Dynamic aspects of the LHRH system associated with ovulation in the little brown bat (Myotis lucifugus).

Adult female bats were collected from natural roosting sites in pre-ovulatory and post-ovulatory conditions. LHRH neurones of these animals were examined using light and electron microscopic immunocytochemistry, and LHRH tissue contents were measured by radioimmunoassay. Comparisons between the two groups of bats revealed that the number of LHRH perikarya detected immunocytochemically, as well as hypothalamic LHRH content, were significantly reduced in post-ovulatory animals. Distributions of immunoreactive perikarya were, however, strikingly similar in both groups. The reduction in immunoreactive cell number observed after ovulation was therefore not restricted to an anatomically defined subset of neurones, but was evident throughout the population. The projection of LHRH-immunoreactive fibres that extend into the pituitary neural lobe in this species also exhibited changes related to endocrine condition. Morphometric indices of fibre density in the neural lobe were significantly reduced in post-ovulatory bats, as was LHRH content of the lower infundibular stalk and neural lobe. Fine structural study of perikarya revealed complex anatomical interactions between LHRH-immunopositive elements, especially in post-ovulatory bats. These interactions included direct apposition of perikarya, as well as more elaborate networks involving various combinations of perikarya and large- and small-caliber processes. These changes in the LHRH system associated with ovulation suggest reduction of stored peptide within perikarya and depletion from terminals within the lower infundibular stem and neural lobe. Parallel reductions in hypothalamic and neural lobe LHRH content during the periovulatory period support the hypothesis that the neural lobe component of the system contributes to control of gonadotrophin secretion in this species. Finally, increased complexity of anatomical contact between components of the LHRH system may be related to activation of this cell population in spring.

GRF immunoreactive neurons in the paraventricular nucleus of the rat: an immunohistochemical study with monoclonal and polyclonal antibodies.

Our study demonstrates a complex GRF neuronal system within the rat hypothalamus. Using both high affinity polyclonal and high specificity monoclonal antibodies to rat (r) GRF, we have substantiated evidence for immunoreactive GRF (GRF-i) perikarya in the parvocellular portion of the paraventricular nucleus. Other hypothalamic areas containing rGRF-positive perikarya include the lateral arcuate nucleus, lateral hypothalamus, perifornical area and dorsomedial nucleus. GRF-i neuronal terminals were seen in the external zone of the median eminence, more rostrally in the periventricular nucleus, and near the suprachiasmatic nucleus and more caudally in the dorsomedial nucleus and ventral premammillary nucleus.

Corticotropin-releasing factor in the dog adrenal medulla is secreted in response to hemorrhage.

To characterize the nature of CRF-like immunoreactivity (CRF-LI) in the dog adrenal, adrenal medullary, adrenal cortical, or hypothalamic tissue was extracted and subjected to RIA after partial purification on C-18 cartridges or Sephadex G-50. Using N- and C-terminal-directed antisera against rat/human (r/h) CRF, significant levels of CRF-LI were found in the adrenal medulla and hypothalamus, but not in the adrenal cortex. Immunocytochemical analysis revealed that CRF-immunoreactive cells were located in the adrenal medulla, many of them concentrated in the vicinity of blood vessels and at the border between adrenal medulla and cortex. However, the cortex was devoid of any CRF-positive cells. On reverse phase HPLC, CRF-LI in the adrenal medulla coeluted with synthetic r/hCRF. In a bioassay system, using dispersed rat anterior pituitary cells, purified adrenal CRF caused a dose-dependent increase in ACTH secretion parallel to the r/hCRF standard, indicating that dog adrenal medulla contains authentic r/hCRF. Evidence of CRF-LI secretion from the adrenal was supported by its presence in adrenal venous, but not in peripheral arterial, plasma. Adrenal venous plasma CRF-LI coeluted with r/hCRF on reverse phase HPLC after affinity chromatographic purification. The CRF-LI secretory rate in conscious trained dogs was 68 +/- 19 pg/min (concentration, 27 +/- 5 pg/ml). In response to 20% hemorrhage, the CRF-LI secretion rate rose 3-fold within 15 min and was associated with increased catecholamine secretion. The existence of a biologically active CRF-like substance in the dog adrenal medulla and its secretion in conjunction with catecholamines after a hemorrhage suggest a physiological role for this peptide other than pituitary or central nervous system regulation.

Luteinizing hormone-releasing hormone neurons in human preoptic/hypothalamus: differential intraneuronal localization of immunoreactive forms.

Luteinizing hormone-releasing hormone (LRH) may be synthesized as part of a larger prohormone, as are several other neuropeptides. In this study, we sought not only to define the distribution and morphological characteristics of LRH neurons within the human preoptic area and hypothalamus, but also to identify sites of initial synthesis, posttranslational conversion to the decapeptide, and storage of LRH in these neurons. Immunoreactive molecular forms were differentiated using a series of antisera with distinct specificities in the peroxidase-antiperoxidase technique. These antisera were capable of detecting the fully processed hormone as well as extended decapeptide sequences. Immunopositive LRH neurons were more abundant in the infundibular area of the hypothalamus than in the preoptic area. Numbers of immunopositive perikarya and subcellular distribution of reaction product varied with binding requirements of the antisera. After treatment with an antiserum that requires the fully processed decapeptide for binding, the reaction product was associated almost entirely with granules in perikarya and processes, while very little was associated with either rough endoplasmic reticulum (RER) or Golgi apparatus. In contrast, with an antiserum capable of detecting extended forms of the decapeptide, the RER and Golgi were labeled in addition to granules. From these data, we infer that in humans, mature decapeptide is present in granules within LRH neuronal perikarya and processes. Furthermore, the molecular forms associated with RER and Golgi may be precursors in which the decapeptide sequence is extended.

Seasonal variations in pituitary LH-gonadotropes of the hibernating bat Myotis lucifugus lucifugus: an immunohistochemical study.

Pituitary gonadotropes were identified throughout the year in the seasonally breeding, hibernating bat Myotis lucifugus lucifugus by means of light microscopic immunohistochemistry. In both male and female bats, these cells were immunoreactive with an antiserum directed to the beta subunit of luteinizing hormone. Some gonadotropes were aggregated near a portion of the infundibular stalk which crosses the anterior lobe, while most were scattered singly in a uniform manner throughout the rest of the pars distalis. This cell population exhibited seasonal variations in both sexes. In males, the proportional volume of the pars distalis occupied by immunoreactive gonadotropes (volume fraction) was significantly reduced in late July, when plasma testosterone levels were approaching their seasonal peak. In females, the volume fraction declined in April, following ovulation, and remained low during pregnancy and lactation. The size and shape of gonadotropes appeared relatively constant throughout the annual reproductive cycle in male bats; the immunoreactive cells were irregular in shape, with cytoplasmic extensions insinuating between and often "cupping" other secretory cell types. In females, the gonadotropes resembled those of males throughout most of the year, except during pregnancy, when these cells became enlarged and ovoid. No evidence of involution was observed in these anterior pituitary cells in either males or females during hibernation.

Luteinizing hormone-releasing hormone (LH-RH) cells and their projections in the forebrain of the bat Myotis lucifugus lucifugus.

Luteinizing hormone-releasing hormone (LH-RH) neurons and their projections were studied by immunocytochemistry in the brains of little brown bats (Myotis lucifugus lucifugus: Chiroptera: Vespertilionidae ) as a first step in the study of relationships between these neurons and the seasonal reproductive events characteristic of this species. The majority of immunoreactive neurons in adult male, adult female, and fetal bats were ovoid bipolar cells with one thin and one thicker process, both of which gave rise to fine varicose fibers. LH-RH-immunoreactive perikarya were concentrated in the region of the arcuate nuclei in all bats examined. Perikarya were also consistently found dispersed in the mammillary region, anterior hypothalamus, preoptic areas, septum, diagonal band of Broca, and olfactory tracts; they were occasionally observed in the dorsal hypothalamus, organum vasculosum of the lamina terminalis (OVLT), habenula, amygdala, and cingulate gyrus. LH-RH-immunoreactive fibers projected heavily to the median eminence, infundibular stalk, and posterior pituitary. In extrahypothalamic areas, these fibers were especially abundant in the stria medullaris/habenula and stria terminalis/amygdala, but also contributed to the diagonal band of Broca and the olfactory tracts. Immunoreactive fibers that may be components of many different pathways clustered in the rostral septum and permeated the medial hypothalamus. LH-RH-containing fibers frequently entered the subfornical organ, but were observed less often in the OVLT and only occasionally in the pineal. The organization of the LH-RH system in the little brown bat resembles that of primates, but differs considerably from that in the rat. Anatomical characteristics of the LH-RH system in bats thus suggest that this animal may be a particularly suitable species for further study of neuroendocrine control of reproductive function as it may relate to primates, including humans.

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.