Proteolytic modification of prolactin by the female rat brain.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses we have identified immunoreactive prolactin (PRL) proteins with molecular weights of 24 and 16 kD in the female rat brain. Because PRL target tissues have been shown to contain enzymes which, in vitro, cleave PRL into a 16-kD PRL fragment, studies were performed to characterize PRL proteolysis in the female rat brain. In vitro proteolysis of PRL was examined by incubating [125]I-PRL with 25,000 g subcellular fractions followed by SDS-PAGE under reducing conditions. At acidic pHs, incubation of PRL with 25,000 g hypothalamic fractions consistently resulted in the generation of a 16-kD fragment. The generation of the 16-kD fragment was time and tissue concentration dependent. Enzyme inhibitor analysis indicated that PRL proteolysis could be blocked by aspartate and serine protease inhibitors, but not sulfhydryl, metalloenzyme or trypsin protease inhibitors. Subcellular localization of hypothalamic PRL proteolytic activity by equilibrium density centrifugation revealed a bimodal distribution of proteolytic activity with modal densities of 1.12 and 1.24 g/ml. Homogenization of the tissue in a hypo-osmotic medium disrupted the high density peak resulting in a single low-density peak at the top of the gradient. These data indicate that subcellular fractions of the rat brain contain enzymes which can cleave PRL into a 16-kD fragment under acidic conditions. The majority of the enzymatic activity is localized in membrane-bound particles with a density similar to subcellular particles which contain PRL.     

Colocalization and hormone regulation of estrogen receptor alpha and N-methyl-D-aspartate receptor in the hypothalamus of female rats

Effects of N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation on neuroendocrine function can be modulated by the steroid hormone milieu. For example, the hypothalamic GnRH neurons, the primary cells regulating reproductive function, are stimulated by NMDAR agonists, and this is greatly potentiated by estrogen. We hypothesized that the actions of glutamate and estrogen may converge at target cells in the brain in which the NMDA and estrogen receptors (ERs) are coexpressed. To this end, we used quantitative stereological techniques to determine the colocalization of the obligatory NMDAR subunit, NR1, and the ERalpha, in the anteroventral periventricular nucleus and the medial preoptic nucleus, two critical regions for reproductive physiology and behavior. We observed extensive colocalization of ERalpha and NR1 in these brain regions (approximately 80%). In the anteroventral periventricular nucleus, treatment of ovariectomized rats with estrogen up-regulated the coexpression, whereas in the medial preoptic nucleus, estrogen had no effect, demonstrating a regional specificity to the estrogen sensitivity. The number of ERalpha cells that did not express NR1 was not altered by estrogen treatment in either brain region. Thus, we speculate that the extensive colocalization of ERalpha and the NMDAR provides an anatomical level at which estrogen and glutamate can act at target cells, and potentially synergize, to influence neuroendocrine and autonomic functions.     

Selective estrogen receptor modulator effects in the rat brain.

The effects in the brain of selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene have not yet been fully elucidated. Based upon the hypothesis that serotonin (5-HT)-steroid hormone interactions are important in mood regulation, we have compared six SERMs (tamoxifen, raloxifene, levormeloxifene, NNC 45-0781, NNC 45-0320, NNC 45-1506) with 17beta-estradiol (E(2)) in terms of their ability to regulate mRNA levels of estrogen receptor (ER)alpha, ER beta, 5-HT(1A) receptor, and 5-HT reuptake transporter (SERT) in the midbrain, amygdala, and hypothalamus of ovariectomized (OVX) rats. Female rats (n = 6/group, 8 groups total) were OVX and allowed to recover for 2 weeks. During the third post-OVX week, rats were injected subcutaneously with E(2) (0.1 mg/kg) or one of the SERMs (5 mg/kg) once per day for 7 days. Twenty-four hours after the last injection, tissue was collected for the determination of mRNA levels by ribonuclease protection assay (RPA). E(2) treatment significantly decreased mRNA levels for ER alpha, ER beta, and SERT in midbrain and ER alpha in hypothalamus. Tamoxifen increased ER beta mRNA levels in hypothalamus, while raloxifene increased ER beta mRNA levels in amygdala. NNC 45-0320 decreased ER alpha mRNA in hypothalamus and decreased ER beta mRNA in amygdala. These results suggest that while SERMs are not full estrogen receptor agonists in the brain, the agonist/antagonist profiles for individual SERMs may differ among brain areas. This raises the possibility of developing new SERMs for selective functions in specific brain areas.     

Changes in ontogenetic expression of estrogen receptor alpha and not of estrogen receptor beta in the female rat reproductive tract

To evaluate ontogenetic expression and localization of estrogen receptor (ER) alpha and beta in fetal female rat reproductive tract, competitive RT-PCR and immunohistochemistry were performed. Expression levels for Müllerian ERalpha, ERbeta1 and ERbeta2 mRNAs were determined by competitive RT-PCR. ERalpha expression on gestational day (GD) 15 x 5 increased 4 x 4-fold by GD 21 x 5, whereas both ERbeta1 and ERbeta2 gene expression were maintained at lower constant levels compared with ERalpha during development. ER immunolocalization was evaluated within three regions along the Müllerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. Nuclear ERalpha was localized predominantly in proximal Müllerian epithelium, and middle and caudal Müllerian mesenchyme on GDs 15 x 5-21 x 5. Staining intensity for ERalpha increased with development in all regions. However, ERbeta immunoreactivity was not detected in any region during prenatal life after separate staining with three different polyclonal anti-rat ERbeta antibodies. These findings provide fundamental information critical for clarifying the species-specific physiological roles of ER subtypes during fetal development and for investigating the tissue-specific mechanisms underlying the prenatal response to estrogen and estrogen receptor agonists.     

A subset of neurons containing immunoreactive prolactin is a target for estrogen regulation of gene expression in rat hypothalamus

Cells whose nuclei accumulated 3H-estradiol were identified autoradiographically in fixed, frozen sections of colchicine-treated rat hypothalamus (n = 3 animals). After autoradiogram development, these sections were subjected to immunocytochemistry using rabbit antirat prolactin antiserum and the avidin-biotinylated horseradish peroxidase method. In the hypothalamus, a substantial subset of the neurons containing immunoreactive prolactin accumulated 3H-estradiol in their nuclei: of 3, 642 immunoreactive cells examined, 1,216 had autoradiographically labeled nuclei, or about 33%. The immunoreactive prolactin neurons with autoradiographically labeled nuclei were located in the medial basal hypothalamus intermingled with immunoreactive prolactin neurons whose nuclei were not labeled autoradiographically. Since hypothalamic immunoreactive prolactin neurons have a rich and widely distributed fiber system, the present results suggest that estrogen, acting through a subset of these neurons, can modify directly the neuronal activity of several brain regions which regulate diverse aspects of the reproductive effort. Also, since immunoreactive prolactin and immunoreactive beta-endorphin exist in the same hypothalamic cell population, opioid peptides derived from pro-opiomelanocortin may mediate some effects of estrogen on the neural circuitry regulating reproduction.     

Effect of estrogen on androgen receptor dynamics in female rat pituitary

We examined the role of estrogen in regulating the number of androgen receptors (AR) in the pituitary gland of the female rat. Female Sprague-Dawley rats (240-280 g) that were ovariectomized for 3 days were used in this study. AR numbers were determined in pituitary cytosol and nuclear fractions by binding and exchange assays using the synthetic ligand 17 alpha-methyl-3H-trienolone. The administration of a single dose of estradiol benzoate [(EB) 10 micrograms/100 g BW] to ovariectomized female rats resulted in a 60% increase in cytosolic AR which was significantly (P less than 0.01) elevated above that of oil-treated controls by 12 h post EB. Cytosolic AR levels remained elevated for as long as 48 h post EB (213% of controls). Saturation analysis of pituitary cytosolic AR revealed a single, high affinity binding site for 17 alpha-methyl-3H-trienolone exhibiting an apparent dissociation constant (Kd) of 0.5 X 10(-10) M in both EB- and oil-treated animals. The administration of cycloheximide (1 mg/kg BW) before EB administration prevented the EB-induced rise in AR when measured 8 h post EB. When dihydrotestosterone (1.5 mg) was injected 24 h after EB or oil, there was a rapid increase in nuclear AR accompanied by a rapid decrease in cytosolic AR. The increase in nuclear AR was significantly greater (P less than 0.05) in EB-pretreated animals vs. oil-treated controls. These observations show that a potential synergism exists between androgen and estrogen in the female rat pituitary and suggest that androgens may play an important role in regulating cyclic pituitary hormone release.     

Fos expression in the female rat brain during the proestrous prolactin surge and following mating

A prolactin (PRL) surge occurs in the female rat during proestrus in response to elevated estradiol levels. The elevated release of ovarian steroids on the day of proestrus is also associated with sexual receptivity. Mating triggers twice-daily PRL surges that supplant the proestrous PRL surge and are responsible for maintaining luteal function during the first half of pregnancy. In order to understand the neuronal mechanisms controlling the proestrous- and mating-induced PRL surges, we examined patterns of Fos expression by immunocytochemistry in specific brain regions as a measure of neuronal activity. Intact female rats were sacrificed at 09.00, 15.00, and 18.00 h on the day of proestrus and the day of diestrus. Brain tissues were also collected at 21.00 h on the day of proestrus from rats receiving intromissions or mounts from males or taken directly from their homecage. On the day of proestrus, the number of neurons expressing Fos in the medial preoptic area (mPOA), medial amygdaloid nucleus (mAMYG), and ventromedial nucleus of the hypothalamus (VMH) was few and was associated with low plasma PRL levels at 09.00 h; however, the number of Fos-positive cells in these brain regions significantly increased at 15.00 and 18.00 h when the proestrous PRL surge occurred. Mating during the evening of proestrus resulted in a dramatic increase in the number of Fos-positive cells in the mPOA, mAMYG, and VMH as well as in the arcuate nucleus (ARC). Nonmated animals in diestrus showed low and unchanged PRL levels and Fos expression in all the brain areas throughout the day. These results suggest that the mPOA, mAMYG, VMH and ARC may be important brain sites for the integration of stimuli associated with proestrous- and mating-induced PRL surges.     

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.     

Differential regulation of estrogen receptor alpha and beta mRNAs in the rat uterus during pregnancy and labor: possible involvement of estrogen receptors in oxytocin receptor regulation

Oxytocin receptor (OTR) mRNA levels in the uterus dramatically increase in the near term human and rat. Estrogen is believed to be a potent stimulator of OTR mRNA expression. However, estrogen does not stimulate rat OTR mRNA expression on day 18 of pregnancy or in progesterone-treated rats. Thus, the regulation of uterine responsiveness to estrogen in the near term rat appears to be an important mediator of estrogen action. To determine the effect of altering uterine responsiveness to estrogen on OTR induction, uterine ERalpha and ER beta mRNA levels were examined by competitive RT-PCR in pregnant and parturient rats, progesterone-treated ovariectomized (OVX) virgin rats and OVX pregnant rats. In pregnant and parturient rats, OTR mRNA levels were highest at 2200-2230 h on day 21 of pregnancy (P21pm) and during labor when compared with other groups. ERalpha mRNA levels significantly increased during labor compared with days 15-21 of pregnancy. Compared with control animals, ERalpha mRNA levels decreased significantly in OVX virgin rats implanted with tubes containing progesterone for one week; 24 h following the removal of the progesterone tubes, ERalpha mRNA levels were found to be similar to control levels. Estrogen treatment following OVX on day 18 of pregnancy caused increased OTR mRNA levels, whereas ovariectomy alone increased ERbeta mRNA but not ERalpha mRNA. Results from the present study suggest that ERalpha and ERbeta mRNA expressions are differentially regulated in the rat uterus. Moreover, during spontaneous labor our findings appear to suggest that ERalpha plays a more prominent role than ERbeta in mediating estrogen action in the induction of uterine OTR mRNA before labor.     

Disruption of estrogen receptor beta gene impairs spatial learning in female mice

Here we provide the first evidence, to our knowledge, that estradiol (E(2)) affects learning and memory via the newly discovered estrogen receptor beta (ERbeta). In this study, ERbeta knockout (ERbetaKO) and wild-type littermates were tested for spatial learning in the Morris water maze after ovariectomy, appropriate control treatment, or one of two physiological doses of E(2). Regardless of treatment, all wild-type females displayed significant learning. However, ERbetaKOs given the low dose of E(2) were delayed in learning acquisition, and ERbetaKOs administered the higher dose of E(2) failed to learn the task. These data show that ERbeta is required for optimal spatial learning and may have implications for hormone replacement therapy in women.     

Binding and cross-linking of iodinated rat prolactin to rat hepatic prolactin receptor

Basal parameters for binding and cross-linking of 125I-rat prolactin (rPRL) to lactogenic (PRL) binding species present in crude membrane fraction (CMF) or detergent-solubilized preparations of rat liver have been investigated. (1) The highest specific binding to CMF was obtained with an incubation time of 50 h at 20 degrees C and with a 50 mM potassium phosphate buffer adjusted to pH 8.5. (2) Cross-linking of 125I-rPRL to binding sites in CMF with disuccinimidyl suberate (DSS) showed the autoradiographic appearance of an Mr 40,000 binding species. (3) No specific binding or cross-linking of rPRL was seen in Triton X-100-solubilized CMF. This is probably due to Triton X-100-induced changes in the physical properties of rPRL. (4) Specific binding of 125I-rPRL was detected in CHAPS-solubilized CMF. Following cross-linking the autoradiographic appearance of a binding species with an Mr value of 40,000 was shown. 125I-hGH was cross-linked to three PRL binding species with Mr 82,000, 40,000 and 35,000 in CHAPS-solubilized preparations. (5) In Golgi-enriched low-density membrane preparation 125I-rPRL was cross-linked to Mr 82,000, 40,000 and 35,000 species. It is proposed that the inability of rPRL to be cross-linked to Mr 82,000 and 35,000 species present in CHAPS-solubilized preparation is the result of CHAPS-induced changes of rPRL binding properties and low solubilizing capacity of CHAPS. (6) In conclusion, this study shows that also the iodinated endogenous hormone, rat prolactin, and not only hGH identifies high and low molecular forms of the rat liver prolactin receptor.