Quantitation of prolactin receptor mRNA in the maternal rat brain during pregnancy and lactation

Prolactin receptor (PRL-R) expression in the brain is increased in lactating rats compared with non-pregnant animals. The aim of the present study was to determine the time-course of changes in PRL-R mRNA levels during pregnancy and/or lactation, and to determine relative levels of the two forms (short and/or long form) of receptor mRNA in specific brain regions. Brains were collected from female rats on dioestrus, days 7, 14 or 21 of pregnancy, day 7 of lactation or day 7 post-weaning. Frozen, coronal sections were cut (300 microm) and specific hypothalamic nuclei and the choroid plexus were microdissected using a punch technique. Total RNA was extracted and reverse transcribed, then first strand cDNA was amplified using quantitative real-time PCR. Results showed an up-regulation of long-form PRL-R mRNA in the choroid plexus by day 7 of pregnancy compared with dioestrus, which further increased on days 14 and 21 of pregnancy and day 7 of lactation, and then decreased to dioestrous levels on day 7 post-weaning. Short-form PRL-R mRNA levels increased on day 14 of pregnancy relative to dioestrus, increased further on day 7 of lactation and decreased on day 7 post-weaning. Changes in mRNA were reflected in increased levels of PRL-R immunoreactivity in the choroid plexus during pregnancy and lactation, compared with dioestrus. In the arcuate nucleus, long-form PRL-R mRNA was increased during pregnancy. In contrast to earlier work, no significant changes in short- or long-form PRL-R mRNA expression were detected in several other hypothalamic nuclei, suggesting that changes in hypothalamic mRNA levels may not be as marked as previously thought. The up-regulation of PRL-R mRNA and protein expression in the choroid plexus during pregnancy and lactation suggest a possible mechanism whereby increasing levels of peripheral prolactin during pregnancy may have access to the central nervous system. Together with expression of long-form PRL-R mRNA in specific hypothalamic nuclei, these results support a role for prolactin in regulating neuroendocrine and behavioural adaptations in the maternal brain.     

Lactation alters gamma-aminobutyric acid neuronal activity in the hypothalamus and cerebral cortex in the rat

Gamma-aminobutyric acid (GABA) neurons terminating in the hypothalamus have been implicated in the neuroendocrine regulation of reproductive hormones, particularly luteinizing hormone (LH) and prolactin. The aim of this study was to examine whether GABAergic neuronal activity in the hypothalamus was modified during lactation, and whether any observed changes correlated with changes in secretion of these hormones. Animals were divided into three experimental groups: diestrous controls, lactating with pups present (with pups), and lactating with pups removed for 4 h (without pups). Animals were decapitated either without treatment, or 60 min after inhibition of GABA degradation by aminooxyacetic acid (AOAA) (100 mg/kg, i.p.). The rate of GABA accumulation in the tissue after AOAA is a measure of GABA turnover. GABA turnover was estimated in 13 microdissected brain regions, and serum prolactin and LH measured by radioimmunoassay. Suckling was associated with significantly increased prolactin and significantly decreased LH compared with diestrous rats. In lactating rats with pups, GABA turnover was significantly increased in the cingulate cortex compared with diestrous rats. GABA turnover was significantly increased in the ventrolateral preoptic nucleus of lactating rats with pups compared with diestrous rats or lactating rats without pups. There was significantly lower GABA turnover in the anterior hypothalamic area, ventromedial and dorsomedial hypothalamic nuclei in lactating rats without pups compared with diestrous rats. There were no significant changes in other brain regions examined. The results demonstrate that activity of GABAergic neurons in specific parts of the hypothalamus and cerebral cortex is altered during lactation.     

Regulation of hypothalamic neuropeptide Y messenger ribonucleic acid expression during lactation: role of prolactin

In the present study, we investigated the role of prolactin (PRL) in the suckling-induced increase in hypothalamic neuropeptide Y (NPY) gene expression in the dorsomedial nucleus of the hypothalamus (DMH) and the caudal portion of the arcuate nucleus of the hypothalamus (ARH-C). Lactating rats were deprived of their eight-pup litters on d 9 postpartum. After 48 h, the animals were randomly divided into two groups: nonsuckled controls and eight pups suckling for 24 h. In addition, some of the suckled animals received two injections of bromocriptine (0.5 mg/rat per injection) to inhibit suckling-induced PRL secretion. Some bromocriptine-treated rats also received ovine PRL (1 mg/rat per injection). In situ hybridization was performed to measure NPY mRNA levels. Suckling for 24 h induced a significant increase in NPY mRNA levels in the DMH and ARH-C. Bromocriptine treatment greatly attenuated the increase of NPY mRNA in the DMH but not in the ARH. Injections of ovine PRL in bromocriptine-treated rats greatly restored DMH NPY mRNA levels but had no additional effects on the ARH NPY expression. Double-label in situ hybridization for NPY and PRL receptor (PRL-R) in the lactating rat brains showed that NPY-positive neurons in the DMH also express PRL-R mRNA. On the contrary, few ARH NPY neurons expressed PRL-R. These data suggest that PRL could act directly on DMH NPY neurons to modulate NPY gene expression during lactation. Thus, the results from the present study demonstrate that NPY neurons in the DMH and ARH are differentially regulated by PRL during lactation.     

Hypothalamic prolactin receptor messenger ribonucleic acid levels, prolactin signaling, and hyperprolactinemic inhibition of pulsatile luteinizing hormone secretion are dependent on estradiol

Hyperprolactinemia can reduce fertility and libido. Although central prolactin actions are thought to contribute to this, the mechanisms are poorly understood. We first tested whether chronic hyperprolactinemia inhibited two neuroendocrine parameters necessary for female fertility: pulsatile LH secretion and the estrogen-induced LH surge. Chronic hyperprolactinemia induced by the dopamine antagonist sulpiride caused a 40% reduction LH pulse frequency in ovariectomized rats, but only in the presence of chronic low levels of estradiol. Sulpiride did not affect the magnitude of a steroid-induced LH surge or the percentage of GnRH neurons activated during the surge. Estradiol is known to influence expression of the long form of prolactin receptors (PRL-R) and components of prolactin's signaling pathway. To test the hypothesis that estrogen increases PRL-R expression and sensitivity to prolactin, we next demonstrated that estradiol greatly augments prolactin-induced STAT5 activation. Lastly, we measured PRL-R and suppressor of cytokine signaling (SOCS-1 and -3 and CIS, which reflect the level of prolactin signaling) mRNAs in response to sulpiride and estradiol. Sulpiride induced only SOCS-1 in the medial preoptic area, where GnRH neurons are regulated, but in the arcuate nucleus and choroid plexus, PRL-R, SOCS-3, and CIS mRNA levels were also induced. Estradiol enhanced these effects on SOCS-3 and CIS. Interestingly, estradiol also induced PRL-R, SOCS-3, and CIS mRNA levels independently. These data show that GnRH pulse frequency is inhibited by chronic hyperprolactinemia in a steroid-dependent manner. They also provide evidence for estradiol-dependent and brain region-specific regulation of PRL-R expression and signaling responses by prolactin.     

Prolactin modulates hypothalamic preproenkephalin,but not proopiomelanocortin,gene expression during lactation

The aim of this study was to examine prolactin (PRL) regulation of preproenkephalin and proopiomelanocortin (POMC) gene expression in the hypothalamus during lactation. In the first experiment, lactating rats were deprived of pups for 3, 6, 12, or 24 h. Preproenkephalin mRNA levels were decreased in the arcuate nucleus (ARC) to 60 or 53% of suckled levels and in the ventromedial nucleus to 70% of suckled levels after 12 or 24 h but were unchanged in the striatum. POMC mRNA levels in the ARC and periarcuate area were increased to 165% of suckled levels within 3 h and remained elevated two- to threefold for 24 h. Subcutaneous administration of bromocriptine to suckled dams markedly suppressed circulating PRL levels and decreased preproenkephalin mRNA signal levels to 38 and 50% of control levels in the arcuate and ventromedial nuclei, respectively. Intravenous administration of oPRL completely reversed this effect. By contrast, bromocriptine with or without administration of ovine PRL (oPRL) did not alter POMC mRNA signal levels in the ARC. Administration of oPRL to pup-deprived dams increased preproenkephalin mRNA levels in the arcuate and ventromedial nuclei and reduced POMC mRNA levels in the ARC to levels similar to suckled control levels. In conclusion, POMC neurons in the ARC appear to be refractory to PRL regulation in the presence of a suckling stimulus, and other components of the suckling stimulus may contribute to the suppression of POMC mRNA levels during lactation. By contrast, PRL provides a regulatory influence for the suckling-induced increase in preproenkephalin mRNA signal levels in arcuate and ventromedial nuclei.     

Quantitative distribution of nuclear androgen receptors in microdissected areas of the rat brain

The binding of androgens to specific high-affinity receptor sites in brain tissue is postulated as an initial event in the mechanism of central androgenic action. In an effort to assess the functional capacity of the androgen receptor system in the central nervous system, we measured the concentration of nuclear (ARn) as well as cytosolic androgen receptors (ARc) in 13 microdissected brain samples from intact male and female Sprague-Dawley rats. Tissues from 6 rats were combined for each determination and androgen receptor contents were measured with single-point in vitro assays that used saturating concentrations of high specific activity 3[H]dihydrotestosterone. We found that ARc levels tended to be higher in females than males although the general patterns of distribution were very similar. As expected, ARn concentrations were significantly higher in males than females. The highest concentrations of ARn (greater than 100 fmol/mg DNA) in males were measured in the ventromedial nucleus of the hypothalamus and medial amygdala; intermediate levels (50-100 fmol/mg DNA) were found in arcuate nucleus-median eminence, medial preoptic nucleus, periventricular preoptic area, bed nucleus of the stria terminalis, anterior hypothalamus, periventricular anterior hypothalamus, lateral septum, and parietal cortex, and low levels (less than 50 fmol/mg DNA) were measured in lateral preoptic nucleus and cortical amygdala. With the exception of the periventricular preoptic area (74 +/- 33 fmol/mg DNA), only very low concentrations of ARn were measured in females. These data provide the first quantitative profile of ARn in discrete brain nuclei and subregions of the rat.     

Sex differences in androgen receptors and aromatase activity in microdissected regions of the rat brain

Males are generally more responsive than females to the behavioral and neuroendocrine actions of androgens. The present experiments were performed to determine whether these differences may result from sex differences in the number of androgen receptors (AR) in specific brain areas. For this reason, AR binding was compared in both cytosol (ARc) and cell nuclear KCl extracts (ARn) from microdissected brain regions of gonadectomized male and female rats treated with doses of testosterone (T) that produced equivalent physiological circulating androgen levels. In addition, microsomal aromatase activity was measured as a biochemical index of tissue responsiveness to T, since estrogen formation in certain brain areas is regulated by androgen. One week after exogenous T administration, males exhibited significantly higher levels of ARn than females in the bed nucleus of the stria terminalis, periventricular preoptic area, and ventromedial nucleus. Males also had significantly higher aromatase levels in these same areas plus the medial preoptic nucleus and anterior hypothalamus. There were no significant differences in ARn concentrations in eight other nuclei that were examined for significant sex differences in ARc levels observed under these experimental conditions. When ARc levels were compared in untreated gonadectomized male and female rats, males had greater levels of ARc in the bed nucleus of the stria terminalis only, indicating that new receptor synthesis may be responsible for the sex differences observed in T-treated rats. These results suggest that sex differences in neural responsiveness to androgens may be due in part to sex differences in ARn occupation in specific brain regions.     

Neurotensin gene expression increases during proestrus in the rostral medial preoptic nucleus: potential for direct communication with gonadotropin-releasing hormone neurons.

Neurotensin (NT)-containing neurons in the rostral portion of the medial preoptic nucleus (rMPN) of the brain may play a key role in regulating the pattern of secretion of GnRH, thereby influencing the reproductive cycle in females. The major goals of this study were to determine whether NT messenger RNA (mRNA) levels in the rMPN exhibit a unique pattern of expression in temporal association with the preovulatory LH surge and to assess whether NT neurons may communicate directly with GnRH neurons. We analyzed NT gene expression in rats using in situ hybridization over the day of proestrus and compared this with diestrous day 1. We also determined whether the high-affinity NT receptor (NT1) is expressed in GnRH neurons using dual-label in situ hybridization and whether this expression varies over the estrous cycle. We found that NT mRNA levels in the rMPN increase significantly on the day of proestrus, rising before the LH surge. No such change was detected on diestrous day 1, when the LH surge does not occur. Furthermore, we observed that a significant number of GnRH neurons coexpress NT1 mRNA and that the number of GnRH neurons expressing NT1 mRNA peaks on proestrus. Together with previous findings, our results suggest that increased expression of NT in the rMPN may directly stimulate GnRH neurons on proestrus, contributing to the LH surge. In addition, our results suggest that responsiveness of GnRH neurons to NT stimulation is enhanced on proestrus due to increased expression of NT receptors within GnRH neurons.     

Inhibition of metastin (kisspeptin-54)-GPR54 signaling in the arcuate nucleus-median eminence region during lactation in rats

Follicular development and ovulation are suppressed during lactation in various mammalian species, mainly due to the suppression of pulsatile GnRH/LH secretion. Metastin (kisspeptin-54), a KiSS-1 gene product, is an endogenous ligand for GPR54, a G-protein-coupled receptor, and suggested to play a critical role in regulating the gonadal axis. The present study therefore aims to determine whether metastin (kisspeptin-54)-GPR54 signaling in discrete brain areas is inhibited by the suckling stimulus that causes suppression of LH secretion in lactating rats. Quantitative RT-PCR revealed that the KiSS-1 mRNA level was significantly lower in the arcuate nucleus (ARC)-median eminence region in lactating ovariectomized (OVX) and estrogen-treated OVX rats than in nonlactating controls. KiSS-1 mRNA in the anteroventral periventricular nucleus was kept at a low level in both lactating and nonlactating rats despite estrogen treatment. GPR54 mRNA levels were significantly lower in lactating than nonlactating rats in the anteroventral periventricular nucleus, but the levels in lactating mothers of the preoptic area and ARC-median eminence were comparable with nonlactating controls. Although KiSS-1 mRNA-expressing cells or metastin (kisspeptin-54) immunoreactivities were densely located in the ARC of nonlactating controls, few were found in the ARC of lactating OVX animals. Various doses of metastin (kisspeptin-54) (0.02, 0.2, and 2 nmol) injected into the third ventricle caused a significant increase in LH secretion in both lactating and nonlactating OVX rats, suggesting that lactating rats are responsive to metastin (kisspeptin-54) stimulus. Thus, the present study demonstrated that KiSS-1 mRNA/metastin (kisspeptin-54) expression is inhibited in the ARC by the suckling stimulus, suggesting that the inhibition is most probably involved in suppressing LH secretion in lactating rats.     

Effect of suckling on prolactin receptor immunoreactivity in the hypothalamus of the rat

Previous studies showed that expression of prolactin (PRL) receptor is increased in numerous hypothalamic nuclei in mid-lactating rats. The increase in PRL receptor expression could be initated by neurohormonal changes during proestrus or pregnancy, or by the suckling stimulus during lactation. The present study investigated whether the PRL receptor expression in numerous hypothalamic nuclei is altered by the suckling stimulus. Three groups (n = 4) of rats on lactation day 10 were used: a continuously suckled group, a nonsuckled group (pups removed for 12 h) and a resuckled group (pups removed for 12 and then resuckled for 9 h). Animals were perfused with 2% paraformaldehyde and brains were sectioned (20 microm) for the immunofluorescence study. Immunoreactivity was semiquantitatively analyzed by counting the immunoreactive cells and measuring the immunostaining intensity in a specific area. Neurons expressing PRL receptors were observed in numerous hypothalamic areas with the highest number being in the arcuate, paraventricular and supraoptic nuclei. The PRL receptor immunofluorescence in several nuclei was significantly decreased in the nonsuckled group, and recovered in the resuckled group. These areas included the ventromedial preoptic, ventrolateral preoptic, lateroanterior hypothalamic, ventrolateral hypothalamic and ventromedial hypothalamic nuclei. PRL receptor immunoreactivity in other areas was not significantly altered by the suckling stimulus. These results demonstrate that expression of PRL receptor in hypothalamic nuclei was differentially affected by the suckling stimulus. PRL receptors in those nuclei which were significantly altered by suckling stimulus may play more critical roles during lactation than those areas which were less sensitive to the suckling stimulus.     

Suckling-induced changes of vasoactive intestinal peptide concentrations in hypothalamic areas implicated in the control of prolactin release

The present study was designed to investigate whether the vasoactive intestinal peptide (VIP) concentration in hypothalamic nuclei, dorsal raphe nucleus (DR) and pituitary lobes of lactating rats changes in physiological situations when prolactin (PRL) secretion is stimulated (suckling) or inhibited (pup separation). In addition VIP levels in blood plasma were determined in both situations. Acute suckling induced changes in VIP concentration only in the rostral part of the anterior hypothalamic (rAHN) and the paraventricular (PVN) nuclei of all the brain areas examined. VIP concentration in the rAHN increased at 5 min from 3.52 +/- 0.30 (mean +/- SEM) to 8.67 +/- 1.91 ng/mg protein (p less than 0.05) but fell to baseline values after 30 min suckling (p less than 0.05; 5 vs. 30 min). Although changes in VIP concentration in the suprachiasmatic nucleus (SCN) did not attain statistical significance, they followed the same trends as the changes of VIP in the rAHN. The opposite pattern of changes was observed in the PVN with a decrease in VIP concentration following 5 min suckling (p less than 0.01). At 30 min the VIP values showed a trend towards 0-min values. Pup removal did not affect VIP concentrations in the rAHN, PVN, SCN, median eminence, supraoptic nucleus and DR. VIP values were not detectable in the arcuate nucleus in any of the experimental situations examined. Lactation increased VIP concentration only in the rAHN and PVN when lactating rats with their pups were compared with virgin female diestrous rats. VIP concentration in the anterior lobe of the pituitary from lactating rats did not change with pup separation or suckling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the effects of castration and steroid replacement on incertohypothalamic dopaminergic neurons in male and female rats

The activities of incertohypothalamic (IH) and tuberoinfundibular (TI) dopamine (DA) neurons were compared in selected brain regions of male and female rats by measuring the rate of DA turnover (alpha-methyltyrosine-induced decline in brain DA concentrations). The rates of DA turnover in regions containing TIDA (median eminence) and rostral IHDA (rostral periventricular and medial preoptic nuclei) neurons were greater in diestrous females than in intact males. In contrast, the rate of DA turnover in the caudal IHDA neurons (medial zona incerta), was greater in intact males than diestrous females. These results indicate that the activities of IHDA neurons, like those of TIDA neurons, differ between the sexes but that the sexual differentiation of IHDA neurons is not homogeneous. Two weeks following orchidectomy, the rates of DA turnover were increased in the median eminence and decreased in the medial preoptic nucleus. Testosterone replacement in orchidectomized males produced opposite effects, causing a decrease in DA turnover in the median eminence and an increase in the medial preoptic nucleus. In female rats, the rates of DA turnover were decreased in the median eminence and medial zona incerta and increased in the medial preoptic nucleus 2 weeks following ovariectomy. Only in the median eminence did 2 days of estrogen replacement in ovariectomized rats produce effects opposite those seen after ovariectomy alone. These data show that the activities of IHDA neurons, as estimated from measurements of DA turnover, can be altered by the removal and replacement of the gonadal steroids.     

The distribution of cells containing estrogen receptor-alpha (ERalpha) and ERbeta messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females

We have used in situ hybridization to compare the distributions of estrogen receptor alpha (ERalpha) and ERbeta messenger RNA (mRNA)-containing cells in the preoptic area and hypothalamus of ewes and rams. Perfusion-fixed brain tissue was collected from luteal phase ewes and intact rams (n = 4) during the breeding season. Matched pairs of sections were hybridized with sheep-specific, 35S-labeled riboprobes, and semiquantitative image analysis was performed on emulsion-dipped slides. A number of sex differences were observed, with females having a greater density of labeled cells than males (P < 0.001) and a greater number of silver grains per cell (P < 0.01) in the ventromedial nucleus for both ER subtypes. In addition, in the retrochiasmatic area, males had a greater (P < 0.05) cell density for ERalpha mRNA-containing cells than females, whereas in the paraventricular nucleus, females had a greater density (P < 0.05) of ERalpha mRNA-containing cells than males. There was a trend (P = 0.068) in the arcuate nucleus for males to have a greater number of silver grains per cell labeled for ERalpha mRNA. In both sexes, there was considerable overlap in the distributions of ERalpha and ERbeta mRNA-containing cells, but the density of labeled cells within each nucleus differed in a number of instances. Nuclei that contained a higher (P < 0.001) density of ERalpha than ERbeta mRNA-containing cells included the preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus, whereas the subfornical organ (P < 0.001), paraventricular nucleus (males only, P < 0.05), and retrochiasmatic nucleus (females only, P < 0.05) had a greater density of ERalpha than ERbeta mRNA-containing cells. The anterior hypothalamic area and supraoptic nucleus had similar densities of cells containing both ER subtypes. The lateral septum and arcuate nucleus contained only ERalpha, whereas only ERbeta mRNA-containing cells were seen in the zona incerta. The sex differences in the populations of ER mRNA-containing cells in the ventromedial and arcuate nuclei may explain in part the sex differences in the neuroendocrine and behavioral responses to localized estrogen treatment in these nuclei. Within sexes, the differences between the distributions of ERalpha and ERbeta mRNA-containing cells may reflect differential regulation of the actions of estrogen in the sheep hypothalamus. Low levels of ERbeta mRNA in the preoptic area and ventromedial and arcuate nuclei, regions known to be important for the regulation of reproduction, suggest that ERbeta may not be involved in these functions.     

Regional induction of c-fos-like protein in rat brain after estradiol administration.

The protooncogene c-fos is induced in rat brain by various forms of physiological stimulation. In this study immunocytochemical staining for a peptide fragment of the c-fos protein was used to assess estradiol's effects on c-fos in rat brain. After estradiol benzoate (100 micrograms/kg) administration to ovariectomized rats, the number of cells staining for c-fos-like protein increased in the anterior medial preoptic area, the medial preoptic area, the medial amygdala nucleus, and the ventromedial nucleus of hypothalamus, all regions rich in estradiol-concentrating cells. This increase peaked between 12-48 h (depending on the region) after estradiol administration and was not observed in several areas with a lower density of estradiol-concentrating cells. In the most affected region, the anterior medial preoptic area, estradiol effects were dose dependent and not altered by progesterone administration. It is not yet clear whether estradiol induces c-fos expression in brain directly or whether c-fos is part of a cascade of mechanisms by which estradiol regulates gene expression.     

Sexually dimorphic effects of testosterone on preoptic area calcitonin gene-related peptide mRNA expression depend upon neuron location and differential estrogen and androgen receptor activation.

Experiments examined activational roles of gonadal steroids on the sexually dimorphic, calcitonin gene-related peptide-expressing neurons of the rat preoptic area. Gonadectomy of male rats followed by treatment with testosterone, dihydrotestosterone, or estrogen demonstrated that the tonic suppressive influence of testosterone on cellular levels of calcitonin gene-related peptide mRNA expression in the medial preoptic nucleus and anteroventral periventricular nucleus occurred through either ER- or AR-mediated mechanisms (P < 0.05). The gonadectomy of adult female rats demonstrated little tonic influence of ovarian steroids upon calcitonin gene-related peptide mRNA levels. However, the administration of male levels of testosterone to ovariectomized rats resulted in reduced calcitonin gene-related peptide mRNA expression within the medial preoptic nucleus (P < 0.05) and, strikingly, a 3-fold induction in calcitonin gene-related peptide mRNA expression in the anteroventral periventricular nucleus (P < 0.01). Testosterone's effects in the medial preoptic nucleus and anteroventral periventricular nucleus of the female required both ER and AR activation. Dual labeling immunocytochemical studies revealed that less than 10% of calcitonin gene-related peptide neurons in the male expressed ARs compared with approximately 50% in the female. These investigations reveal that sexually differentiated region- and steroid receptor-specific mechanisms function in association with the sex differences in circulating gonadal steroids to maintain the sexually dimorphic nature of calcitonin gene-related peptide expression in the preoptic area of the adult rat.