Localization of the VIP2 receptor protein on GnRH neurons in the female rat.

In mammals, the timing and occurrence of the preovulatory LH surge critically depends on the proper functioning of the suprachiasmatic nucleus (SCN). Recent evidence suggests that vasoactive intestinal polypeptide (VIP) conveys time of day information from the SCN to GnRH neurons. However, it is not completely clear whether this action is exerted directly at the level of the GnRH neuron. To determine if GnRH neurons are direct targets for VIP, triple-label immunofluorescence was utilized to simultaneously localize GnRH, VIP and VIP2 receptor protein. The present results demonstrate that about 40% of all GnRH neurons analyzed contain VIP2 receptor immunoreactivity and that VIP-containing processes were seen in close apposition to a significant number of VIP2 receptor-positive GnRH neurons. GnRH neurons that exhibit immunoreactivity for the VIP2 receptor are located predominantly in the OVLT region and the rostral preoptic area. In the median eminence, where the majority of GnRH neurons terminate, VIP2 receptor immunoreactivity was absent. In summary, these findings indicate that VIP can communicate directly with 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.

Fetal grafts containing suprachiasmatic nuclei restore the diurnal rhythm of CRH and POMC mRNA in aging rats.

We assessed whether fetal tissue containing the suprachiasmatic nuclei (SCN) can restore age-related changes in the diurnal rhythm of hypothalamic corticotropin-releasing hormone (CRH) and anterior pituitary proopiomelanocortin (POMC) mRNA. Young, middle-aged, and middle-aged SCN-transplanted rats were killed at seven times of day. In young rats, CRH mRNA exhibited a diurnal rhythm in the dorsomedial paraventricular nuclei but not in other subdivisions of the nuclei. No rhythm was detected in aging rats. SCN transplants restored a rhythm in CRH mRNA, but the timing was not precisely the same as in young animals. POMC mRNA exhibited a daily rhythm in young rats. Aging abolished the rhythm and decreased the average mRNA level; fetal transplants restored the rhythm, but the amplitude remained attenuated. These data are the first demonstration that fetal tissue can restore the diurnal rhythm of a neuroendocrine axis that is driven by the SCN. We conclude that the neuroendocrine substrate from the aging host remains capable of responding to diurnal cues to express diurnal rhythmicity in CRH/POMC mRNA when fetal SCN transplants confer the appropriate signals.

Menopause: the aging of multiple pacemakers.

Menopause signals the permanent end of menstrual cyclicity in a woman's life. Its impact reaches far beyond just the reproductive system. An understanding of the factors that interact and govern the process of aging in the reproductive system will help us to develop strategies for alleviating the negative aspects of menopause and may help us to better comprehend the process of biological aging.

Prolactin receptor gene expression in specific hypothalamic nuclei increases with age.

Increasing prolactin levels or increasing responsiveness to prolactin may contribute to reproductive aging by influencing the secretory patterns of hypothalamic GnRH, pituitary gonadotropins, and/or ovarian steroids. Some studies have documented changes in the levels of prolactin in peripheral plasma. The goal of this study was to determine whether prolactin receptor mRNA levels in the brain change with aging, which may lead to increasing responsiveness to prolactin. Young (2-4 months) and middle-aged (9-11 months), demonstrating 3 consecutive estrous cycles, and old (16-19 months) and very old (20-21 months) rats, exhibiting repeated pseudopregnancies, were bilaterally ovariectomized. They were implanted with Silastic capsules containing estradiol-17 beta one week later, and killed 2 days after capsule implantation. Changes in prolactin receptor gene expression were assessed using in situ hybridization. The level of prolactin receptor mRNA in choroid plexus, periventricular area of the preoptic nucleus, and arcuate nucleus increased significantly by the time the animals were old. In the lateral ventromedial nucleus, prolactin receptor gene expression did not change significantly during aging, even in the oldest group of rats. These findings suggest that changes in the prolactin receptor gene may influence the ability of prolactin to exert effects and may allow older animals to be more responsive to prolactin than young rats.

Effect of lactation on hypothalamic preproenkephalin gene expression.

Enkephalin appears to modulate several aspects of reproductive function in female rats. The purpose of this study was to determine if lactation influences preproenkephalin gene expression in one or more hypothalamic nuclei known to be involved in maternal or reproductive behavior and prolactin secretion. Lactating rats were killed on day 3 (LAC 3) or day 10 (LAC 10) of lactation. Controls consisted of regular 4-day cycling rats that were killed on diestrous day 1, with 9 to 12 females per group. We used in situ hybridization histochemistry to assess preproenkephalin gene expression in individual cells in the medial preoptic nucleus, anterior, medial and posterior arcuate nucleus, magnocellular and parvocellular aspects of paraventricular nucleus, and ventromedial nucleus. Preproenkephalin mRNA in the anterior arcuate nucleus increased to reach significance (P < 0.05) at day 10 of lactation. Levels in the medial arcuate nucleus increased significantly (P < 0.001) by day 3 of lactation (LAC 3) and remained elevated on day 10 (LAC 10). No significant differences between lactating and control rats were detected in preproenkephalin mRNA levels in the posterior arcuate nucleus, medial preoptic nucleus or in the ventromedial nucleus. Substantial levels of preproenkephalin mRNA were found in the paraventricular nucleus, particularly in a limited region of the magnocellular portion. However, these levels did not change with lactation. These data provide evidence for differential regulation of the preproenkephalin gene during lactation. This change may contribute to lactational hyperprolactinemia and suppressed GnRH secretion, leading to reproductive acyclicity.

Prolactin receptor mRNA localization in the hypothalamus by in situ hybridization.

Prolactin receptors may mediate the action of prolactin in the brain to influence behavior and neuroendocrine secretions. We recently demonstrated prolactin receptor gene expression in the anterior and medial basal hypothalamus and not in the cortex by the reverse transcription-polymerase chain reaction. In this paper, we localize the prolactin receptor gene expression to individual cells with in situ hybridization. Several steps in the in situ hybridization method were modified to increase sensitivity by using (i) probes complementary to the coding sequence of the extracellular binding domain common to both long and short prolactin receptor, (ii) more stringent hybridization and wash conditions to reduce background and (iii) higher specific activity, more complex and saturating amounts of probe. We detected prolactin receptor gene expression in cells of the periventricular area of the preoptic nucleus, medial preoptic nucleus, supraoptic nucleus, rostral arcuate nucleus and choroid plexus. Cortical brain tissue, which has been demonstrated previously by reverse-transcription polymerase chain reaction to be lacking in prolactin receptor mRNA, did not have any detectable signal for the receptor mRNA and was used as an indication of background levels of signal. The mean area of silver grains over labeled cells in periventricular area of the preoptic nucleus, medial preoptic nucleus, supraoptic nucleus, arcuate nucleus, lateral ventromedial nucleus was at least 10 times greater than the background in the cortex of the same brain section.

The effect of time of day on levels of hypothalamic proopiomelanocortin primary transcript, processing intermediate and messenger ribonucleic acid in proestrous and estrous rats.

Several lines of evidence from different laboratories suggest that hypothalamic beta-endorphinergic activity decreases around the time of initiation of the LH surge and may increase on estrus to extinguish the expression of the daily neuronal signal for the surge. In several hormone systems, factors that stimulate or suppress hormone release also stimulate or repress transcription of the hormone gene and translation of the messenger RNA encoding the hormone. Therefore, information about neurohormone activity may be inferred from data on changes in the levels of RNA species encoding these neurohormones. We used a solution hybridization/RNase protection assay to test the hypotheses that 1) the abundance of primary transcript of the hypothalamic POMC gene decreases at the time of initiation of the proestrous LH surge and 2) levels of POMC primary transcript (and by inference, levels of beta-endorphin neuronal activity and secretion) increase on estrus. 96 rats exhibiting at least two consecutive 4-day estrous cycles were killed at either 0600 or 1300 h on proestrus and estrus. Dissections of the medial basal hypothalamus were pooled into 4 samples at each time-point (6 rats per sample) and RNA was extracted from nuclear and cytoplasmic fractions separately. We measured levels of POMC primary transcript, processing intermediate and fully spliced mRNA in the nuclear fractions and POMC mRNA in cytoplasmic fractions. Compared to 0600 h, levels of POMC primary transcript decreased significantly during the afternoons of both proestrus and estrus (P < 0.05). Levels of nuclear processing intermediate RNA and cytoplasmic mRNA followed the same trend but the afternoon declines did not reach statistical significance. We conclude from these data that the afternoon decline in POMC gene expression is not unique to the day of proestrus and we speculate that an afternoon decline in beta-endorphinergic neuronal activity may instead be a component of the daily signal for the LH surge.

Neuroendocrine ageing: its impact on the reproductive system of the female rat.

Numerous changes occur with age at all levels of the reproductive axis. Clearly, changing ovarian function plays a critical role in the cessation of reproductive cycles. Likewise, many changes in the function of neurotransmitters, gonadotrophin-releasing hormone and gonadotrophin itself appear to contribute to the ageing of the reproductive axis. It appears that the effects of ageing on neural time-keeping mechanisms may be important in the cascade of events that lead to reproductive dysfunction. The use of modern neurobiological and molecular methods to assess neural function within small regions of the brain should deepen our understanding of the complex interactions between neuroendocrine systems that underlie female reproductive cycles and cause them to change with age.

Detection of prolactin receptor (PRL-R) mRNA in the rat hypothalamus and pituitary gland.

Prolactin receptor (PRL-R) mRNAs exist in several tissues where prolactin is known to act including the liver, testes, prostate, ovary, mammary gland, adrenal gland and kidney. PRL also acts at the level of the hypothalamus and pituitary gland to feed back and regulate its own secretion and the secretion of other anterior pituitary hormones. Therefore, we hypothesized that PRL-R mRNA would exist in these target tissues as well. Total RNA was extracted from rat anterior and medial basal hypothalamus, anterior and posterior pituitary gland, cerebral cortex, skeletal muscle and liver. After reverse transcribing total RNA with Murine-MLV reverse transcriptase and random or oligo(dT) pmers, the polymerase chain reaction (PCR) was performed. PCR products were then analyzed by ethidium bromide staining. Using primers that flanked the coding region for the extracellular binding domain we detected PRL-R mRNA in the anterior and medial basal hypothalamus, anterior and posterior pituitary gland, as well as in the liver, but not in the cerebral cortex or skeletal muscle. In addition, when we used primers that distinguish the long and short forms of the PRL-R mRNA, both forms of the PRL-R mRNA were detectable in the same tissues. Our data suggest that PRL may feed back at the level of the hypothalamus and pituitary gland through the same short and/or long PRL-R mRNA that mediate PRL action in the peripheral tissues.

Aging progressively decreases the densities and alters the diurnal rhythms of alpha 1-adrenergic receptors in selected hypothalamic regions.

Associated with the decline in reproductive function that occurs with age in female rats are a diminished ability to secrete LH in response to ovariectomy as well as alterations in the preovulatory LH surge mechanisms. The noradrenergic system, an important regulator of LH release, exhibits age-related alterations in the pattern of neurotransmitter release. In addition, the density of alpha 1-receptors changes in intact old rats. To determine whether age-related changes in the pulsatile pattern of LH secretion are associated with declines in the densities and/or a loss of diurnal rhythm of alpha 1-receptors, we measured the density of alpha 1-receptors at various times of day in young, middle-aged, and old ovariectomized rats. By middle-age, mean concentrations of alpha 1-receptors decline in the median eminence and suprachiasmatic nucleus. By old age, mean concentrations of alpha 1-receptors decline in all regions of the hypothalamus examined but not in the dorsal lateral septum. In addition, the diurnal rhythms in alpha 1-receptors, which occur in the medial preoptic nucleus and the suprachiasmatic nucleus of young rats, are lost by middle-age. These data indicate that progressive alterations in the mean concentrations and diurnal rhythms of alpha 1-receptors occur with age in brain regions important for the regulation of reproductive functions and may contribute to the age-associated deficits in LH secretion.

Estrogen and progesterone regulate opiate receptor densities in multiple brain regions.

The negative and positive feedback, actions of estrogen and progesterone on the hypothalamus may be mediated in part by the endogenous opiate system. Estrogen and progesterone suppress physiological responses to the administration of opiate peptides. To determine whether this desensitization to opiates involves receptor down-regulation, we measured the density of naloxone-binding sites in hypothalamic regions regulating gonadotropin release and/or sexual behavior. Using autoradiographic techniques, we measured the density of naloxone-binding sites at various times of the day in intact proestrous, ovariectomized, or ovariectomized rats treated with estrogen or estrogen plus progesterone. Ovariectomy increased naloxone-binding sites compared to intact proestrous rats in all hypothalamic regions examined. Treatment with estrogen decreased and treatment with estrogen plus progesterone decreased even more the densities of naloxone-binding sites. This steroid-induced suppression of opiate receptors appears to have important physiological repercussions and provides a basis for the steroid-induced differential sensitivity to exogenous opiates that has been observed previously.

Hyperprolactinemia decreases naloxone binding in the arcuate nucleus of ovariectomized rats.

Hyperprolactinemia suppresses endogenous prolactin (PRL) secretion and inhibits LH release in ovariectomized rats. Opiate peptides appear to mediate the suppressive effects of hyperprolactinemia on both endogenous PRL and LH secretion. A mechanism by which hyperprolactinemia may change the ability of opiates to influence PRL and LH is by altering the density of opiate receptors. We, therefore, examined the effect of hyperprolactinemia on the density of naloxone binding sites in hypothalamic regions that are important in the regulation of PRL and LH secretion. Female rats, ovariectomized for 4 days, were treated with ovine prolactin (oPRL) every 8 h for 2 days, and naloxone binding sites were measured using autoradiographic procedures. oPRL treatment suppressed the concentration of naloxone binding sites throughout the arcuate nucleus but had no effect in the median eminence, suprachiasmatic nucleus, and medial preoptic nucleus. There is evidence that the tuberoinfundibular dopaminergic neurons of the arcuate nucleus are directly influenced through opiate receptors. We propose that the observed decrease in the density of opiate receptors may occur on dopaminergic neurons. This theory provides an explanation for a mechanism for the suppression of endogenous PRL and LH by hyperprolactinemia: a decrease in opiate receptors will decrease opiate suppression of dopamine neurons allowing dopamine activity to increase. Increase in dopamine release are known to decrease PRL and LH secretion in ovariectomized rats. Alternatively, decreased naloxone binding may result from homologous down-regulation of receptors due to increased opiate activity. If opiate activity increases, it may directly inhibit LHRH neurons and may suppress the activity of inhibitory neurons leading to increased dopamine activity.

Age-associated alterations in catecholaminergic concentrations, neuronal activity, and alpha 1 receptor densities in female rats.

The density of alpha 1-adrenergic receptors and catecholamine concentrations and neuronal activity were assessed in selected estrogen-responsive regions of the hypothalamus and pineal glands. The results of this study demonstrate that the densities of alpha 1-adrenergic receptors and norepinephrine and epinephrine concentrations and activity rate constants are altered in aged rats. The direction and extent of the change depend on the specific brain region and reproductive state of the animals. No widespread decline with increased age was observed in any of the parameters measured.

Hypothalamic monoamines and their catabolites in relation to the estradiol-induced luteinizing hormone surge.

Monoamines and non-conjugated catabolites (serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 3,4-dihydroxyphenyl-acetic acid (DOPAC), homovanillic acid (HVA), 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), norepinephrine (NE), and dopamine (DA] were measured in the medial basal hypothalamus (MBH) and preoptic area (POA) of ovariectomized (OVX) and OVX estradiol (E2)-treated rats using high-performance liquid chromatography with electrochemical detection. These E2 treatments were sufficient to induce an LH surge. The use of MHPG/NE ratios as estimates of NE release was validated in the rat hypothalamus by the major decreases of MHPG after injection of the alpha 2-adrenergic agonist, clonidine, and by MHPG increases after the alpha 2-antagonist, yohimbine. The ratio, MHPG/NE, decreased between morning and afternoon in the MBH but not in the POA; there were no differences between OVX and E2-treated rats. Previous studies using a variety of methods indicate that NE turnover increases during LH surges. The present data suggest that unconjugated MHPG is not a sensitive measure of NE release in the rat hypothalamus, but can detect the large changes produced by stimulating or inhibiting the alpha 2-adrenergic autoreceptor. The ratios of DOPAC/DA and 5-HIAA/5-HT in the MBH decreased consistently between morning and afternoon in OVX rats, with or without E2 treatment. This suggests that the release of DA and 5-HT decreases during the day regardless of steroidal milieu.

The role of the hypothalamus in aging of the female reproductive system.

We have investigated the role of neuroendocrine and neurochemical changes in the age-related deterioration of cyclic female reproductive function. During middle age the timing and amplitude of the proestrous and estradiol-induced LH surge is altered. We have found that the diurnal pattern of norepinephrine turnover is altered in critical hypothalamic areas known to regulate the release of LHRH. These changes may contribute to alterations in the timing and the amplitude of LH release, which may, in turn, affect the ability of rats to maintain regular estrous cycles.

Effects of age on beta 1- and beta 2-adrenergic receptors in the brain assessed by quantitative autoradiography.

Aging female rats undergo a loss of reproductive cyclicity that has been correlated with alterations in norepinephrine neuronal activity in specific hypothalamic regions involved in the regulation of gonadotropin secretion. We explored the possibility that deteriorating reproductive function may also be due to changes in the density of receptors which bind and mediate responsiveness to norepinephrine. Existing evidence suggests that beta-adrenergic receptors mediate norepinephrine's inhibitory effect on luteinizing hormone (LH). Therefore, we determined whether changes in beta-adrenergic receptor densities occur in specific hypothalamic nuclei associated with reproductive function. Furthermore, we wished to determine whether age affects beta 1- and beta 2-adrenergic receptor densities differentially and whether age-related changes in receptor densities are related to hormonal status and/or vaginal smear cytology of the animals. Old constant estrous rats, old persistent diestrous rats, young cycling rats on estrus, and young cycling rats on diestrus were killed at 1000 h and brain beta 1- and beta 2-adrenergic receptor densities were measured using topical autoradiographic and computerized image analysis methods which permit a high level of neuroanatomical discrimination. We analyzed beta 1- and beta 2-adrenergic receptor densities in 7 brain areas including 4 hypothalamic regions known to be important in reproductive function. The pineal gland, the molecular layer of the cerebellum and the caudate putamen were examined because they contain high concentrations of beta-adrenergic receptors which have been reported to change with age. Serum luteinizing hormone was measured in the same rats. Serum LH concentrations were the same in all rats regardless of vaginal smear cytology and age.(ABSTRACT TRUNCATED AT 250 WORDS)

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.