Rapid inhibition of female sexual behavior by gonadotropin-inhibitory hormone (GnIH)

Gonadotropin-releasing hormone (GnRH) is largely responsible for the initiation of sexual behaviors; one form of GnRH activates a physiological cascade causing gonadal growth and gonadal steroid feedback to the brain, and another form is thought to act as a neurotransmitter to enhance sexual receptivity. In contrast to GnRH, gonadotropin-inhibitory hormone (GnIH) inhibits gonadotropin release. The distribution of GnIH in the avian brain suggests that it has not only hypophysiotropic actions but also unknown behavioral actions. GnIH fibers are present in the median eminence (ME) and are in apparent contact with chicken GnRH (cGnRH)-I and -II neurons and fibers. In birds, cGnRH-I regulates pituitary gonadotropin release, whereas cGnRH-II enhances copulation solicitation in estradiol-primed females exposed to male song. In the present study, we determined the effects of GnIH administered centrally to female white-crowned sparrows. A physiological dose of GnIH reduced circulating LH and inhibited copulation solicitation, without affecting locomotor activity. Using rhodaminated GnIH, putative GnIH binding sites were seen in the ME close to GnRH-I fiber terminals and in the midbrain on or close to GnRH-II neurons. These data demonstrate direct effects of GnIH upon reproductive physiology and behavior, possibly via separate actions on two forms of GnRH.     

Differential regional distribution of gonadotropin-releasing hormones in amphibian (clawed toad, Xenopus laevis) brain

In most vertebrate species two forms of gonadotropin-releasing hormone (GnRH) are present in the brain, and their differential distribution suggests they have different functional roles. The regional distribution and relative concentrations of GnRH molecular forms in the brain of adult clawed toad (Xenopus laevis) were determined using high performance liquid chromatography and radioimmunoassay with a library of region-specific GnRH antisera. Four immunoreactive forms of GnRH were detected: mammalian, hydroxyproline mammalian, chicken II, and an unidetified form of GnRH. Mammalian GnRH was distributed throughout the brain, and hydroxyproline mammalian was present in the forebrain, midbrain (excluding hypothalamus), and hypothalamus. Chicken GnRH II also occurred throughout the brain, but was present in greater amounts in the hindbrain and midbrain (excluding hypothalamus). An unidentified form of GnRH with properties of salmon GnRH was detected in the forebrain. Considering the relative proportions of mammalian GnRH and chicken GnRH II in the major brain areas, the concentration of mammalian GnRH was high in the forebrain, midbrain (excluding hypothalamus), and in particular in the hypothalamus, and very little chicken GnRH II was present in these areas. In the hindbrain, chicken GnRH II predominated and the concentration of chicken GnRH II was highest in the medulla. These findings suggest: (1) mammalian GnRH is the prime regulator of gonadotropin release from the pituitary, and (2) chicken GnRH II has an extrapituitary role.     

Inhibitory and stimulatory interactions between endogenous gonadotropin-releasing hormones in the African catfish (Clarias gariepinus)

In the brain of all vertebrate classes, chicken (c) GnRH-II ([His(5), Trp(7),Tyr(8)]GnRH, cGnRH-II) is expressed in the mesencephalon. In addition, at least one other form of GnRH is expressed in the preoptical area/hypothalamus. In the human pituitary stalk and the mouse median eminence, cGnRH-II is present together with mammalian GnRH. Similarly, in the pituitary of several teleost fish (e.g., goldfish and eel, but not salmon or trout), a teleost GnRH is found together with cGnRH-II. These GnRHs are not colocalized in the same cells. Hence, these GnRH peptides may differentially regulate gonadotropin secretion and, in addition, may exert their effects simultaneously. The current study therefore investigated the effects of combinations of the two forms of GnRH present in the African catfish (Clarias gariepinus) pituitary-cGnRH-II and catfish GnRH ([His(5),Asn(8)]GnRH, cfGnRH)-on the cytosolic free calcium concentration ([Ca(2+)](i)) in single, Fura-2-loaded catfish gonadotrophs, as well as their effects on both in vitro and in vivo LH secretion. Both inhibitory and stimulatory effects of combinations of cfGnRH and cGnRH-II on [Ca(2+)](i) were observed, which were mirrored by their effects on both in vitro and in vivo LH secretion. The following pattern became apparent. The effect of intermediate or maximal effective cfGnRH doses was inhibited by the simultaneous presence of subthreshold or borderline effective cGnRH-II doses. Conversely, subthreshold or borderline effective concentrations of cfGnRH enhanced the effects of intermediate and maximal concentrations of cGnRH-II. In addition, combinations of cfGnRH and cGnRH-II concentrations that were equally active when tested separately showed an additive effect. The observed interactions between the two GnRHs may be of particular physiological relevance in the control of seasonal LH levels in the African catfish, as well as in other teleost species. Moreover, the occurrence of mutual inhibitory and stimulatory interactions between endogenous GnRHs may be a widespread aspect of GnRH action in vertebrates.     

Characterization and localization of gonadotropin-releasing hormone in the brain and pituitary of the three-spined stickleback,Gasterosteus aculeatus

Radioimmunoassay (RIA) studies on highperformance liquid chromatography (HPLC) fractions of brain extracts of the three-spined stickleback, Gasterosteus aculeatus, provided evidence for at least two forms of gonadotropin-releasing hormone (GnRH). One form showed chromatographic and immunological properties similar to that of synthetic salmon GnRH (sGnRH). A second, unidentified form of GnRH eluted in the same position as chicken GnRH I (cGnRH-I); however, it did not cross-react in a cGnRH-I RIA. Furthermore, it cannot be excluded that chicken GnRH II (cGnRH-II) and maybe one other unidentified form are present in the stickleback. The distribution of GnRH in the brain of breeding adult male sticklebacks was studied by use of immunohistochemistry. Two antisera against sGnRH and antisera against mGnRH and cGnRH-II were applied on cryosections and visualized using the peroxidase-antiperoxidase method. Staining patterns were similar after incubations with all four antisera. Immunoreactive fibers were found in most parts of the brain. Three distinct groups of GnRH-immunoreactive perikarya were found in the nucleus olfactoretinalis, in the nucleus anterior periventricularis, and in the nucleus lateralis tuberis. Moreover, weakly stained cells occurred in a periventricular position in the midbrain. The proximal pars distalis of the pituitary, housing the gonadotropic cells, was richly innervated by GnRH-positive fibers. In the pars intermedia and in the rostral pars distalis, immunoreactive fibers were absent.     

Differential distribution of two molecular forms of gonadotropin-releasing hormone in discrete brain areas of goldfish (Carassius auratus)

Two molecular forms of gonadotropin-releasing hormone (GnRH) were identified in the extracts of various brain areas, spinal cord and pituitary in female and male goldfish and had chromatographic and immunological properties similar to [His5, Trp7, Tyr8]-GnRH (cGnRH-II) and [Trp7,Leu8]-GnRH (sGnRH). Radioimmunoassay using different GnRH antisera after high pressure liquid chromatography did not reveal significant peaks of mammalian GnRH, [Gln8]-GnRH and [Tyr3,Leu5,Glu6,Trp7,Lys8]-GnRH in the brain extracts. The proportion of cGnRH-II-like immunoactivity to sGnRH-like immunoactivity was higher in the caudal brain areas compared to the rostral areas. The differential distribution of two GnRH forms suggest that the different GnRH forms may have different physiological functions.     

Changes in immunoreactivity to anti-cGnRH-I and -II are associated with photostimulated sexual status in male quail

In sexually active males exposed to long-day (LD) photoperiod, perikarya in the olfactory bulb, lobus parolfactorius, n. accumbens, and preoptic region were immunoreactive (ir) to an antiserum against gonadotropin-releasing hormone (anti-cGnRH-I), and a cluster of ir-perikarya was found in the caudal-most septal area. Ir-perikarya in these brain areas of sexually inactive short-day (SD) males were located within more discrete areas than those in LD brain, which were more scattered in appearance. Absolute cell numbers were similar between LD and SD brains. Ir-fibers in LD brains were mostly in the external median eminence, along the lateral ventricle to septum (especially in and about the n. accumbens), in the septal-preoptic area, along the third ventricle, and at the n. commissure palli. There were fewer ir-fibers in SD brain. Many small dark ring-like ir-structures were found in the hyperstriatum, hippocampus, and n. taeniae. Interpreted as being ir-terminals on non-ir perikarya, these were not observed in SD males. cGnRH-II ir-perikarya were observed in only two areas regardless of reproductive status: (1) ventral to the substantia grisea centralis and caudal to the oculomotor complex, and (2) scattered in and about the lateral hypothalamus. Ir-fibers occurred in the habenular area, hyperstriatum, hippocampus, parahippocampal area, cortex piriformis, and n. taeniae. cGnRH-II ir-fibers occurred in the external median eminence but were less intensely stained than cGnRH-I ir-fibers. These fibers in SD males were similar except in the diencephalon, where scattered swellings were observed. Thus, the appearance and distribution of anti-cGnRH-I and -II ir-structures change with the sexual status of male quail, but changes in immunoreactivity to anti-cGnRH-I appear to be more widespread.     

Homologous desensitization of gonadotropin-releasing hormone (GnRH) receptors in the goldfish pituitary: effects of native GnRH peptides and a synthetic GnRH antagonist.

Gonadotropin-releasing hormone (GnRH) stimulates release of gonadotropin hormone (GTH) through interaction with high affinity receptors in the goldfish pituitary. In the present study, we investigated desensitization of two native GnRH peptides, [Trp7, Leu8]-GnRH (sGnRH) and [His5, Trp7, Tyr8]-GnRH (cGnRH-II), using superfused fragments of goldfish pituitary in vitro. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses given every 60 min) resulted in dose-dependent secretion of GTH from the goldfish pituitary; cGnRH-II had a greater GTH release potency and displayed a greater receptor binding affinity than sGnRH. Both sGnRH and cGnRH-II-induced GTH release were partially inhibited by concomitant treatment with either [D-Phe2, Pro3, D-Phe6]-GnRH or [D-pGlu1, D-Phe2, D-Trp3.6]-GnRH. These antagonists had greater receptor binding affinities than the native peptides, with no stimulatory action on GTH release in the absence of the GnRH agonists. Continuous treatment with either sGnRH or cGnRH-II (10(-7) M), rapidly desensitized pituitary GTH release in a biphasic fashion; initially there was a rapid increase in GTH release of approximately 10-20-fold (phase 1), followed by a sharp decline in GTH release, reaching a stable concentration 2-3-fold above the basal level (phase 2). Further stimulation of the pituitaries with sGnRH or cGnRH-II (10(-7) M) (second treatment) after 60 min recovery resulted in a significantly lower sGnRH or cGnRH-II-induced GTH release compared to that observed during the initial treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further data on the origin of gonadotropin releasing hormone in the median eminence of the rat hypothalamus

Gonadotropin releasing hormone (GnRH) content of the two halves of the median eminence of the rat hypothalamus was determined by radioimmunoassay three weeks after three different unilateral knife cuts around the preoptic area. A unilateral cut in front or above the area caused a more than 25% decrease in the GnRH content of the two halves of the median eminence. A cut lateral to the preoptic region had only a slight effect similar to that observed after sham operations. The data suggest that probably more than 50% of the rat median eminence GnRH derives from outside the preoptic-suprachiasmatic region. The GnRH fibres projecting to the median eminence but arising from outside the preoptic region, probably mainly from GnRH perikarya in the limbs of the diagonal band of Broca and septum, enter this area partly from rostral and partly from above, but not from lateral direction. partly from rostral and partly from above, but not from lateral direction. Several of these fibres probably cross before terminating in the median eminence.     

Localization of avian LHRH-immunoreactive neurons in the hypothalamus of the domestic fowl,Gallus domesticus,and the Japanese quail,Coturnix coturnix

The localization of LHRH-containing perikarya and nerve fibers in the hypothalami of the domestic fowl and Japanese quail was investigated by means of the specific immunoperoxidase ABC method, using antisera against chicken LHRH-I ([Gln8]-LHRH), chicken GnRH-II ([His5-Trp7-Tyr8]-LHRH[2-10]) and mammalian LHRH ([Arg8]-LHRH). Chicken LHRH-I-immunoreactive perikarya were sparsely scattered in the nucleus preopticus periventricularis (POP), nucleus filiformis (FIL) and nucleus septalis medialis (SM), and in bilateral bands extending from these nuclei into the septal area in both species. A few reactive perikarya were also observed in the nucleus accumbens (Ac) and lobus parolfactorius (LPO). Numerous cLHRH-I-immunoreactive fibers were widely scattered in the preoptic, septal and tuberal areas, and were densely concentrated in the external layer of the median eminence and in organum vasculosum of the lamina terminalis (OVLT) in both species. Anti-mammalian LHRH serum cross-reacted weakly with perikarya and fibers immunoreactive to anti-cLHRH-I serum in normal chicken and quail. Anti-cGnRH-II[2-10] serum immunoreacted with magnocellular neurons distributed in the rostral end of the mesencephalon along the midline close to the nervus oculomotorius (N III). These perikarya were apparently different from cLHRH-I immunoreactive neurons. No immunoreactive cells and fibers against anti-cGnRH-II[2-10] were observed in the hypothalamus and median eminence of the chicken or quail. Anti-cGnRH-II[2-10] bound specifically with cGnRH-II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoassay of gonadotrophin-releasing hormone in brain tissue of Booroola Merino ewes

The presence of a fecundity gene (F) in Booroola Merino ewes increases the ovulation rate. To test how F gene expression affects the gonadotrophin-releasing hormone (GnRH) concentration in hypothalamic or extrahypothalamic regions of the brain, GnRH was measured by radioimmunoassay in acetic acid extracts of various brain tissues from Booroola ewes which were homozygous (FF), heterozygous (F+) or non-carriers (++) of the F gene. The GnRH concentration in brain tissues from FF, F+ and ++ animals which had been ovariectomized 5 months previously was also evaluated. No significant F gene-specific differences were noted in any of the brain areas tested, in intact or ovariectomized animals. However, in ovariectomized ewes, the concentrations of GnRH increased about 2-fold in the median eminence of the hypothalamus, remained unchanged in the medial basal hypothalamus and dropped to less than 10% of the values in intact ++ animals in the preoptic area. These studies suggest that the changed pituitary sensitivity and increased gonadotrophin release in Booroolas carrying the F gene(s) is not attributable to increased hypothalamic GnRH concentrations in these animals.     

Expression of the GnRH and GnRH receptor (GnRH-R) genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland of anestrous and luteal phase ewes

Data exists showing that seasonal changes in the innervations of GnRH cells in the hypothalamus and functions of some neural systems affecting GnRH neurons are associated with GnRH release in ewes. Consequently, we put the question as to how the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland is reflected with LH secretion in anestrous and luteal phase ewes. Analysis of GnRH gene expression by RT-PCR in anestrous ewes indicated comparable levels of GnRH mRNA in the preoptic area, anterior and ventromedial hypothalamus. GnRH-R mRNA at different concentrations was found throughout the preoptic area, anterior and ventromedial hypothalamus, stalk/median eminence and in the anterior pituitary gland. The highest GnRH-R mRNA levels were detected in the stalk/median eminence and in the anterior pituitary gland.During the luteal phase of the estrous cycle in ewes, the levels of GnRH mRNA and GnRH-R mRNA in all structures were significantly higher than in anestrous ewes. Also LH concentrations in blood plasma of luteal phase ewes were significantly higher than those of anestrous ewes.In conclusion, results from this study suggest that low expression of the GnRH and GnRH-R genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland, amongst others, may be responsible for a decrease in LH secretion and the anovulatory state in ewes during the long photoperiod.     

Interactions between gonadotropin-releasing hormone (GnRH) and orexin in the regulation of feeding and reproduction in goldfish (Carassius auratus)

Links between energy homeostasis and reproduction have been demonstrated in vertebrates. As a general rule, abundant food resources favor reproduction whereas low food availability induces an inhibition of reproductive processes. In both mammals and fish, gonadotropin-releasing hormone (GnRH) and orexin (OX) are hypothalamic neuropeptides that play critical roles in the regulation of sexual behavior and appetite, respectively. In order to assess possible interactions between orexin and GnRH in the control of feeding and reproduction in goldfish, we examined the effects of chicken GnRH (cGnRH-II) intracerebroventricular (ICV) injection on feeding behavior and OX brain mRNA expression as well as the effects of orexin ICV injections on spawning behavior and cGnRH-II brain mRNA expression. Treatment with cGnRH-II at doses that stimulate spawning (0.5 ng/g or 1 ng/g) resulted in a decrease in both food intake and hypothalamic orexin mRNA expression. Treatment with orexin A at doses that stimulate feeding (10 ng/g) induced an inhibition of spawning behavior and a decrease in cGnRH-II expression in the hypothalamus and optic tectum-thalamus. Our results suggest that the anorexigenic actions of cGnRH-II in goldfish might be in part mediated by OX and that orexin inhibits reproductive behavior in part via the inhibition of the GnRH system. Our data suggest the existence of a coordinated control of feeding and reproduction by the orexin and GnRH systems in goldfish.     

Direct evidence for the co-expression of URP and GnRH in a sub-population of rat hypothalamic neurones: anatomical and functional correlation

Urotensin-II-related peptide (URP) is an eight amino-acid neuropeptide recently isolated from rat brain and considered as the endogenous ligand for the GPR14 receptor. Using single and double immunohistochemical labelling, in situ hybridization and ultrastructural immunocytochemistry, we explored the cellular and subcellular localization of URP in the male rat brain. URP peptide was detected in numerous varicose fibres of the median eminence (ME) and organum vasculosum laminae terminalis (OVLT) as well as in neuronal cell bodies of the medial septal nucleus and diagonal band of Broca where corresponding mRNA were also detected. Combining in situ hybridization with immunohistochemistry, we showed that cell bodies of the rat anterior hypothalamus contained both URP mRNA and GnRH peptide. In addition, double ultrastructural immunodetection of URP and GnRH peptides clearly revealed, in the median eminence, the co-localization of both peptides in the same neuronal processes in the vicinity of fenestrated portal vessels. This remarkable cellular and subcellular distribution led us to test the effect of URP on the GnRH-induced gonadotrophins release in the anterior pituitary, and to discuss its putative role at the level of the median eminence.     

Distribution of gastrin and CCK-like peptides in rat brain

Summary The distribution of gastrin and CCK-like peptides in the rat brain was studied by immunocytochemistry using an antiserum reacting equally well with both groups of peptides. Immunoreactive nerve cell bodies were detected in all cortical areas, in the hippocampus where they were particularly numerous, in the mesencephalic central gray and in the medulla oblongata. After colchicine treatment immunoreactive material appeared also in cell bodies of the magnocellular hypothalamic system. Immunoreactive nerve fibers were widely distributed in the brain. Particularly dense accumulations were seen in the hippocampus near the ventral surface of the brain, in the caudate nucleus, in the interpeduncular nucleus, the parabrachial nucleus, the dorsal part of the medulla oblongata and in the dorsal horn of the spinal cord. In the hypothalamus immunoreactive nerve fibers were observed in all nuclei, being most frequent in the ventromedial, dorsal and lateral hypothalamic nuclei. A rich supply of nerve fibers was seen in the outer zone of the median eminence and in the neurohypophysis. From previous immunochemical analysis it appears that the peptide demonstrated in most parts of the brain is identical with CCK-8. In the neurosecretory cell bodies of the hypothalamus, the median eminence and the neurohypophysis, however, the immunoreactive material is probably identical with gastrin.     

Activity of vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in positions 5, 7 and 8 in the goldfish pituitary.

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp7, Leu8]m-GnRH; s-GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr8, His8 and Leu8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His5, Trp7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three GnRH systems in the brain and pituitary of a pleuronectiform fish,the barfin flounder Verasper moseri

To clarify the possible function of gonadotropin-releasing hormone (GnRH) in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri, the distribution of three forms of GnRH in various areas of the brain was examined by radioimmunoassay, and the localization of GnRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary was determined by immunocytochemistry. The dominant form in the pituitary was seabream GnRH (sbGnRH), levels of which were much higher than those of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In contrast, sbGnRH levels were extremely low in all other brain areas examined. Levels of sGnRH and cGnRH-II were high in the anterior and posterior part of the brain, respectively. sbGnRH-ir cell bodies were located in the preoptic area, whereas sbGnRH-ir fibers were localized mainly in the preoptic area-hypothalamus-pituitary and formed a distinctive bundle of axons projecting to the pituitary. sGnRH-ir cell bodies were located in the ventromedial part of the rostral olfactory bulbs and in the terminal nerve ganglion (the transitional area between the olfactory bulb and the telencephalon). cGnRH-II-ir cell bodies were localized to the midbrain tegmentum. sGnRH-ir and cGnRH-II-ir fibers were observed throughout the brain except in the pituitary gland. These results indicate that sbGnRH is responsible for the neural control of the reproductive endocrinology of the barfin flounder (hypothalamo-hypophysial system), and that sGnRH and cGnRH-II function as neurotransmitters or neuromodulators in the brain.     

Short photoperiod-induced gonadal regression: effects on the gonadotropin-releasing hormone (GnRH) neuronal system of the white-footed mouse, Peromyscus leucopus

The peroxidase-antiperoxidase method was used to determine quantitatively the effect of short photoperiod-induced gonadal regression on the immunoreactive gonadotropin-releasing hormone (GnRH) neuronal system of female Peromyscus leucopus. In mice exposed to either long (16L:8D) or short (8L:16D) photoperiod, immunoreactive cell bodies were loosely organized into six groups: olfactory peduncle, diagonal band of Broca, septum, preoptic area (POA), anterior hypothalamus (AH), and basal hypothalamus. The POA and AH contain the largest number of cell bodies, which supply the major GnRH innervation to the median eminence (ME) and several extrahypothalamic brain sites. Exposure to short photoperiod increased the number of immunoreactive cell bodies within the anterior hypothalamus and preoptic area (AHPOA) and also increased the optical density for staining of immunoreactive cell bodies in the AHPOA and olfactory peduncle. The ME of mice exposed to short photoperiod had a higher density of GnRH fibers relative to that of mice exposed to long photoperiod, and the content of GnRH fibers in the rostral ME was correlated with the optical content for immunostaining of cell bodies in the AHPOA. These results are evidence that gonadal regression induced by short photoperiod (mediated by the pineal gland) involves alterations of GnRH neuronal activity. Notably, data from this study are consistent with the hypothesis that suppressed release of GnRH from neurovascular terminals in the ME, rather than lack of availability of the decapeptide, promotes gonadal regression.     

Novel localization of NMDA receptors within neuroendocrine gonadotropin-releasing hormone terminals

About 1000 hypothalamic neurons synthesize and release gonadotropin-releasing hormone (GnRH), the master molecule of reproduction in all mammals. At the level of the median eminence at the base of the brain, where GnRH and other hypothalamic releasing hormones are secreted into the capillary system leading to the anterior pituitary gland, there is non-synaptic regulation of neurohormone release by a number of central neurotransmitters. For example, glutamate, the major excitatory amino acid in the brain, directly regulates GnRH release from nerve terminals via NMDA receptors (NMDARs). Moreover, the effects of glutamate action on GnRH secretion are potentiated by estrogens, and this relates to the physiologic control of ovulation by the hypothalamus. We sought to determine the ultrastructural relationship between GnRH neuroterminals and NMDARs, and this regulation by estradiol. Using immunofluorescent confocal microscopy, postembedding immunogold electron microscopy, fractionation, and Western blotting, we demonstrated: (i) GnRH is localized in large dense-core vesicles of neurosecretory profiles/terminals, (ii) the NMDAR1 subunit is found primarily on large dense-core vesicles of neurosecretory profiles/terminals, (iii) there is extensive colocalization of GnRH and NMDAR1 on the same vesicles, and (iv) estradiol modestly but significantly alters the distribution of NMDAR1 in GnRH neuroterminals by increasing expression of NMDAR1 on large dense-core vesicles. Western blots of fractionated median eminence support the presence of NMDAR1 in subcellular fractions containing large dense-core vesicles. These data are the first to show the presence of the NMDAR on neuroendocrine secretory vesicles, its co-expression with GnRH, and its regulation by estradiol. The results provide a novel anatomical site for the NMDAR and may represent a new mechanism for the regulation of GnRH release.