Joan C. King

Immunocytochemical localization of estrogen-induced progestin receptors in guinea pig brain

By using a combination of monoclonal antibodies to progestin receptors and a double-bridge peroxidase-antiperoxidase technique, a sensitive immunocytochemical method was developed for visualizing progestin receptor immunoreactivity (PR-IR) in brains of estrogen-primed guinea pigs. PR-IR neurons were observed throughout the hypothalamus and preoptic area. They were seen in largest numbers in the arcuate nucleus, periventricular preoptic regions, medial preoptic nucleus, medial preoptic area and in the ventrolateral area of the hypothalamus. Lower numbers of PR-IR positive cells were detected in the bed nucleus of stria terminalis, paraventricular nucleus and lateral hypothalamus with scattered cells seen throughout the hypothalamus and preoptic area. The PR-IR was mostly intranuclear with lighter staining in neuronal processes. Electron microscopy confirmed the predominantly intranuclear localization and further demonstrated that the reaction product was dispersed throughout the nucleus, but not associated with the nucleolus. Few PR-IR cells were observed in the absence of estradiol priming, and the reaction product was much lighter than in the presence of estradiol. Progesterone injection was without apparent effect on intensity of the PR-IR.

LHRH neurons and their projections in humans and other mammals: Species comparisons

Using light microscopic immunocytochemistry, we have identified LHRH neurons and their projections in humans, monkeys, ferrets, bats and rats. In all these species, LHRH neurons project to the vascular contact zone of the ME, but positions of perikarya vary. This cell population, confined largely to rostral forebrain regions in rats, expands into the medial basal hypothalamus in humans, rhesus monkeys, ferrets and bats. Accompanying this expansion is an augmentation of extrahypothalamic LHRH projections. In rats, LHRH projections are primarily confined to the ME and OVLT. In humans, monkeys, ferrets and bats, however, there are also substantial projections to the posterior pituitary, habenular complex and amygdala. Although the significance of these extrahypothalamic projections is unknown, LHRH may function at some of these sites as a neuromodulator. Humans, monkeys, ferrets and bats further differ from rats in the apparent presence of mature decapeptide within perikarya. Whether variations in the dynamics of maturation of LHRH are related to differences in location of these neurons is currently under investigation.

EXAMINATION OF GUINEA PIG LUTEINIZING HORMONE-RELEASING HORMONE GENE REVEALS A UNIQUE DECAPEPTIDE AND EXISTENCE OF TWO TRANSCRIPTS IN THE BRAIN

We sequenced the complementary DNA (cDNA) encoding guinea pig LHRH from an expression library derived from the preoptic area-anterior hypothalamus. Data from in situ hybridization and RNase protection assays verified that the cloned cDNA was complementary to guinea pig LHRH messenger RNA. The architecture of the deduced precursor resembles that of LHRH precursors identified in other species. In contrast, the predicted sequence of the decapeptide differs from mammalian LHRH by two amino acid substitutions in positions 2 and 7. This is a novel finding, because the amino acid sequence that comprises LHRH decapeptide is identical in all mammals studied to date. Moreover, the predicted substitution in amino acid position 2 is unique among vertebrates. A second observation of potential significance is the existence of two subspecies of LHRH messenger RNA differing only in the length of their 3′ untranslated regions. These two transcripts were verified by sequence analysis of positive clones from the cDNA librar...

Day-night variation in prepro vasoactive intestinal peptide/peptide histidine isoleucine mRNA within the rat suprachiasmatic nucleus☆

Neurons within the suprachiasmatic nuclei of the hypothalamus (SCN) appear to function as a circadian clock that controls the timing of many physiological systems. The SCN contain several chemically distinct neuronal subpopulations, including a large group of interneurons within the ventrolateral SCN that exhibit co-localizable immunoreactivity for both vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The purpose of the present study was to determine whether VIP/PHI neurons within the rat SCN exhibit rhythmicity in the cellular levels of the messenger RNA encoding the precursor from which both VIP and PHI are derived. Using both quantitative in situ and solution hybridization prepro-VIP/PHI mRNA levels early in the dark phase were demonstrated to be significantly higher than those 5 h after the onset of the daily light period. Since no statistically reliable (P greater than 0.05) day-night variation was observed in the levels of prepro-VIP/PHI mRNA within cortex, these data suggest that the rhythmicity in prepro-VIP/PHI mRNA is an intrinsic property of VIP/PHI-containing SCN neurons, or rhythmically driven by local synaptic events within the SCN.

Corticotropin releasing hormone mRNA is elevated on the afternoon of proestrus in the parvocellular paraventricular nuclei of the female rat

We investigated changes during the estrous cycle in cellular levels of corticotropin-releasing hormone (CRH) mRNA in parvocellular neurons of the hypothalamic paraventricular nucleus, using in situ hybridization. Intact female rats with 4 day cycles were sacrificed at 11 different times during the cycle at 09.00 h and 16.00 h on each day, with additional collection times at 14.00 h, 18.00 h, and 20.00 h on the day of proestrus. Twelve microns coronal sections of fresh-frozen brains were made through the paraventricular nuclei (PVN) and placed on gelatin-coated slides. A 48 base oligodeoxynucleotide probe complementary to the coding region for rat CRH was used to measure CRH mRNA. There was a sharp increase (P less than 0.01) in CRH mRNA in the ventral PVN between 14.00 and 16.00 h on the day of proestrus, at the approximate time of the ovulatory surge. Following this rise, there was an even larger decline (P less than 0.01) between P 16.00 h and P 20.00 h. Levels of CRH mRNA did not change greatly on other days of the cycle, nor were there significant changes in the dorsal PVN. Given the known effects of CRH on GnRH secretion, these changes occur at a time when they could serve to modulate the midcycle luteinizing hormone (LH) surge.

Cells containing immunoreactive estrogen receptor-α in the human basal forebrain

The distribution of estrogen receptor protein-α (ER-α)-containing cells in the human hypothalamus and adjacent regions was studied using a monoclonal antibody (H222) raised against ER-α derived from MCF-7 human breast cancer cells. Reaction product was found in restricted populations of neurons and astrocyte-like cells. Neurons immunoreactive for ER-α were diffusely distributed within the basal forebrain and preoptic area, infundibular region, central hypothalamus, basal ganglia and amygdala. Immunoreactive astrocyte-like cells were noted within specific brain regions, including the lamina terminalis and subependymal peri-third-ventricular region. These data are consistent with the location of estrogen receptors in the basal forebrain of other species and the known effects of estrogens on the cellular functions of both neurons and supporting elements within the human hypothalamus and basal forebrain.

Dynamic changes in LHRH neurovascular terminals with various endocrine conditions in adults.

Luteinizing hormone-releasing hormone (LHRH) release is required for ovulation in mammals. Although evidence for the direct action of gonadal steroids on LHRH neurons has been minimal, their importance in inducing the preovulatory surge of LHRH is unequivocal. We have identified a subgroup of LHRH neurons with progestin receptors in guinea pigs. Given their central position, these neurons may constitute foci of initial activity, which are amplified throughout the population of LHRH neurons, resulting in increased LHRH neurosecretion on the afternoon of proestrus. Additionally, gonadal steroids may regulate LHRH secretion at the level of the terminals. Using immunoelectron microscopy and image analysis, we have illustrated the dramatic influence of gonadal steroids on individual LHRH terminals in the median eminence of rats. Indirectly, gonadal steroids may modulate LHRH release by modulating glial elements. Using double-label fluorescence confocal microscopy, we illustrate that LHRH terminals in the median eminence are encased by end-feet of tanycytes. Acting on glial elements, gonadal steroids may regulate access of LHRH terminals to the basal lamina and influence the amount of the neuropeptide reaching the portal vessels. We propose that during the preovulatory surge, LHRH release is coordinated by synergistic mechanisms operating at the level of particular subgroups of neuronal perikarya and/or discrete regions of the median eminence. These synergistic actions may ensure that LHRH is released in a precipitous fashion, to induce the surge of LH from the pituitary, required for ovulation.

Human brain contains vasopressin and vasoactive intestinal polypeptide neuronal subpopulations in the suprachiasmatic region

The suprachiasmatic nuclei (SCN) and retinohypothalamic tract ( RHT ) in the anterior hypothalamus have been postulated to play an important role in the timing of daily biological rhythms in mammals. Although physiological studies have described circadian rhythms in man, the presence of an RHT or SCN has not been conclusively demonstrated in the human brain. Immunocytochemical identification of distinct ventral vasoactive intestinal polypeptide (VIP) containing and dorsal vasopressin containing neuronal subpopulations in the human suprachiasmatic region provides correlative evidence of neuronal clusters which are homologous to discrete cell groups in the SCN of other mammalian species. Manipulation of the circadian system has been used to treat some affective illnesses and other physiological timing disorders. Characterization of the neural substrates underlying human circadian rhythms could be useful in the development of future treatment modalities and is essential for understanding normal human circadian organization.

A Subgroup of LHRH Neurons in Guinea Pigs with Progestin Receptors Is Centrally Positioned within the Total Population of LHRH Neurons

Although the role of gonadal steroids in inducing the LH surge is undisputed, the mechanism(s) whereby steroids induce the release of the hypothalamic luteinizing hormone-releasing hormone (LHRH) remain(s) enigmatic. In this study we examined the issue of the presence of steroid receptors in LHRH neurons using a mammalian species that has a true luteal phase, namely, guinea pigs. Progestin receptors (PR) were localized in LHRH neurons of ovariectomized guinea pigs administered estradiol (10-20 micrograms estradiol benzoate) for 3-4 days, using several different immunocytochemical protocols. The subgroup of LHRH neurons containing PR, although small, was strategically positioned within the core of the total population of LHRH neurons. This central position was visualized in simultaneous views of three-dimensional computer reconstructions of the populations of LHRH/PR neurons and LHRH neurons. The subgroup of LHRH/PR neurons formed a thread permeating the population of LHRH neurons. We propose that in guinea pigs, LHRH neurons containing progestin receptors, are foci of activity, capable of activating a larger component of the LHRH population of cells in certain endocrine conditions, such as prior to the LH surge.

Neuronal systems immunoreactive with antiserum to lamprey gonadotropin-releasing hormone in the brain of Petromyzon marinus.

SummaryThe role of gonadotropin-releasing hormone (GnRH) in mammalian reproduction has been studied extensively; however, the role of a structurally different, but related, decapeptide is not well characterized in the most primitive class of vertebrates, Agnatha. Utilizing an antiserum directed to the recently characterized lamprey GnRH, we examined immunoreactive neuronal perikarya and nerve fibers in sections from the brain of the sea lamprey, Petromyzon marinus, using the unlabeled peroxidase-antiperoxidase method. Neuronal perikarya and fibers were immunopositive with antisera generated to lamprey GnRH and also to certain antisera generated to mammalian GnRH. Immunopositive neuronal perikarya were detected in an arcshaped population extending from ventral to dorsal preoptic areas. Fibers from these cells projected to the neurohypophysis via the preoptico-hypophyseal tract, but in addition also protruded into the third ventricle. Additionally, some fibers coursed along the external surface of the brain, and may also release GnRH into meningeal compartments. The presence of fully processed, mature decapeptide is indicated within neuronal perikarya, as well as in projecting nerve fibers and terminals. No reaction product was detected in sections incubated with an antiserum to the interior amino acid sequences of mammalian LHRH. This finding supports the structure reported for lamprey GnRH by Sherwood et al. (1986).