone secretions, and estrogen and progesterone-induced Fos expression in gonadotropin-releasing hormone neurons. The number of CRH-ir neurons in the preoptic area did not change, whereas CRH-ir neurons in the BSTLD significantly decreased in estrogen-primed ovariectomized rats exposed to testosterone during the neonatal period. These results suggest that the sexual differentiation of CRH neurons in the BSTLD is a result of testosterone exposure during the critical period and the BSTLD is more fragile than the preoptic area during sexual differentiation. Furthermore, sex differences in CRH in the preoptic area may not be caused by testosterone during this period.

An endogenous dopaminergic tone acting on dopamine D3 receptors may be involved in diurnal changes of tuberoinfundibular dopaminergic neuron activity and prolactin secretion in estrogen-primed ovariectomized rats

The diurnal rhythm of tuberoinfundibular dopaminergic (TIDA) neuron activity, i.e., high in the morning and low in the afternoon, is prerequisite for the afternoon prolactin (PRL) surge in proestrous and estrogen-primed ovariectomized (OVX) female rats. Whether dopamine acts via D(3) receptors in regulating the rhythmic TIDA neuron activity and PRL secretion in estrogen-primed OVX (OVX+E(2)) rats is the focus of this study. Intracerebroventricular (icv) injection of a D(3) receptor agonist, PD128907 (0.1-10 μg/3 μl), in the morning significantly reduced the basal activity of TIDA neurons and increased plasma PRL level. The effects of PD128907 were reversed by co-administration of U99194A, a D(3) receptor antagonist, but not by raclopride, a D(2) receptor antagonist. To determine whether endogenous dopamine acts on D(3) receptors involved in the diurnal changes of the activities, we used both U99194A, a D(3) receptor antagonist, and an antisense oligodeoxynucleotide (ODN) against D(3) receptor mRNA in the study. U99194A (0.1 μg/3 μl, icv) given at 1200 h significantly reversed the lowered TIDA neuron activity and the afternoon PRL surge at 1500 h. Moreover, OVX+E(2) rats pretreated with the antisense ODN (10 μg/3 μl, icv) for 2 days had the same effects as the D(3) receptor antagonist on TIDA neuron activity and the PRL surge. The same treatment with sense ODN had no effect. In conclusion, an endogenous DA tone may act on D(3) receptors to inhibit TIDA neuron activity and in turn stimulate the PRL surge in the afternoon of OVX+E(2) rats.

The CRH-R1 receptor mediates luteinizing hormone, prolactin, corticosterone and progesterone secretion induced by restraint stress in estrogen-primed rats

Acute stress has been shown to modify hypothalamus–pituitary–gonadal (HPG) axis activity. Corticotropin-releasing hormone (CRH), the principal regulator of the hypothalamus–pituitary–adrenal (HPA) axis, has been implicated as a mediator of stress-induced effects on the reproductive axis. The role of the specific CRH receptor subtypes in this response is not completely understood. In the current study, we investigated the role of the CRH-R1 receptor on luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), progesterone (P) and corticosterone (CT) secretion in stress-induced responses under the influence of estrogen (E2). Estrogen-primed ovariectomized rats (estradiol cypionate, 10μg sc) received an i.v. administration of antalarmin (0.1 or 1mg/kg), a selective CRH-R1 antagonist, or vehicle before restraint stress for 40min. Seven blood samples were collected from two experimental groups (one from 10:00h to 14:00h and the other from 10:00h to 18:00h). An increase of plasma LH induced by restraint acute-stress was followed by alteration of the secretion pattern in the estrogen-induced afternoon surge. In a similar manner, we observed a suppression of the afternoon surge in plasma FSH, a delay of E2-induced PRL secretion, and an increase in plasma P and CT. Antalarmin attenuated stress-induce LH increase, decreased CT and P secretion and blocked the stress effects on PRL secretion. These findings suggest that CRH-R1 mediates, at least in part, the restraint stress effects on the HPA, PRL, and reproductive axes.

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.

Fourth ventricular alloxan injection suppresses pulsatile luteinizing hormone release in female rats.

Previous studies have suggested the presence of a glucose-sensing mechanism in the hindbrain that appears to regulate reproductive function as well as feeding behavior. The ependymocytes lining the ventricular wall of the hindbrain showed immunoreactivities to pancreatic glucokinase (GK), a key enzyme for glucose sensing in pancreatic B cells. Our goal in the present study was to test whether the GK-immunopositive ependymocytes in the wall of the fourth cerebroventricle (4V) play a role in regulating gonadal activity. Our approach was to determine the effect of injecting alloxan, a GK inhibitor, into the 4V on pulsatile luteinizing hormone (LH) secretion. Estrogen-primed ovariectomized rats received an injection of alloxan (10 or 20 microg/animal) into the 4V and blood samples were collected every 6 min for 3 h for measurement of blood LH, corticosterone and glucose levels. Pulsatile LH secretion was suppressed after alloxan injection and all pulse parameters were significantly (P<0.05) inhibited by 20 microg alloxan. Plasma corticosterone levels were increased significantly (P<0.05) by 20 microg alloxan, suggesting that LH pulse suppression by alloxan may be at least partly mediated by activation of the hypothalamo-pituitary-adrenal axis. The present results suggest that acute suppression of GK activity in the hindbrain inhibits pulsatile LH secretion in female rats, and supports the idea that GK-immunopositive ependymocytes may sense glucose levels in the cerebrospinal fluid and play a role in regulation of LH secretion.

Effects of glucose and related substrates on the recovery of the electrical activity of gonadotropin-releasing hormone pulse generator which is decreased by insulin-induced hypoglycemia in the estrogen-primed ovariectomized rat

We investigated the effect of glucose and its related substrates on the recovery of pulsatile luteinizing hormone (LH) secretion which was suppressed by insulin in estrogen-primed ovariectomized rats. We also examined the effect of glucose on the electrical activity of the gonadotropin-releasing hormone (GnRH) pulse generator which was suppressed by insulin. The intravenous (i.v.) injection of insulin (5 units/rat) suppressed the pulsatile LH secretion for 3 h in estrogen-primed ovariectomized rats. This suppressive effect of insulin on the LH secretion was rapidly reversed by the i.v. injection of glucose and mannose but not by the injection of lactate and saline. Fructose could recover the LH secretion suppressed by insulin, but took a longer time than glucose did. By monitoring the electrical activity of the GnRH pulse generator, we found that i.v. injection of insulin suppressed the pulsatile LH secretion by decreasing the activity of the GnRH pulse generator. Again, the i.v. injection of glucose, but not saline, immediately recovered the decrease in the electrical activity of the GnRH pulse generator. Fructose could recover the activity of the GnRH pulse generator, but it took a longer time than glucose did. We suggest that glucose availability, but not simply a metabolic state such as the ATP level, is an essential factor for maintaining the electrical activity of the GnRH pulse generator which is responsible for pulsatile LH secretion.

Circadian change of dopaminergic neuron activity: effects of constant light and melatonin.

Twenty-four hour profiles of tuberoinfundibular (TI), nigrostriatal and mesolimbic dopaminergic (DA) neuronal activities were assessed in estrogen-primed ovariectomized rats using DOPAC and DOPA levels in terminal regions of DA neurons. Significant decreases in DOPAC and DOPA levels in the median eminence were observed at 17.00 and 21.00 h, which corresponded with higher serum prolactin levels. DOPAC or DOPA levels in the striatum and nucleus accumbens were, however, significantly higher during the dark (21.00-05.00 h) phase. In rats kept under conditions of continuous light, no late afternoon decline in median eminence DOPA was observed; this decline could be reinstated by repeated injections of melatonin between 18.00 and 01.30 h for 3 days. In summary, circadian rhythms of central DA neurons were shown and melatonin may play an entraining role.

Regulation of Gonadotropin-Releasing Hormone and Luteinizing Hormone Secretion by AMPA Receptors

Recent work has demonstrated that glutamate functions as a major transmitter involved in the regulation of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in female animals, although the specific receptors and mechanisms mediating its effects have not been completely worked out. The purpose of the present study, therefore, was to examine the role of the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)-type glutamate receptor in the control of GnRH and LH secretion in female animals. Toward this end, the dose- and steroid-dependent effects of AMPA on GnRH and LH secretion in female rats were investigated using both in vitro and in vivo approaches, and the role of AMPA receptors in the production of the steroid-induced LH surge was also assessed. The results of the study revealed that central administration of AMPA resulted in a stimulation of LH release in the estrogen-primed ovariectomized adult rat. AMPA was also found to potently stimulate GnRH release in vitro from mediobasal hypothalamic (MBH) fragments obtained from estrogen-primed ovariectomized adult rats, and this effect was blocked by the selective AMPA receptor antagonist, NBQX. The mechanism of action of AMPA appeared to differ from that of N-methyl-D-aspartate (NMDA) as AMPA, in contrast to NMDA, failed to elevate nitric oxide synthase activity in the hypothalamus. The effect of AMPA on LH secretion was demonstrated to be steroid dependent, as central administration of AMPA stimulated LH release in estrogen-primed ovariectomized rats, but was inhibitory to LH release in non-estrogen-primed ovariectomized rats. In contrast, AMPA stimulated GnRH release equally well from MBH fragments obtained from estrogen-primed or non-estrogen-primed ovariectomized rats. The different effects of AMPA on LH release may be due to different pituitary sensitivities between the two models, or alternatively, AMPA may stimulate the release of LH inhibitory factors in the ovariectomized rat in the absence of estrogen. Finally, a physiological role for AMPA receptors in the production of the steroid-induced LH surge was suggested, based on the finding that central administration of the selective AMPA receptor antagonist, NBQX, into the third cerebroventricle significantly attenuated the steroid-induced LH surge in the ovariectomized adult female rat.

Evidence that brain nitric oxide synthase is the major nitric oxide synthase isoform in the hypothalamus of the adult female rat and that nitric oxide potently regulates hypothalamic cGMP levels.

Recent studies suggest that nitric oxide (NO) may function as a neurotransmitter in the hypothalamus. In order to provide further evidence supporting this contention, we examined: (1) whether the hypothalamus displays significant NO synthase (NOS) activity and whether the activity is inhibited by an NOS inhibitor, (2) whether the different NOS isoforms [brain (b)-NOS, endothelial (e)-NOS and macrophage (m)-NOS] are expressed in the various nuclei of the hypothalamus of the random cycling adult female rat, (3) whether the NO donor molecule, sodium nitroprusside (SNP), regulates the heme-containing enzyme, guanylate cyclase in the preoptic area and medial basal hypothalamus of the random cycling adult female rat as well as the ovariectomized steroid (estradiol-17 beta)-treated rat. The results of the study showed that the preoptic area (POA) and medial basal hypothalamus (MBH) of the adult female rat displays significant NOS activity which can be dose-dependently inhibited by an NOS inhibitor. All three NOS isoform mRNA transcripts were present in the hypothalamus, with the order of expression being b-NOS > e-NOS > m-NOS. Immunohistochemical localization using monoclonal antibodies to the specific NOS isoform proteins revealed that b-NOS represented the major form of NOS in the hypothalamus based on density and distribution of immunostaining. b-NOS immunostaining was especially dense in the organum vasculosum laminae terminalis (OVLT), medial preoptic area (MPOA), supraoptic nucleus, and moderately dense in the arcuate nucleus/median eminence. The pattern and density of b-NOS staining closely mirrored our previously reported pattern of NADPH-diaphorase staining in the hypothalamus, and a polyclonal antibody to b-NOS yielded a similar staining pattern as that observed for the monoclonal antibody. In contrast to the dense staining observed for b-NOS in the hypothalamus, we observed no specific staining for m-NOS in the hypothalamus. e-NOS immunostaining, on the other hand, was present in the hypothalamus, but to a much lesser extent than b-NOS. Light e-NOS staining was observed in the OVLT, MPOA, supraoptic nucleus and arcuate nucleus/median eminence. That NO can regulate guanylate cyclase as a potential mediator of its effects was demonstrated using SNP which dose-dependently elevated cGMP levels in the POA and MBH of random cycling rats and estrogen-primed ovariectomized rats. The effect of SNP was due to its NO donor ability as it was blocked by the NO scavenger molecule, hemoglobin. Interestingly, hemoglobin alone caused a 50-60% reduction in basal cGMP levels, suggesting that endogenously produced NO regulates basal guanylate cyclase activity. Taken as a whole, the present study demonstrates that b-NOS is the major NOS isoform in the hypothalamus and it also provides evidence that cGMP may be a mediator of NO effects in the female hypothalamus as evidenced by the potent ability of SNP to elevate cGMP levels in the POA and MBH.

Bombesin acts in the suprachiasmatic nucleus to affect circadian changes in tuberoinfundibular dopaminergic neuron activity and prolactin secretion

Central administration of bombesin has been shown to block the afternoon PRL surge. A circadian change in the activity of tuberoinfundibular dopaminergic (TIDA) neurons that coincides with the onset of the PRL surge has also been found. Whether bombesin acts on the TIDA neuron to affect the PRL surge and where bombesin acts were the focuses of this study. Bombesin (0.75 microgram/3 microliters.rat) was first injected into the lateral cerebroventricle of estrogen-primed ovariectomized rats at 1200 h through preimplanted cannulas. Both the PRL surge and the circadian changes in TIDA neuron activity (using both 3,4-dihydroxyphenylacetic acid concentration and 3,4-dihydroxyphenylalanine accumulation in the median eminence as indexes) were blocked by the treatment. In suprachiasmatic nucleus-lesioned rats, however, both rhythms were absent, and the injection of bombesin exhibited no significant effect. Using microinjection of bombesin (50 ng/0.2 microliter) bilaterally into either the suprachiasmatic or arcuate nuclei at 1200 h, only the former injection effectively blocked both TIDA neuron activity and the PRL surge. We conclude that bombesin may act on the rhythm generation center, the suprachiasmatic nucleus, to exert its effect to disrupt TIDA neuron activity and the PRL surge.

Bombesin acts in the suprachiasmatic nucleus to affect circadian changes in tuberoinfundibular dopaminergic neuron activity and prolactin secretion.

Central administration of bombesin has been shown to block the afternoon PRL surge. A circadian change in the activity of tuberoinfundibular dopaminergic (TIDA) neurons that coincides with the onset of the PRL surge has also been found. Whether bombesin acts on the TIDA neuron to affect the PRL surge and where bombesin acts were the focuses of this study. Bombesin (0.75 microgram/3 microliters.rat) was first injected into the lateral cerebroventricle of estrogen-primed ovariectomized rats at 1200 h through preimplanted cannulas. Both the PRL surge and the circadian changes in TIDA neuron activity (using both 3,4-dihydroxyphenylacetic acid concentration and 3,4-dihydroxyphenylalanine accumulation in the median eminence as indexes) were blocked by the treatment. In suprachiasmatic nucleus-lesioned rats, however, both rhythms were absent, and the injection of bombesin exhibited no significant effect. Using microinjection of bombesin (50 ng/0.2 microliter) bilaterally into either the suprachiasmatic or arcuate nuclei at 1200 h, only the former injection effectively blocked both TIDA neuron activity and the PRL surge. We conclude that bombesin may act on the rhythm generation center, the suprachiasmatic nucleus, to exert its effect to disrupt TIDA neuron activity and the PRL surge.

Acute stress suppresses the N-methyl-D-aspartate-induced luteinizing hormone release in the ovariectomized estrogen-primed rat.

The effects of N-methyl-D-aspartate (NMDA) and luteinizing hormone releasing hormone (LH-RH) on luteinizing hormone (LH) secretion were examined in ovariectomized estrogen-primed rats under nonstressed and acutely stressed conditions. The basal LH levels were significantly elevated 15 min after the onset of acute immobilization stress, but were not altered in emotionally stressed or nonstressed rats. Intravenous injections of 10 and 40 mg/kg NMDA significantly elevated serum LH levels by 161 and 212%, respectively, from baseline within 10 min in nonstressed animals. However, the NMDA-induced LH release was significantly reduced when tested 30 min after the onset of acute immobilization stress. Acute emotional stress, which did not affect the baseline LH, also suppressed the LH release response to NMDA, suggesting that the reduced LH responses to NMDA in stressed animals was not due to the elevated baseline level. Pituitary LH release responses to LH-RH were not affected by acute immobilization. We conclude from these results: (1) acute immobilization stress exerts both stimulatory and inhibitory effects on LH release, while acute emotional stress has only an inhibitory effect in estrogen-primed ovariectomized rats; (2) this inhibition occurs at the suprapituitary level, and (3) it involves a suppression of the responsiveness of the hypothalamic LH-RH neuronal system to the excitatory amino acid input.

Evidence that nitric oxide may mediate the ovarian steroid-induced luteinizing hormone surge: involvement of excitatory amino acids.

The involvement of excitatory N-methyl-D-aspartate (NMDA) receptors in the hypothalamic control of pituitary LH secretion is well recognized. Recent evidence shows that nitric oxide (NO), a free radical gas, may act as neurotransmitter in the brain, and its efflux is stimulated by activation of NMDA receptors. Studies were undertaken to determine whether NO is involved in the hypothalamic release of LHRH and in the LH surge induced by progesterone (P) in estrogen-primed ovariectomized rats. Rats were ovariectomized and 2 weeks later received estradiol benzoate (30 micrograms sc) at 1000 h. Two days later, P was injected at 1000 h to potentiate the estradiol benzoate-induced LH surge in the afternoon. Serial blood samples were collected at hourly intervals from 1400-1800 h via an intraatrial cannula implanted the day before P injection. Additionally, at various times before onset of the LH surge at 1400 h, the rats were injected sc with one of three inhibitors of NO synthase, the enzyme that generates NO. Control, saline-injected rats showed unambiguous LH surges in the afternoon. However, either a single injection at 1000 h of NG-methyl-L-arginine (20 mg/kg) or three injections at 1000, 1200, and 1400 h of either Nw-nitro-L-arginine methyl ester (NAME, 40 mg/kg) or Nw-nitro-L-arginine (60 mg/kg) to inhibit NO efflux markedly suppressed the P-induced LH surge in the afternoon. To ascertain whether suppression of LH surge was due to blockade of hypothalamic LHRH release, a series of in vitro studies were performed in steroid-primed rats. First we examined the effects of sodium nitroprusside (NPS), a compound that spontaneously generates and releases NO. NPS increased basal and KCl-induced LHRH release in vitro from the medial basal hypothalamus-preoptic area and median eminence fragments. No direct effect of NO at the pituitary level was seen, since NPS did not alter basal or LHRH-induced LH in vitro release from hemipituitaries. In addition, we tested the effects of NAME on NMDA-induced LHRH release in vitro from the median eminence-arcuate nucleus fragments. As expected, NMDA alone (50 mM) induced a significant increase in LHRH release. Addition of NO synthase inhibitor, NAME (1 or 10 mM) to suppress NO efflux, significantly diminished the NMDA-induced LHRH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Presence of NMDA Receptor mRNA in the Anterior Pituitary of the Female Rat: Steroid Modulation and Changes during Gonadotropin Surge Induction

N-methyl-d-aspartate (NMDA) receptors have been implicated in the regulation of anterior pituitary hormone secretion. A part of the regulatory effect of NMDA on pituitary hormone release could be due to a direct regulatory effect at the pituitary. The present study examined whether mRNA for the NMDA receptor is expressed in the anterior pituitary of female rats. Additionally, the effect of different steroids on pituitary NMDA receptor mRNA levels was also examined. Using Northern analysis, hybridization of the NMDA R1 cDNA probe to female rat anterior pituitary RNA yielded a 4.0-kb band, demonstrating the presence of the mRNA for the NMDA receptor in the pituitary. In the ovariectomized immature rat, estradiol treatment was found to significantly suppress pituitary NMDA receptor mRNA levels. Conversely, pituitary NMDA receptor mRNA levels were elevated by the treatment of estrogen-primed ovariectomized rats with progesterone. The increase of pituitary NMDA receptor mRNA levels by progesterone occurred at a time when progesterone induces an LH surge. Using the pregnant mare serum gonadotropin-primed immature rat, pituitary NMDA receptor mRNA levels were found to increase in a progressive manner throughout the afternoon of proestrus to reach a peak at 2000 h—the time of peak preovulatory LH surge levels. In conclusion, the present study demonstrates that the mRNA for the NMDA receptor is expressed in the anterior pituitary. The NMDA receptor mRNA in the pituitary is regulated by steroids with peak levels paralleling gonadotropin surge expression. As a whole, the present study provides further evidence that the pituitary could be an important site of action for neuroendocrine effects of excitatory amino acids.

Estrogen-induced calbindin-D 9k gene expression in the rat uterus during the estrous cycle: late antagonistic effect of progesterone.

Progesterone modulates estrogen-stimulated responses in the uterus. Calbindin-D 9k (CaBP9k), a 17 beta-estradiol-responsive gene expressed in the uterus, was used as a marker to examine the interactions between endogenous progesterone and estradiol in the rat. The variations in uterine CaBP9k messenger RNAs (mRNAs) during the rat estrous cycle indicated that CaBP9k gene expression was greatest during the estrogen-dominated phases (proestrus and estrus) and became totally repressed during diestrus, when progesterone predominates. Estradiol was found to be the major controlling factor of CaBP9k gene expression in vivo, progesterone antagonizing estrogen-induced CaBP9k gene expression. The inhibitory role of progesterone was further examined in two experiments. Mature cyclic rats were injected with the progesterone antagonist RU486 before the progesterone surge of proestrus, and the estrous cycle was mimicked in ovariectomized rats by sequential injections of estrogen and progestin. Progesterone did not appear to be involved in the rapid decrease in CaBP9k mRNA during estrus but was implicated in the down-regulation of the estrogen-stimulated CaBP9k gene expression at the end of estrus and during diestrus. This delayed effect of progesterone was confirmed in the ovariectomized rat model. CaBP9k mRNA accumulation in estrogen-primed ovariectomized rats was suppressed by estrogen followed 1 h later by the progesterone agonist R5020. This effect occurred more than 24 h after progestin treatment. The inhibition of the estrogen-induced CaBP9k gene expression in the rat uterus by progesterone is certainly mediated by the progesterone receptor, because progesterone had no effect without estrogen priming or when the antagonist RU486 was used. The delayed progesterone effect probably does not involve depletion of nuclear estrogen receptors, the major rapid mechanism proposed for estrogen inhibition by progesterone in the rodent uterus, or control of estrogen receptor synthesis, as shown by Northern blot analysis of estrogen receptor mRNA.

Central Administration of Bombesin Blocks the Estrogen-Induced Afternoon Prolactin Surge

The effect of central administration of bombesin (BB), a neuropeptide originally isolated from the skin of frogs, on the estrogen-induced afternoon prolactin (PRL) surge was studied. Two doses of BB, 0.15 and 0.75 micrograms/rat, were injected into the lateral cerebroventricle of estrogen-primed ovariectomized rats at 12.00 h through preimplanted cannulae. Serial blood samples were obtained through preimplanted intra-atrial catheters during the whole afternoon hours. The small dose of BB delayed the afternoon PRL surge for an hour, but did not prevent it from occurring. The large dose of BB, however, blocked the surge completely. When the same effective dose of BB was co-administered with a BB antagonist, [Leu13-psi(CH2NH) Leu14]-BB (L psi L-BB), it no longer inhibited the PRL surge. The afternoon PRL surge was also reinstated in BB-treated rats by giving a single injection of sulpiride (1 microgram/rat, i.v.), a dopamine antagonist, at 14.00 h in the same afternoon. These results suggest that BB, by acting through specific BB receptors, can inhibit the PRL surge possibly through activating the dopaminergic systems.

Rat anterior pituitary estrogen receptor mRNA levels after estrogen and progesterone treatment

Previous work from our laboratory has demonstrated that progesterone can induce a rapid decrease in estrogen receptor (ER) binding in the anterior pituitary of the rat. This effect of progesterone on ER correlates well with the loss of estrogen action. The objectives of this study were to determine whether the rapid decrease in ER binding in the rat pituitary after a single injection of estradiol and the decrease in ER binding induced by progesterone in the estrogen-primed rat could be due to a suppression of mRNA levels for the estrogen receptor. Ovariectomy for 14 days decreased ER mRNA levels and injection of 2 μg of estradiol in ethanol:saline for 3 days increased ER mRNA levels. A single injection of 1 μg of estradiol brought about no change in ER mRNA levels in the pituitary of ovariectomized rats within 1 h of administration. Five micrograms of estradiol increased the ER mRNA levels, whereas 10 μg of estradiol brought about a decrease in ER mRNA levels. Thus, the reported decrease in pituitary ER binding after 1 and 5 μg of estradiol was not accompanied by a similar change in ER mRNA levels, suggesting that the decrease in ER binding may be due to accelerated processing of ER or to decreased translation of ER mRNA. When 2 μg of estradiol was injected into estrogen-primed ovariectomized rats, the recovery of ER binding at 18 h was preceded by an increase in ER mRNA levels at 12 h, suggesting new ER synthesis during the recovery process. Progesterone (0.8 mg/kg body wt), in the absence of estradiol-priming, increased ER mRNA levels in the pituitary at 1, 6, and 12 h and decreased them at 18 h. In estrogen-primed animals, the changes in ER mRNA levels brought about by progesterone combined with estradiol were similar to those observed with estradiol alone, with the exception that ER mRNA levels were elevated in the progesterone-treated group at 1 h, a time when ER binding was further suppressed. Thus the progesterone-induced decrease in ER binding was not due to a suppression of ER mRNA levels. In estrogen-primed rats 0.8 and 4.0 mg/kg body wt doses of progesterone increased ER mRNA levels, whereas the 2.0 mg/kg body wt dose did not. These results indicate that estradiol alone has a time- and dose-dependent effect on ER mRNA levels in the anterior pituitary. Progesterone by itself and in the presence of estrogen priming has a dose-related effect on anterior pituitary ER mRNA levels. Furthermore, the effect of progesterone on decreasing ER binding appears to be due either to a post-transcriptional effect and/or to accelerated receptor processing.

Role of 5α-reduction in progesterone's ability to release FSH in estrogen-primed ovariectomized rats

In ovariectomized estrogen-primed rats, progesterone as well as 5α-dihydroprogesterone (5α-DHP) are capable of inducing the release of gonadotropins. This study examined the need of 5α-reduction as a prerequisite for the action of progesterone. The 5α-reductase inhibitor, N, N-diethyl-4-methyl-3-oxo-4-aza-5 α-androstane-17 β-carboxamide was injected at a 1 or 2 mg dose/rat 2 h prior to an injection of 0.4 or 0.8 mg progesterone/kg body weight at 0900 h to immature ovariectomized, estrogen-primed rats and serum was analyzed for LH and FSH at 1500 h. Pituitary and hypothalamic 5α-reductase activity was measured at the time of progesterone administration and at the time of the surge by incubating tissue homogenates with [ 3H]progesterone. Substrate, ([ 3H]progesterone) and product ([ 3H]5α-DHP), were separated by reverse phase HPLC. The pituitary 5α-reductase activity was not blocked at 1500 h. However, both pituitary and hypothalamic 5α-reductase was blocked at the time of progesterone administration. No effect was seen by acute administration of the 5α-reductase inhibitor upon either the 0.4 or 0.8 mg progesterone/kg-induced release of LH and FSH. There was, however, a specific, significant inhibition of progesterone-induced FSH but not LH release when the 5α-reductase inhibition was sustained throughout the afternoon of the gonadotropin surge. These results indicate a biologically significant role for the irreversible 5α-reduction of progesterone in the modulation of the release of FSH.