Plasma prolactin and luteinizing hormone profiles during the estrous cycle of the female rat: effects of surgically induced persistent estrus

Experiments were carried out to investigate the hypothalamic control mechanism for prolactin (PRL) and luteinizing hormone (LH) secretion in the female rat. Anterior medial preoptic nucleus (AMPO) or suprachiasmatic nucleus (SCN) lesions were produced by passing 5-10 microA of direct current (tip negative). Persistent estrus (PE) began as early as 6 days and as late as 30 days after electrolytic lesioning. Blood samples obtained during diestrus, proestrus and estrus revealed well-described profiles of plasma PRL and LH in sham-lesioned animals, indicating that our cannulation and blood sampling procedure had no adverse effects on the plasma hormone levels. Individual sham-operated animals sampled on successive or alternate proestrous afternoons showed precise timing of the PRL and LH surges. However, when a shift occurred in the PRL surge a comparable shift would also occur in the LH surge, indicating a coupling between the mechanisms regulating the PRL and the LH surge. The AMPO-, SCN- or combine-lesioned PE animals exhibited low basal levels of plasma PRL and LH. Small secretory bursts occurred one to three times during the 6-hour sampling periods. Animals with incomplete SCN lesions had plasma PRL titers significantly higher than the other 3 groups. Plasma progesterone levels were significantly lower in the PE animals (p less than 0.01), whereas plasma estrogen levels were not significantly different from proestrous controls. These experiments indicate that during the afternoon of proestrus, the surges of plasma PRL and LH are very precise in the time of onset. Moreover, the mechanisms controlling the surge of PRL and LH are temporally coupled.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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

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

Control of the estradiol-induced prolactin surge by the suprachiasmatic nucleus

In the present study we investigated how the suprachiasmatic nucleus (SCN) controls the E(2)-induced PRL surge in female rats. First, the role of vasopressin (VP), a SCN transmitter present in medial preoptic area (MPO) projections and rhythmically released by SCN neurons, as a circadian signal for the E(2)-induced PRL surge was investigated. Using a reverse microdialysis technique, VP was administered in the MPO during the PRL surge, resulting in a suppression of the surge. VP administration before the surge did not affect PRL secretion. Also, administration of a V1a receptor antagonist before the surge was ineffective. Second, lesions of the SCN were made that resulted in constant basal PRL levels, suggesting that with removal of the SCN a stimulatory factor for PRL secretion disappeared. Indeed, the PRL secretory response to blockade of pituitary dopamine receptors was significantly reduced in SCN-lesioned animals. These data suggest that the afternoon decrease of VP release in the MPO by SCN terminals enables the PRL surge to occur, and may thus be a circadian signal for the PRL surge. Simultaneously the SCN is involved in the regulation of the secretory capacity of the pituitary, possibly via specific PRL-releasing factors.     

Gonadotropin-releasing hormone gene expression during the rat estrous cycle: effects of pentobarbital and ovarian steroids.

Although hypothalamic GnRH release is known to be modulated by neural and hormonal factors, the relationship between altered GnRH secretion and GnRH synthesis remains unclear. In an attempt to address this question, we examined GnRH gene expression in the rat hypothalamus using in situ hybridization histochemistry. An 25S-labeled antisense RNA probe was used to identify neurons expressing GnRH mRNA in an area that included the diagonal band of Broca, the organum vasculosum of the lamina terminalis, and the preoptic area. The number of GnRH mRNA-expressing cells was determined at various times during the rat estrous cycle. During proestrus, the number of GnRH mRNA-expressing cells decreased somewhat at 1400-1600 h, increased significantly at 1800 h (the time of the LH surge), then gradually returned to basal levels at 2200 h. Expression did not change substantially at other times during the estrous cycle. To understand this close temporal relationship between the LH surge and increased GnRH mRNA levels, we examined GnRH gene expression in proestrous animals in which the LH surge was blocked with pentobarbital. Pentobarbital treatment blocked the increase in the number of GnRH mRNA-expressing cells normally observed at 1800 h in saline-treated controls, suggesting that the increase in GnRH gene expression is closely coupled to secretion of GnRH from the hypothalamus. Finally, we addressed the question of whether ovarian steroids have direct effects on GnRH gene expression by examining GnRH mRNA levels in ovariectomized steroid-treated rats at a time before (1100 h) and a time after (1800 h) hypothalamic GnRH hypersecretion. At 1100 h, no significant changes were observed, but at 1800 h, estrogen-treated rats showed a significant increase in both the number of GnRH mRNA-expressing cells and serum LH levels. This suggests that estrogen influences GnRH gene expression indirectly, perhaps by altering hypothalamic GnRH release. Our results in each of these models suggest that GnRH mRNA levels increase in response to GnRH hypersecretion at the time of the LH surge.     

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.     

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.     

Endogenous excitatory amino acid involvement in the preovulatory and steroid-induced surge of gonadotropins in the female rat

The physiological role of N-methyl-D-aspartate (NMDA) receptors in the regulation of preovulatory and steroid-induced surges of gonadotropins in the female rat was examined. The specific and potent noncompetitive NMDA receptor antagonist MK801 was used for blockade of NMDA neurotransmission. MK801 treatment completely inhibited the ability of progesterone to induce LH and FSH surges in the estrogen-primed ovariectomized rat. Administration of MK801 on proestrus in the immature female rat primed with PMSG resulted in a significant attenuation of the proestrous LH, FSH, and PRL surge and a corresponding attenuation of ovulation. Similarly, in the adult cycling female rat, MK801 administration on proestrus led to a significant attenuation of the proestrous LH and PRL surges. Mean FSH levels were lower in MK801-treated adult rats than in vehicle-treated rats, but this effect was not significant. In the estrogen-primed ovariectomized immature rat, the agonist NMDA caused a rapid (less than 10 min) elevation of LH and FSH in vivo. The gonadotropin-releasing effect of NMDA may be mediated at the level of the hypothalamus, since the medial basal hypothalamus/preoptic area of NMDA-treated rats killed 3 and 5 min post-NMDA had a significantly greater release of GnRH in vitro than that of vehicle-treated rats. In conclusion, these findings demonstrate that the preovulatory gonadotropin surge in the female rat is dependent on NMDA neurotransmission for its expression and add further evidence for a critically important role for NMDA receptors in the physiological regulation of gonadotropin secretion in the female rat.     

Persistent estrus and blockade of progesterone-induced LH release follows lesions which do not damage the suprachiasmatic nucleus

Very small electrolytic lesions were made over the anterior or posterior portion of the optic chiasm in mature female rats showing normal estrous cycles. Lesions over the posterior portion of chiasm destroyed the suprachiasmatic nucleus of the hypothalamus (SCN) while the anterior lesions destroyed a small neural structure, here designated as the medial preoptic nucleus (MPN). Both lesions were effective in inducing persistent vaginal estrus, but when animals were ovariectomized and treated with exogenous and progesterone it was found that lesions including the MPN alone, but not the SCN alone, eliminated the positive feedback effects of this steroid regimen on LH release.     

Dorsal raphe lesion alters the estrous cycle and the preovulatory gonadotropin release

The purpose of this study was to examine the role that the dorsal raphe (DR)-median eminence (ME) serotonergic projection may have in the proestrous gonadotropin and prolactin (PRL) release. DR electrolytic lesions were performed in cycling rats during the first day of diestrus. The effect of DR lesions after 48-72 h of survival (short-term lesioned animals) or after 35-40 days of survival (long-term lesioned animals) on estrous cyclicity, preovulatory gonadotropin and PRL releasing pattern, ovulation and serotonin (5-HT) content of the ME were studied. Following DR lesions the estrous cycle became irregular, remaining in the diestrus phase for several days. Preovulatory gonadotropin release in short-term lesioned animals was increased; on the contrary, in long-term lesioned rats a delay in the surge of these two hormones and a decrease in LH secretion were detected. Long-term lesioned animals also showed a diminished secretion of PRL. The number of ova did not differ between control and lesioned animals. DR lesions in both short- and long-term lesioned rats reduced 5-HT levels in the ME by about 50% and nullified the normal 5-HT decline during the afternoon of proestrus. Our results suggest that the DR exerts a stimulatory influence on the preovulatory gonadotropin release by means of its 5-HT projection to the ME. As the pattern of hormonal secretion in lesioned animals remains similar to that of controls, it may be suggested that this pathway acts as a fine modulator of the mechanisms involved in the regulation of LH and FSH release in cycling rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation of preoptic area glutamate release correlates with reduced luteinizing hormone secretion in middle-aged female rats

Glutamate (Glu) and its receptors are involved in the maturation and maintenance of the neural mechanisms governing the preovulatory LH surge of young, reproductive-aged rodents and nonhuman primates. Little is known about the role of Glu in the delayed onset and reduced peak amplitude of the LH surge that characterizes female rodents during early reproductive senescence. The present study tested the hypothesis that the delayed and attenuated LH surge observed in middle-aged female rats is associated with altered hypothalamic Glu release. We used intracerebral microdialysis in young (3-4 months) and middle-aged (9-11 months) female rats to monitor changes in medial preoptic area Glu release and jugular vein catheters to monitor changes in serum LH levels. All animals were ovariectomized and injected with estradiol and progesterone in doses sufficient to produce a robust LH surge in most (approximately 70%) young rats. In both young and middle-aged females that surged, extracellular Glu levels were higher than in those that did not surge. Among animals that surged, the onset of the LH surge was significantly delayed, and the amplitude of the surge was significantly reduced in middle-aged compared with young rats. Middle-aged females also had significantly reduced extracellular Glu levels throughout the day of the LH surge when compared with young females. These data strongly suggest that age-related hypothalamic dysfunction contributes to reproductive aging independent of gonadal failure. We propose that reduced medial preoptic area Glu transmission contributes to reproductive aging by attenuating excitatory input to GnRH neurons.     

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.     

In vivo release of prolactin-releasing peptide in rat hypothalamus in association with luteinizing hormone and prolactin surges.

Prolactin (PRL)-releasing peptide (PrRP) is a novel hypothalamic peptide reported to be a potent and specific stimulator of PRL secretion. This author recently reported that PrRP might play a significant role in mediating the steroid-induced PRL surge in the rat. In order to examine the secretory profile of PrRP in the rat hypothalamus before and during the luteinizing hormone (LH) and PRL surges, this study employed the push-pull perfusion technique and determined the in vivo release of PrRP and also of gonadotropin-releasing hormone (GnRH) in ovariectomized rats primed with estradiol and progesterone. In the medial preoptic area (MPOA) where the GnRH neuronal perikarya exist, GnRH release was increased prior to the initiation of the LH surge, and PrRP also started rising even earlier than GnRH. In the median eminence-arcuate nucleus complex (ME-ARC), where GnRH neuronal fibers terminate, GnRH secretion started increasing before the commencement of the LH surge, but the release of PrRP did not change significantly. These results suggest that PrRP may play a role in mediating the steroid-induced LH surge by activating GnRH neurons in the MPOA. A possible involvement of PrRP in the PRL surge was not suggested from the present data. The lack of a significant alteration in PrRP release in the ME-ARC may argue against a direct hypophysiotropic action of the peptide.     

The hypothalamic-pituitary axis of streptozotocin-induced diabetic female rats is not normalized by estradiol replacement

Studies in diabetic rats have found abnormalities at the hypothalamic, pituitary, and/or ovarian level but have not controlled for changes in estrogen levels induced by diabetes. The purpose of this investigation was to study the effect of diabetes on the hypothalamic-pituitary axis in ovariectomized rats treated with estradiol (E2). Ovariectomized 60 day old female rats were assigned to control (C, n = 42), diabetic (D, n = 47) or insulin-treated diabetic (DI, n = 16) groups. Diabetes was induced with an injection of streptozotocin in the D and DI groups. In the C, D, and DI groups, estrogen was replaced by implanting blank, 5 micrograms or 20 micrograms E2 pellets sc. Pituitary LH responsiveness to GnRH was assessed in C and D animals. Anterior hypothalamic and midhypothalamic concentrations of proGnRH and GnRH, pituitary LH and FSH and serum levels of LH, and E2 were measured by RIA. Anterior hypothalamic proGnRH concentrations were decreased in diabetic rats treated with 5 micrograms E2 compared to 5 micrograms E2 control animals (P less than 0.05). Midhypothalamic GnRH concentrations were also reduced in D vs. C animals despite comparable estrogen therapy (P less than 0.004). GnRH-stimulated LH levels were greater in E2-treated diabetic females than in similarly treated control rats (P less than 0.001). D and DI animals were more sensitive than controls to the inhibitory effect of estrogen on basal LH levels. Pituitary LH and FSH content was lower in 20 micrograms E2-replaced animals but was not influenced by the diabetic state. These data demonstrate a diabetes-induced decrease in hypothalamic proGnRH and GnRH concentration which is not corrected with E2 replacement. The hyper-responsiveness of the diabetic rat pituitary to GnRH also suggests a chronic lack of GnRH stimulation from the hypothalamus but a continued ability of the pituitary to respond to GnRH.     

Evidence that neurotensin participates in the central regulation of the preovulatory surge of luteinizing hormone in the rat

Neurotensin (NT) has been implicated in the central regulation of LH and PRL secretion in the rat. We investigated the importance of NT release to the neural events that trigger the preovulatory LH surge and coincident PRL surge, using as our animal model ovariectomized (OVX) rats treated with estrogen and progesterone to induce reliable and robust surges. To interfere with the action of endogenous NT in the basal forebrain, we administered a NT antiserum (NTAS) in a series of bilateral microinjections aimed at the anterior border of the medial preoptic area. One week after OVX, rats bearing cerebral guide cannulae received Silastic capsules (3 x 15 mm; sc) containing 17 beta-estradiol. Two days later, beginning at 0830 h, conscious rats were administered either NTAS or control serum bilaterally in a series of four 100-nl injections spaced at 30-min intervals. After an initial blood sample, rats received progesterone (4 mg, sc) at 1200 h; blood samples were then taken at 1-h intervals from 1400-2100 h. Blood samples were obtained from conscious, freely moving rats via a chronic atrial catheter implanted previously. Plasma levels of LH and PRL were measured by RIA, and the location of microinjection sites was verified histologically. Administration of NTAS caused a 66% reduction in the magnitude of the LH surge without altering its timing, whereas the PRL surge was unaffected. These results provide strong evidence that NT in the basal forebrain participates in the steroid-induced LH surge and suggest that NT plays a role in the preovulatory LH surge.     

Progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by inhibiting the surge of gonadotropin-releasing hormone.

Previous studies indicate an elevation of circulating progesterone blocks the positive feedback effect of a rise in circulating estradiol. This explains the absence of gonadotropin surges in the luteal phase of the menstrual or estrous cycle despite occasional rises in circulating estradiol to a concentration sufficient for surge induction. Recent studies demonstrate estradiol initiates the LH surge in sheep by inducing a large surge of GnRH secretion, measurable in the hypophyseal portal vasculature. We tested the hypothesis that progesterone blocks the estradiol-induced surge of LH and FSH in sheep by preventing this GnRH surge. Adult Suffolk ewes were ovariectomized, treated with Silastic implants to produce and maintain midluteal phase concentrations of circulating estradiol and progesterone, and an apparatus was surgically installed for sampling of pituitary portal blood. One week later the ewes were allocated to two groups: a surge-induction group (n = 5) in which the progesterone implants were removed to simulate luteolysis, and a surge-block group (n = 5) subjected to a sham implant removal such that the elevation in progesterone was maintained. Sixteen hours after progesterone-implant removal (or sham removal), all animals were treated with additional estradiol implants to produce a rise in circulating estradiol as seen in the follicular phase of the estrous cycle. Hourly samples of pituitary portal and jugular blood were obtained for 24 h, spanning the time of the expected hormone surges, after which an iv bolus of GnRH was injected to test for pituitary responsiveness to the releasing hormone. All animals in the surge-induction group exhibited vigorous surges of GnRH, LH, and FSH, but failed to show a rise in gonadotropin secretion in response to the GnRH challenge given within hours of termination of the gonadotropin surges. The surges of GnRH, LH, and FSH were blocked in all animals in which elevated levels of progesterone were maintained. These animals in the surge-block group, however, did secrete LH in response to the GnRH challenge. We conclude progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by acting centrally to inhibit the surge of GnRH secreted into the hypophyseal portal vasculature.     

Effect of naloxone on luteinizing hormone, follicle-stimulating hormone, and prolactin secretion in the different phases of the estrous cycle

It is becoming increasingly clear that the effects exerted by opioid agonists and antagonists on the release of gonadotropins and of PRL may vary according to the endocrine milieu. To investigate this issue further, female rats with a regular 4-day estrous cycle have been injected sc with the opioid antagonist naloxone at different hours of the day, during each of the various days of the estrous cycle. The animals were killed 20 min after the sc administration of naloxone (2.5 mg/kg dissolved in 0.9% saline solution) at 1000 and 1600 h on the first and second day of diestrus and at 1000, 1200, 1400, 1600, 1800, and 2000 h on proestrus and estrus. Animals were killed by decapitation, and trunk blood was collected and assayed for LH, FSH, and PRL. The data obtained from naloxone-treated animals were compared to those derived from controls injected sc with 0.9% saline solution and killed at the same time intervals. The sc injections of naloxone stimulated LH release in every phase of the estrous cycle; however the magnitude of the responses was highly variable. Increases of the order of 700-1.000% were observed during the 2 days of diestrus, at 1000 and 1400 h of the day of proestrus, and at 1600, 1800, and 2000 h of the day of estrus. Much higher responses (of the order of 2.700-3.300%) were observed at 1600 h of the day of proestrus and at 1000, 1200, and 1400 h of the day of estrus. The LH response to naloxone appeared to be obliterated at 1800 and 2000 h of the day of proestrus. Serum levels of FSH and PRL were not affected by the treatment at any of the time intervals considered. These findings suggest that, in normally cycling adult female rats, naloxone exerts a stimulatory effect on LH release during each day of the estrous cycle; that the stimulatory effect of naloxone is minimal at the time of the spontaneous proestrous LH surge; and that the effect of naloxone on LH release is, on the contrary, maximal just before the spontaneous proestrous LH surge and up to 1400 h of the day of estrus. The observation that naloxone does not affect FSH and PRL release underlines once more that the central mechanisms controlling LH, FSH, and PRL secretion are different.     

Effects of hyperprolactinemia on estrous cyclicity, serum luteinizing hormone, prolactin, estradiol, and progesterone concentrations, and catecholamine activity in microdissected brain areas

The purpose of the present study was to determine the duration of PRL treatment required to suppress estrous cyclicity and preovulatory LH surges and to ascertain whether such treatment affects cyclicity by altering catecholamine activity and/or ovarian steroid secretion. Rats exhibiting 4-day estrous cycles were treated with ovine PRL (oPRL) or vehicle beginning on diestrous day 1 at 0900 h, diestrous day 2 at 2400 h, proestrus at 0600 h, or proestrus at 1200 h. Jugular veins of rats were cannulated to the level of the right atrium on diestrous day 2. Unrestrained rats were bled on proestrus. Preovulatory LH surges and ovulation were completely blocked, and vaginal cytology remained leukocytic on the expected day of proestrus and estrus when oPRL treatment was begun on diestrous day 1. Such treatment elevated progesterone levels beginning on diestrous day 2 and suppressed the preovulatory rise in estradiol observed on proestrous morning and afternoon in control rats. To determine the effect of oPRL on catecholamine activity, alpha-methylparatyrosine was administered to groups of oPRL- or vehicle-treated rats at 0900 or 1500 h on diestrous day 1, diestrous day 2, or proestrus. Animals were killed 0, 45, or 90 min later. Norepinephrine and dopamine concentrations were measured in the median eminence, suprachiasmatic nucleus, medial preoptic nucleus, striatum, ventral aspect of the stria terminalis, and posterior pituitary gland by radioenzymatic assay. Controls exhibited increased norepinephrine turnover rates in the median eminence, suprachiasmatic nucleus, and medial preoptic nucleus on proestrous afternoon concomitant with preovulatory LH surges. In contrast, oPRL-treated rats showed no such increase. In addition, median eminence dopamine turnover rates were elevated beginning on the afternoon of diestrous day 1 in oPRL-treated rats compared to control values. No other differences in norepinephrine and dopamine turnover rates were observed in oPRL-treated rats compared with controls in any other brain area on any day examined. Thus, the data indicate that elevated PRL concentrations have profound effects on reproductive cyclicity by disrupting ovarian steroid secretion and essential preovulatory neurochemical events in selected brain areas involved in the regulation of LHRH.     

Further evidence that preoptic anterior hypothalamic GABAergic neurons are part of the GnRH pulse and surge generator

The in vivo release rates of GABA from the preoptic/anterior hypothalamic area (PO/AH) of ovariectomized rats were assessed by means of a focal perfusion cannula system. Seven days after surgery all animals received a sc silastic capsule implant containing either estradiol-17 beta (E2) or corn oil, and they were perfused 3 days later. Perfusate fractions were sampled at 5-min intervals and blood was collected every 10 min over a period of 5 h. In ovariectomized animals PO/AH GABA release was pulsatile without any diurnal rhythm. Prior to frequency analysis by means of the pulsar-programme, LH and GABA values were z-transformed. Significant LH peaks of all examined ovariectomized rats were superimposed and GABA data were arranged accordingly. It became evident that LH episodes are preceded by a significant reduction of preoptic anterior hypothalamic GABA release. The secretion patterns of GABA and LH were profoundly affected by E2 replacement. During early noon when LH levels were low, constantly elevated hypothalamic GABA release rates were observed in E2-substituted rats in comparison to ovariectomized rats. GABA release rates fell significantly during the E2-induced LH surge. Our previous demonstration of the existence of a large number of estrogen-respective GABAergic neurons in the PO/AH is suggestive of these neurons changing their activity in response to estrogen treatment. We conclude that these estrogen-respective GABAergic neurons are involved in the generation of GnRH pulses as well as in the generation of the so-called positive feedback effect of E2 on LH release.(ABSTRACT TRUNCATED AT 250 WORDS)