Early expression of chicken gonadotropin-releasing hormone-1 in the developing chick.

Gonadotropin-releasing hormone (GnRH), which is essential for reproductive function, is made by neurones that migrate from the nasal region into the brain during early embryonic development. This migration begins in chick when the olfactory pit is formed. This is approximately the time that GnRH neurones can be detected immunocytochemically. The present study investigated (i). how early in development the GnRH gene is expressed and (ii). the sites of its expression. Accordingly, reverse transcriptase-polymerase chain reaction (PCR) and in situ hybridization were performed on chick embryos before gastrulation up until the stage by which GnRH neurones have begun to migrate into the central nervous system. Primers were made to the 5'- and 3'-UTR region of the message for cGnRH-I, the form of the peptide that is essential for reproductive function in the chicken. PCR product was found in all stages and the sequences of products from all stages were identical. Thus, the GnRH gene is expressed continuously throughout embryonic development. In situ hybridization with a digoxygenin labelled riboprobe revealed staining along the primitive streak immediately before gastrulation. In later stages, cGnRH-I gene expression was seen in association with the anterior neural ridge. The expression was subsequently restricted to a narrow, clearly defined region, which is associated with the presumptive nasal cavity and olfactory placode. Later, GnRH neurones could be seen in their migratory routes by both in situ hybridization and immunocytochemistry. Expression of the GnRH gene has been described in preimplantation stages in mammals and there is evidence that the neuropeptide plays a role in formation and maintenance of the placenta. What role (if any) it may play in early avian development remains unknown. The demonstration of sites of GnRH expression during the early period of neurulation suggests that GnRH neurones arise before olfactory placode formation.

Some hypothalamic hamartomas contain transforming growth factor alpha, a puberty-inducing growth factor, but not luteinizing hormone-releasing hormone neurons.

Activation of LH-releasing hormone (LHRH) secretion, essential for the initiation of puberty, is brought about by the interaction of neurotransmitters and astroglia-derived substances. One of these substances, transforming growth factor alpha (TGFalpha), has been implicated as a facilitatory component of the glia-to-neuron signaling process controlling the onset of female puberty in rodents and nonhuman primates. Hypothalamic hamartomas (HH) are tumors frequently associated with precocious puberty in humans. The detection of LHRH-containing neurons in some hamartomas has led to the concept that hamartomas advance puberty because they contain an ectopic LHRH pulse generator. Examination of two HH associated with female sexual precocity revealed that neither tumor had LHRH neurons, but both contained astroglial cells expressing TGFalpha and its receptor. Thus, some HH may induce precocious puberty, not by secreting LHRH, but via the production of trophic factors--such as TGFalpha--able to activate the normal LHRH neuronal network in the patient's hypothalamus.

Role of the cysteine-rich domain of the t-SNARE component, SYNDET, in membrane binding and subcellular localization.

Wild-type syndet is efficiently recruited at the plasma membrane in transfected AtT-20 cells. A deletion at the cysteine-rich domain abolishes palmitoylation, membrane binding, and plasma membrane distribution of syndet. Syndet, SNAP-25A, and SNAP-25B share four cysteine residues, of which three, Cys2, Cys4, and Cys5, are absolutely conserved in all three homologs. Mutations at any pair of cysteines within cysteines 2, 4, and 5 shift syndet from the cell surface into the cytoplasm. Thus, at least two cysteines within the conserved triplet are necessary for plasma membrane localization. Syndet C1S/C3S, with substitutions at the pair Cys1 and Cys3, distributes to the plasma membrane, a Golgi-like compartment, and the cytosol. We conclude that Cys1 and Cys3 are not absolutely necessary for membrane binding or plasma membrane localization. Our results show that the cysteine-rich domain of syndet plays a major role in its subcellular distribution.

Synchronized neuronal networks: the GnRH system.

The anatomical substrate for coordinated release from the dispersed gonadotropin-releasing hormone (GnRH) neuronal population remains obscure. There is physiological evidence that the GnRH hormone itself has a role in tonic inhibition or modulation of GnRH function. This has led to the hypothesis that there is an ultrashort negative feedback mechanism subserved by axon collaterals acting back on the GnRH neurons. Recent ultrastructural studies have revealed GnRH synapses on GnRH neurons and their processes. Furthermore, there are alterations in the frequency of these synapses with the age and hormonal condition of the animal. Another candidate for coordination of neuronal activity for which there is some evidence in the magnocellular system, is the gap junction. Recently, physiological and anatomical evidence for gap junctional modifications among an immortalized GnRH-secreting cell line (GT1) has been reported. However, at present there is no immunocytochemical or ultrastructural evidence for gap junctions between GnRH neurons. A third and highly unorthodox anatomical relationship between (among) these cells has been suggested by serial ultrastructural reconstructions of pairs of GnRH neurons in close association. In some regions, GnRH neuronal processes can be seen to extend from each member of a pair of GnRH neurons. These meet and merge, forming an intercellular bridge. This phenomenon has been observed in several pairs of GnRH neurons in rat and monkey. The important caveat in making these observations is that techniques employed to demonstrate sites of antigenicity can severely compromise the ultrastructural integrity of membrane components. For this reason, further verification of the existence of intercellular bridges is being pursued. Should their existence be confirmed, they would be prime candidates for the coordination of secretory events among the scattered GnRH neuronal population.

Embryonic development of the gonadotropin-releasing hormone (GnRH) system in the chick: a spatio-temporal analysis of GnRH neuronal generation, site of origin, and migration.

We present a quantitative immunocytochemical study of GnRH migration by developmental stage. GnRH peptide was detected in cells of the olfactory epithelium at stage 19. Migration was initiated a few hours later at stage 20. Of interest is the observation that GnRH neurons paused at the central nervous system border for 3 days, entering the brain at stage 29. The major expansions of the GnRH population occurred at two points; stages 26 and 42. In one animal a third population expansion occurred after hatching, with the number of GnRH cells reaching 6600. To determine the site of origin of GnRH cells, embryos were exposed to tritiated thymidine and killed 5 h later. Most GnRH cells incorporated label in the olfactory epithelium; however, some autoradiographically labeled GnRH cells, possessing a neuronal morphology, were found in the olfactory nerve and the forebrain, suggesting that some GnRH neurons divide as they migrate. A cumulative labeling method employing tritiated thymidine was used to examine the birth date of GnRH neurons. Postmitotic GnRH cells were first detected at stages 19-21. At stage 24, a peak in GnRH neurogenesis preceded the increase in GnRH neurons expressing their peptide at stage 26. After stage 24, there was a gradual addition of postmitotic cells to the population through stage 35. A pulse-chase paradigm indicated that birth date did not influence the final GnRH cell distribution. Injections at stage 29, when 10% of the GnRH neurons are born, generated double labeled cells in all locations where placode-derived GnRH neurons reside.

Expression of Rab3D N135I inhibits regulated secretion of ACTH in AtT-20 cells.

Rab proteins are small molecular weight GTPases that control vesicular traffic in eucaryotic cells. A subset of Rab proteins, the Rab3 proteins are thought to play an important role in regulated exocytosis of vesicles. In transfected AtT-20 cells expressing wild-type Rab3D, we find that a fraction of the protein is associated with dense core granules. In the same cells, expression of a mutated isoform of Rab3D, Rab3D N135I, inhibits positioning of dense core granules near the plasma membrane, blocks regulated secretion of mature ACTH, and impairs association of Rab3A to membranes. Expression of Rab3D N135I does not change the levels of ACTH precursor or the efficiency with which the precursor is processed into ACTH hormone and packaged into dense core granules. We also find that cells expressing mutated Rab3D differentiate to the same extent as untransfected AtT-20 cells. We conclude that expression of Rab3D N135I specifically impairs late membrane trafficking events necessary for ACTH hormone secretion.

Syndet is a novel SNAP-25 related protein expressed in many tissues.

SNAP-25 is a synaptosomal associated protein localized at the plasma membrane of nerve terminals. SNAP-25 associates with syntaxin 1 and vesicle-associated membrane protein-2 (VAMP-2) and is thought to form a complex essential for neurotransmitter release. We have identified syndet, a novel protein related to the family of SNAP-25 isoforms. Like SNAP-25, syndet has regions with high probability of forming coiled coils, a cysteine rich-domain, and lacks a signal sequence or transmembrane domains. Syndet is tightly bound to membranes, possibly by acylation within the cysteine-rich domain. Syndet is expressed in non-neuronal tissues. In adipocytes, syndet is found at the plasma membrane and in an intracellular compartment. The identification of syndet supports the hypothesis that multiple SNAP-25 related proteins ensure specificity of vesicle fusion at the cell surface.

GnRH perikarya in medial basal hypothalamus of pubertal female rhesus macaque are ensheathed with glia.

A previous study in our laboratory revealed that the cell bodies of gonadotropin releasing hormone (GnRH) neurons in the preoptic area (POA) of early to midpubertal female rhesus monkeys were extensively invested with thick glial processes. Because the medial basal hypothalamus (MBH) plays a critical role in the control of pulsatile and cyclic gonadotropin release in the primate, we have now focused on the ultrastructural milieu of GnRH neurons of this region in the same sample of monkeys. The ensheathment of the perikarya of GnRH neurons in the MBH with such glial processes was more pronounced than in the POA. Whereas the mean proportion of the cell membrane covered by these glia was 57% in the POA, it was 72% in the MBH. In addition, the cell bodies of GnRH neurons in the MBH of the pubertal monkey (unlike those in the POA) were less well innervated than were those in the adult cycling monkey, further highlighting differences between these brain regions. Differences in the anatomical milieu of the MBH between immature monkeys, in which GnRH release is still relatively quiescent, and adult cycling monkeys are consistent with the hypothesis that GnRH neurons within the MBH are under particular constraint in the immature animals. The functional significance of these observations must, however, await further studies.

Effects of ovariectomy on GnRH neuronal morphology in rhesus monkey (Macaca mulatta).

Gonadotropin releasing hormone (GnRH) neurons are typically simple, fusiform cells; however, over the course of prepubertal development increasing numbers take on a 'spiny' appearance. Following gonadectomy there is a decrease in the frequency of these spiny GnRH neurons. These observations which were made in the rat suggest that GnRH neurons are directly affected by the gonadal steroid milieu, though they do not themselves contain receptors for these steroidal hormones. In that there are important species differences in the hypothalamic-pituitary-gonadal axis between rats and primates, the present study was undertaken to determine whether a reduction in ovarian hormones would produce similar changes in the morphology of GnRH neurons in the monkey. A further aim was to determine whether such changes were localized to a specific brain region. Immunocytochemically defined GnRH neurons were compared in adult rhesus macaques which had been ovariectomized for 6 weeks to 2 years (n = 7) and intact, cycling animals (n = 8). Within the intact group, there were significantly more spiny GnRH neurons in the medial basal hypothalamus (MBH) than in the preoptic area (POA) (about 50% of the total in the MBH compared to 33% in the POA). Following ovariectomy the frequency of spiny cells in the MBH dropped to less than 30%, but was not significantly reduced in the POA. These results suggest that changes in systemic gonadal steroid levels result in changes in the morphology of GnRH neurons preferentially in the MBH, a region that is considered critical in the generation of GnRH pulsatile release in the monkey.

Novel associations among gonadotropin-releasing hormone neurons.

GnRH is secreted in bursts into the hypophyseal portal vasculature by a small dispersed population of neurons. The means by which the activity of these intrinsically pulsatile cells is coordinated are unknown. This study was initiated as a continuation of our examination of the synaptic input to these cells and their anatomical relationships. Brain tissue from female rhesus monkeys and male and female rats was prepared for the immunocytochemical demonstration of GnRH. At the light microscopic level, GnRH neurons were occasionally found to be in close apposition. Such pairs (or small groups) were randomly distributed throughout the population of GnRH neurons from the diagonal band of Broca through the anterior hypothalamic area in rats and monkeys and in the medial basal hypothalamus in monkeys. The percentage of neurons found in such associations was small (2-7% in rats and 3-15% in monkeys) and was independent of the hormonal condition of the animal. GnRH neurons, either singly or in pairs, were serially sectioned for electron microscopic examination. The sparsity of synaptic input to the cell body that we had reported earlier on the basis of random sampling was confirmed. No soma had more than a dozen synapses, but none totally lacked innervation. The most significant result of serial reconstruction was the discovery of intercellular bridges or passageways between contiguous pairs of GnRH neurons. These were formed by the fusion of processes extending from the two cells or by fusion and opening of passageways in the membranes along regions of contiguity between the two cells. They were found in four of seven pairs of neurons examined in the rat and in four of eight pairs in the monkey. This syncytial arrangement along with GnRH-GnRH synaptic interactions could contribute to the coordination of dispersed influences on these neurons and the propagation of coordinated pulsatile release of GnRH.

Glial ensheathment of GnRH neurons in pubertal female rhesus macaques.

During the period of development, prior to full sexual maturity, gonadotropin hormone-releasing hormone (GnRH) neurons are fully capable of synthesizing and processing the GnRH decapeptide. Nonetheless, the secretion of the hormone is not adequate to stimulate adult patterns of gonadotropin release. The present study was undertaken to examine ultrastructural characteristics of the GnRH neuron and its relationship to its environment in early-midpubertal female rhesus monkey. The neurons bore all the ultrastructural immunocytochemical characteristics of those in mature animals, but quantitative morphometrics revealed that they were extensively apposed by glial processes. Such ensheathment was described earlier in ovariectomized adult animals and was found to be reversible by administration of gonadal steroids. The density of synaptic input to GnRH neurons in the pubertal animals did not differ significantly from that of adult intact or ovariectomized animals from a previous study. Chemical identification will be required to determine whether there are age or hormonal differences in the innervation of these neurons. These results provide anatomical evidence in support of indications from other studies that the ovarian steroidal milieu affects GnRH-glial relationships. Further testing will be required to determine whether the attainment of sexual maturity in the female rhesus macaque is dependent upon a reduction in glial ensheathment of GnRH neurons.

Gonadotropin-releasing hormone neurons in the rhesus macaque are not immunoreactive for the estrogen receptor.

The issue of whether gonadotropin-releasing hormone (GnRH) neurons in the primate contain the estrogen receptor was examined by immunocytochemistry using prepubertal and adult (intact and ovariectomized) female rhesus macaques. No GnRH neurons were found to contain nuclei that were immunoreactive for the estrogen receptor. These results confirm in primates what has been reported in other species and leave open the question of how the effects of gonadal steroids on GnRH neurons are mediated.

Comparison of ultrastructural characteristics of gonadotropin-releasing hormone neurons in prepubertal and adult male rats.

Gonadotropin-releasing hormone neurons from prepubertal (29-day-old) and adult (three-month-old) male rats were demonstrated immunocytochemically using the LR1 antibody, and prepared for electron microscopic examination. Gonadotropin-releasing hormone neurons were equally immunoreactive in the two age groups, but there were heavy deposits of reaction product in the outer nuclear envelope of these neurons in prepubertal animals. Point count stereology on electron micrographic montages of gonadotropin-releasing hormone neurons at x25,000 was used to compare the relative proportion of cytoplasm containing various subcellular organelles. More of the cytoplasm was occupied by Golgi apparatus and secretory vesicles in the prepubertal animals. The representation of mitochondria was equal in the two age groups, while there were more lysosomes in the gonadotropin-releasing hormone neurons from adult animals. The density of synaptic input to the neurons was estimated using quantitative morphometrics on electron micrographs of three levels of section through the neuron, magnified x25,000. The percentage of the perikaryal membrane with synaptic contacts was greater in the gonadotropin-releasing hormone neurons from adults. Most strikingly, there were gonadotropin-releasing hormone terminals on gonadotropin-releasing hormone soma of these neurons in prepubertal animals, but not in the adults. The highly immunoreactive outer nuclear envelope and relative larger representation of Golgi and secretory vesicles in gonadotropin-releasing hormone neurons in prepubertal animals suggest that these cells are actively synthesizing peptides, including gonadotropin-releasing hormone. The large representation of Golgi apparatus may also reflect the active biosynthesis of membrane in association with the elaboration of neuronal processes.(ABSTRACT TRUNCATED AT 250 WORDS)

FOS expression in the gonadotropin-releasing hormone (GnRH) neuron does not increase during the ovarian steroid-induced GnRH surge in the rhesus monkey.

The purpose of this study was to investigate the expression of the immediate early gene, c-fos, in GnRH neurons in female rhesus monkeys as a function of generation of the LH surge. Adult monkeys were either intact (n = 6) or ovariectomized (n = 10). Intact animals received estradiol benzoate (EB; 330 micrograms in oil, sc; n = 5) or oil (n = 1). Ovariectomized animals received either EB (n = 5) or EB, followed by progesterone (P; 2.5 ml in oil, im; n = 4), or oil (n = 1). Animals were killed from 31-75 h after EB treatment. Blood samples were collected to document LH release in response to steroid treatment. A surge of LH was initiated in most animals that received EB alone or EB plus P about 30 h after steroid treatment. Animals were perfused with 4% paraformaldehyde, and brain blocks encompassing the region known to contain the majority of GnRH neurons (septum through the medial basal hypothalamus) were cut on the vibratome. Sites of FOS and GnRH immunoreactivities were demonstrated using double labels with a variety of chromogens. Regardless of the time in the surge, there were very few GnRH neurons with FOS immunoreactivity in their nuclei (0-9%). FOS-positive nuclei were seen in many other neurons in various brain regions, including the suprachiasmatic and supraoptic nuclei. There were no differences in FOS expression in GnRH neurons in intact and ovariectomized animals or in steroid- or oil-treated animals. These results suggest that FOS activation in GnRH neurons is not associated with the initiation of the secretory GnRH stimulus to the LH surge in the rhesus monkey. If confirmed, these data suggest that the GnRH nerve terminal may be the primary site for the control of the GnRH surge.

Biosynthesis of gonadotropin-releasing hormone during the rat estrous cycle: a cellular analysis.

In an attempt to understand the regulatory events that control the synthesis of gonadotropin-releasing hormone (GnRH) during the estrous cycle of the rat, we undertook a cellular analysis of time of translation of GnRH mRNA into protein. A specific antiserum, Rb 1076, which recognizes both the extended proGnRH as well as the processed form of the decapeptide was used for immunocytochemical staining. A cell was considered to be actively translating the pro-GnRH mRNA if elements of the rough endoplasmic reticulum (RER), including the outer nuclear envelope, were filled with reaction product. A synthetically quiescent cell contained only immunopositive neurosecretory granules. Cycling rats were killed at various times and all GnRH cells scored as being RER positive (+) or negative (-). On the morning of estrus almost all GnRH neurons in 5 out of 6 of the animals studied were synthesizing their unique peptide. The immunostaining in many of the cells at this time was very pale, suggesting a prior depletion. This was the only time point examined where near uniformity among individuals in a group was observed. At all other times considerable heterogeneity was observed among animals within a group. For example, at 17.30 h on the afternoon of proestrus 50% or more of the GnRH neurons were RER+ in half of the animals; the other half had values of 16-45% RER+. Synthetically active and inactive cells were found in close proximity in all animals. No regional differences were observed; all GnRH cell subpopulations from the level of the diagonal band of Broca through the hypothalamus reflected the population as a whole.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural changes in gonadotropin-releasing hormone neurons as a function of age and ovariectomy in rats.

In this study we examined the effects of aging on various aspects of the ultrastructure of gonadotropin-releasing hormone neurons in female rats, including the density of synaptic input and the volume fraction of various subcellular organelles. In addition, we explored the possibility that removal of estrogen might provide a protective effect on the aging of the gonadotropin-releasing hormone neuron as exposure to gonadal steroids alters the time course of reproductive aging. Our experimental groups included four- and 18-20-month-old virgin female rats divided as follows: young intact, young short-term ovariectomized, old intact, old short-term ovariectomized and old long-term ovariectomized. Brain tissue was processed for immunocytochemical detection of gonadotropin-releasing hormone neurons and selected cells from the preoptic area were chosen for electron microscopic examination. The percentage of plasma membrane containing synaptic modification was quantified using a morphometrics program, and the volume fraction of lysosomes/lipofuscin, rough endoplasmic reticulum and Golgi apparatus were estimated using point count stereology. Whereas we had previously found a significant increase in the density of synaptic input to gonadotropin-releasing hormone neurons in aged virgin male rats, the density of synaptic input to gonadotropin-releasing hormone cells in the virgin female was not affected by age. The volume fraction of lysosomes/lipofuscin was increased in all age groups. Aging produced a dramatic decrease in the volume fraction of rough endoplasmic reticulum as well as a decrease in Golgi, suggesting a general decrease in biosynthetic activity of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased synaptic input to gonadotropin-releasing hormone neurons in aged, virgin, male Sprague-Dawley rats.

Using double-label ultrastructural immunocytochemistry, we found the synaptic input to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area of aged (20 months old), virgin, male Sprague-Dawley rats to be denser than that in young adults (3 months old). These results confirmed earlier observations on F-344 virgin male rats. The aging F-344 rat, however, is prone to testicular tumor and so it was essential to see if the phenomenon was reproducible in another rat strain. In the first study, a portion of the increase in synaptic density was due to an increase in the proportion of synapses containing pleiomorphic vesicles, frequently associated with the neurotransmitter GABA. We tested the possibility directly using a double-label protocol for GnRH and glutamic acid decarboxylase (GAD). However, in the present study the density of input by GABA did not change with age. This inhibitory amino acid represented about 10% of the total innervation in young animals; but, in aged animals, because the total synaptic input was greater, GABA represented only about 4% of the innervation. Synaptic vesicles within GAD-immunoreactive terminals were uniformly clear and spherical, suggesting that pleiomorphic vesicle shape is not an appropriate criterion for GABAergic innervation.

Reproductive history affects the synaptology of the ageing gonadotropin-releasing hormone system in the male rat.

This study is an examination of the density of synaptic input to gonadotropin-releasing hormone (GnRH) neurons in young adult and aged retired breeder male rats. In earlier experiments on aged virgin male rats we observed an increase in synaptic input to this specific neuronal population, ascribable in part to synapses containing flattened vesicles, suggesting GABAergic input. The present study utilized retired breeders in order to dissect the effects of ageing from those associated with reproductive behavioral history. Tissue from the preoptic area was treated for the simultaneous electron microscopic immunocytochemical demonstration of GnRH with tetramethylbenzidine and glutamic acid decarboxylase (the essential enzyme in the production of GABA) using 3,3'-diaminobenzidine. Estimates of the density of synaptic input to the soma of GnRH neurons were made by calculating the percentage of perikaryal membrane with postsynaptic modification. Five GnRH neurons per animal were measured using computerized morpho-metrics and differences in the percent of membrane with synaptic modification between experimental groups were tested using the Mann-Whitney U non-parametric statistic. There was no difference in the total density of synaptic input to GnRH neurons in the young and old animals, or in the proportion of this input that was immunoreactive for glutamic acid decarboxylase. Similar measurements were made on random, non-identified neurons in the same region and a significant decrease with ageing in total synaptic input was found, though the glutamic acid decarboxylase component was unchanged. The present results are in contrast to our earlier findings on virgin males and suggest that reproductive behavioral experience affects the connectivity of GnRH neurons.

Effects of gonadal steroids on the ultrastructure of GnRH neurons in the rhesus monkey: synaptic input and glial apposition.

The secretion of the gonadotropins is modulated by the gonadal steroids, but the means by which these effects are mediated are not well understood. The present anatomical study was undertaken to investigate the possibility that the GnRH system responds to alterations in the gonadal steroid environment with reversible changes in synaptic input and glial wrapping such as have been observed in other neuroendocrine systems. The ultrastructure of GnRH neurons was studied in the preoptic area and medial basal hypothalamus of rhesus monkeys in various steroid conditions including five intact cycling, four long-term ovariectomized animals, two long-term ovariectomized animals with steroid replacement (LtOVX+), and two animals replaced with steroid at the time of ovariectomy (StOVX+). Electron micrographic montages of GnRH neuronal profiles were analyzed using computerized morphometrics, and the percentages of the length of perikaryal membrane immediately apposed by glial processes and that with postsynaptic modification were calculated. Ovariectomy resulted in a significant increase in the apposition of glial processes to GnRH perikaryal membranes and a significant decrease in their innervation in both brain regions. There was also a higher incidence of GnRH neurons with immunostaining confined to secretory granules and a decrease in the volume of nucleoli, both of which could be interpreted as indications that GnRH peptide synthesis was reduced in ovariectomized animals. After an ovarian steroid replacement regimen which mimicked two menstrual cycles, the innervation of GnRH neurons was increased and the glial ensheathment was partially reduced. This was true for both the LtOVX+ and StOVX+ steroid-replacement groups. GnRH neurons in the medial basal hypothalamus received more synaptic input than did those in the preoptic area, regardless of the steroid condition of the animal. The degree of glial ensheathment of GnRH neurons in the preoptic area became significantly greater than that in the medial basal hypothalamus after ovariectomy. These observations suggest there may be differences in the role of GnRH neurons in these two brain regions. These immunocytochemical ultrastructural studies provide strong evidence that alterations in the gonadal steroid milieu can produce morphological changes in the GnRH neuron and its immediate environment in the primate.

Light and electron microscopic immunocytochemical analysis of antibodies directed against GnRH and its precursor in hypothalamic neurons.

A battery of antibodies directed against different portions of the precursor to gonadotropin-releasing hormone (GnRH), as well as to the mature decapeptide, were characterized immunocytochemically in two ways. Absorption experiments were used to determine the epitope recognized by each antiserum. Electron microscopic immunocytochemistry was then used to define the subcellular organelles that contained reaction product when tissue was incubated with these reagents. These latter observations helped to determine if the antibody recognized the epitope as part of the intact precursor or only after it had been cleaved from parent protein. Our results demonstrate that the GnRH precursor is routed from the rough endoplasmic reticulum through the Golgi apparatus to the secretory vesicles. Furthermore, we show that initial cleavage and processing of the GnRH precursor begin in the cell soma. These antibodies should be useful in the future in determining changes in processing of precursor in animals that differ in endocrine function.