Gonadotrophin-releasing Hormone Cells Research Articles

Distribution of GnRH and Kisspeptin Immunoreactivity in the Female Llama Hypothalamus.

Llamas are induced non-reflex ovulators, which ovulate in response to the hormonal stimulus of the male protein beta-nerve growth factor (β-NGF) that is present in the seminal plasma; this response is dependent on the preovulatory gonadotrophin-releasing hormone (GnRH) release from the hypothalamus. GnRH neurones are vital for reproduction, as these provide the input that controls the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland. However, in spontaneous ovulators, the activity of GnRH cells is regulated by kisspeptin neurones that relay the oestrogen signal arising from the periphery. Here, we investigated the organisation of GnRH and kisspeptin systems in the hypothalamus of receptive adult female llamas. We found that GnRH cells exhibiting different shapes were distributed throughout the ventral forebrain and some of these were located in proximity to blood vessels; sections of the mediobasal hypothalamus (MBH) displayed the highest number of cells. GnRH fibres were observed in both the organum vasculosum laminae terminalis (OVLT) and median eminence (ME). We also detected abundant kisspeptin fibres in the MBH and ME; kisspeptin cells were found in the arcuate nucleus (ARC), but not in rostral areas of the hypothalamus. Quantitative analysis of GnRH and kisspeptin fibres in the ME revealed a higher innervation density of kisspeptin than of GnRH fibres. The physiological significance of the anatomical findings reported here for the ovulatory mechanism in llamas is still to be determined.

Presence of Oxytocin Receptors in the Gonadotrophin‐Releasing Hormone (GnRH) Neurones in Female Rats: A Possible Direct Action of Oxytocin on GnRH Neurones

Gonadotrophin-releasing hormone (GnRH) neurones constitute the final output pathway of a neuronal network that controls the preovulatory luteinising hormone (LH) surge and ovulation. Throughout the reproductive cycle, several neurotransmitters stimulate and inhibit the activity of GnRH neurones, including oxytocin. The central administration of oxytocin antiserum abolishes the pro-oestrous LH surge whereas oxytocin stimulates GnRH secretion from hypothalamic explants suggesting an oxytocin central action. Within the GnRH neuronal population in the rat, GnRH cells in the medial preoptic area (MPOA) are activated at the time of the LH surge. Thus, we hypothesised that GnRH neurones in the MPOA may express oxytocin receptors, and that oxytocin neurones in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) may be differentially activated during the oestrous cycle. Oxytocin receptors mRNA was detected in the MPOA using reverse transcription-polymerase chain reaction. In animals in either metoestrus or pro-oestrus, double-label immunofluorescence indicated that approximately 10% of GnRH neurones in the MPOA coexpressed oxytocin receptors and that a few oxytocin fibres are located in the vicinity of these GnRH neurones. However, other neurones positive for the oxytocin receptors were found near GnRH neurones. At both oestrous stages, double-label immunofluorescence revealed that approximately 30% of oxytocin neurones in the SON were Fos-positive whereas oxytocin neurones in the PVN were consistently Fos-negative. Together, these data suggest that oxytocin may directly control neuronal activity in a subpopulation of GnRH neurones. Moreover, both oxytocin neuronal activity and the oxytocin receptor expression on GnRH cells are not influenced by oestrogen.

Projections to the Preoptic Area from the Paraventricular Nucleus, Arcuate Nucleus and the Bed Nucleus of the Stria Terminalis Are Unlikely to Be Involved in Stress-Induced Suppression of GnRH Secretion in Sheep

Stress compromises reproductive function and the major physiological system activated during stress is the hypothalamo-pituitary-adrenal axis. Corticotrophin-releasing hormone and arginine vasopressin (AVP), which are produced in neurones of the paraventricular nucleus (PVN), drive the hypothalamo-pituitary-adrenal axis and are also implicated in the suppression of the reproductive axis. We used retrograde tracing and Fos labelling to map the projections from the PVN to the preoptic area (POA) where most gonadotrophin releasing hormone (GnRH) neurones are found. Fluorogold (FG) injections were made into the POA of gonadectomised male and female sheep (n = 5/sex), the animals were stressed and the brains recovered for histochemistry. All animals responded to stress with an increase in the number of Fos-labelled nuclei in the PVN. Few retrogradely labelled cells of the PVN were activated by stress. Dual labelling showed that very few FG-labelled cells also stained for corticotrophin-releasing hormone, none for AVP or enkephalin. Dual labelling for FG and Fos in the bed nucleus of the stria terminalis (BNST) and the arcuate nucleus showed that no FG-labelled cells in the BNST and only few in the ARC were activated by stress. No sex differences were observed in the activation of FG-labelled cells in any of the nuclei examined. We conclude that, although cells of the PVN, BNST and/or arcuate nucleus may affect reproduction via the GnRH cells of the POA, this is unlikely to involve direct input to the POA. If cells of these regions are involved in GnRH suppression during stress, this may occur via interneuronal pathways.

Prenatal programming of the female reproductive neuroendocrine system by androgens

It has been clear for several decades that the areas of the brain that control reproductive function are sexually dimorphic and that the 'programming actions' of the male gonadal steroids are responsible for sex-specific release of the gonadotrophins from the pituitary gland. The administration of exogenous steroids to fetal/neonatal animals has pinpointed windows of time in an animals' development when the reproductive neuroendocrine axis is responsive to the organisational influences of androgens. These 'critical' periods for sexual differentiation of the brain are trait- and species-specific. The neural network regulating the activity of the gonadotrophin releasing hormone (GnRH) neurones is vital to the control of reproductive function. It appears that early exposure to androgens does not influence the migratory pathway of the GnRH neurone from the olfactory placode or the size of the population of neurones that colonise the postnatal hypothalamus. However, androgens do influence the number and the nature of connections that these neurones make with other neural phenotypes. Gonadal steroid hormones play key roles in the regulation of GnRH release acting largely via steroid-sensitive intermediary neurones that impinge on the GnRH cells. Certain populations of hormonally responsive neurones have been identified that are sexually dimorphic and project from hypothalamic areas known to be involved in the regulation of GnRH release. These neurones are excellent candidates for the programming actions of male hormones in the reproductive neuroendocrine axis of the developing female.

Colocalisation of Dynorphin A and Neurokinin B Immunoreactivity in the Arcuate Nucleus and Median Eminence of the Sheep

Dynorphin A (DYN)-containing cells play a key role in conveying the negative feedback influence of progesterone upon pulsatile gonadotrophin-releasing hormone (GnRH) secretion in the ewe. A very high percentage of DYN cells in the arcuate nucleus express the progesterone receptor; another population of arcuate nucleus cells that also express steroid receptors in the sheep are those that express the tachykinin peptide, neurokinin B (NKB). Both DYN and NKB fibres have been shown to form close contacts with ovine GnRH cells. Therefore, the present study tested the hypothesis that neurones expressing NKB and DYN represent the same neuronal population in the arcuate nucleus. Confocal microscopic analysis of brain sections processed for dual immunofluorescence revealed that a large majority of DYN neurones in the arcuate nucleus were also immunoreactive for NKB. Likewise, a similar majority of NKB neurones in the arcuate nucleus were immunoreactive for DYN. By contrast, DYN cells in the preoptic area and anterior hypothalamus did not colocalise with NKB, nor did DYN cells in the paraventricular or supraoptic nuclei. Fibres that stained positively for both DYN and NKB were seen in the arcuate nucleus, where they formed close appositions with DYN/NKB-positive neurones, and in the external zone of the median eminence. Taken together with previous findings, these data suggest that a subpopulation of arcuate nucleus neurones coexpressing DYN and NKB mediate the negative feedback influence of progesterone on pulsatile GnRH secretion in the ewe and may also be involved in other feedback actions of gonadal steroids.

New insights in developmental origins of different GnRH (gonadotrophin-releasing hormone) systems in perciform fish: an immunohistochemical study in the European sea bass ( Dicentrarchus labrax)

The knowledge of the roles and origins of different gonadotrophin-releasing hormone (GnRH) systems could greatly contribute to improve the understanding of mechanisms involved in the physiological control of early development, puberty and spawning. Thus, in this study, we have analyzed the distribution of the cells expressing salmon GnRH, seabream GnRH and chicken GnRH-II forms in the brain and pituitary of developing sea bass using specific antibodies to their corresponding GnRH-associated peptides. The first prepro-chicken GnRH-II-immunoreactive cells arose in the germinal zone of the third ventricle at 4 days after hatching, increasing their number from days 10 to 30, in which they adopted their adult position. The prepro-chicken GnRH-II-immunoreactive fibers became conspicuous in the first week and from day 26 they reached almost all brain areas, especially the hindbrain, being never detected in the pituitary. First prepro-salmon GnRH-immunoreactive cells were detected in the olfactory placode at day 7 after hatching and reached the olfactory bulbs at day 10. Migrating prepro-salmon GnRH cells arrived at the ventral telencephalon at day 15, and became apparent in the preoptic area from day 45. The prepro-salmon GnRH innervation was more evident in the forebrain and increased notably between 10 and 30 days, at which fibers already extended from the olfactory bulbs to the medulla. A few prepro-salmon GnRH-immunoreactive fibers were observed in the pituitary from day 30. The prepro-seabream GnRH-immunoreactive cells were first detected at day 26 in the rostral olfactory bulbs. On day 30, prepro-seabream GnRH-immunoreactive cells were also present in the ventral telencephalon, reaching the preoptic area and the hypothalamus at 45 and 60 days, respectively. The prepro-seabream GnRH innervation appeared restricted to the ventral forebrain, increasing notably during the sixth week, when fibers also reached the pituitary. A significant prepro-seabream GnRH innervation was not detected in the pituitary until day 60.

Noradrenergic projections from the A1 field to the preoptic area in the brain of the ewe and Fos responses to oestrogen in the A1 cells.

Previous studies have shown that there is a population of noradrenergic cells in the caudal A1 field of the brainstem of the ewe that contain oestrogen receptors and project to the preoptic area, where gonadotrophin releasing hormone (GnRH) neurones are located. There are some discrepancies in the literature regarding the extent of this projection and the location of the cells in the A1 region. The issue has been a focus of attention because the positive feedback response to oestrogen that causes the ovulatory GnRH/luteinizing hormone surge may originate from this brainstem region. The aim of the present study was to determine the extent of the projections to the preoptic area and to determine whether the caudal A1 cells are activated by oestrogen. Eleven ovariectomized ewes received an injection of the retrograde tracer FluoroGold into the preoptic hypothalamus and four of these also received an i.m. injection of oestrogen 2 h before tissue collection. A further three sheep received i.m. oil injections to act as controls for those receiving oestrogen. Dopamine-beta-hydroxylase (DBH)-positive, retrogradely labelled cells were found within the A1 field in sheep that received preoptic FluoroGold injections. Cells in the vicinity of the A2 and A6 fields, that were retrogradely labelled with FluoroGold, were not DBH-positive. Thus, cells in the A1 field provide a direct noradrenergic projection to the preoptic area and may be involved in the control of the secretion of GnRH in this species. Cells that project to the preoptic hypothalamus from more rostrally located areas of the brainstem are not noradrenergic. In the animals that received oestrogen, double-labelling immunohistochemistry was performed throughout the A1 field for FluoroGold, DBH and Fos. DBH cells of the A1 field expressed Fos only in the oestrogen-treated animals and not in the oil-treated animals. There was a decline in the number of DBH cells that were retrogradely labelled from the caudal region of A1 towards obex. There was a similar gradient in the number of cells that were double-labelled for Fos and FluoroGold. We conclude that there is a population of noradrenergic cells in the caudal A1 field that project to the preoptic area; this is a larger group of cells than previously reported. Oestrogen elicits an acute Fos response in these cells, which may be involved in the time-delayed positive feedback response on GnRH cells. The caudal-to-rostral gradient in the labelling with FluoroGold and Fos in DBH-positive cells is similar to that seen previously for oestrogen receptor in DBH-positive cells in the A1 field.

Noradrenergic Projections from the A1 Field to the Preoptic Area in the Brain of the Ewe and Fos Responses to Oestrogen in the A1 Cells

AbstractPrevious studies have shown that there is a population of noradrenergic cells in the caudal A1 field of the brainstem of the ewe that contain oestrogen receptors and project to the preoptic area, where gonadotrophin releasing hormone (GnRH) neurones are located. There are some discrepancies in the literature regarding the extent of this projection and the location of the cells in the A1 region. The issue has been a focus of attention because the positive feedback response to oestrogen that causes the ovulatory GnRH/luteinizing hormone surge may originate from this brainstem region. The aim of the present study was to determine the extent of the projections to the preoptic area and to determine whether the caudal A1 cells are activated by oestrogen. Eleven ovariectomized ewes received an injection of the retrograde tracer FluoroGold into the preoptic hypothalamus and four of these also received an i.m. injection of oestrogen 2 h before tissue collection. A further three sheep received i.m. oil injections to act as controls for those receiving oestrogen. Dopamine‐β‐hydroxylase (DBH)‐positive, retrogradely labelled cells were found within the A1 field in sheep that received preoptic FluoroGold injections. Cells in the vicinity of the A2 and A6 fields, that were retrogradely labelled with FluoroGold, were not DBH‐positive. Thus, cells in the A1 field provide a direct noradrenergic projection to the preoptic area and may be involved in the control of the secretion of GnRH in this species. Cells that project to the preoptic hypothalamus from more rostrally located areas of the brainstem are not noradrenergic. In the animals that received oestrogen, double‐labelling immunohistochemistry was performed throughout the A1 field for FluoroGold, DBH and Fos. DBH cells of the A1 field expressed Fos only in the oestrogen‐treated animals and not in the oil‐treated animals. There was a decline in the number of DBH cells that were retrogradely labelled from the caudal region of A1 towards obex. There was a similar gradient in the number of cells that were double‐labelled for Fos and FluoroGold. We conclude that there is a population of noradrenergic cells in the caudal A1 field that project to the preoptic area; this is a larger group of cells than previously reported. Oestrogen elicits an acute Fos response in these cells, which may be involved in the time‐delayed positive feedback response on GnRH cells. The caudal‐to‐rostral gradient in the labelling with FluoroGold and Fos in DBH‐positive cells is similar to that seen previously for oestrogen receptor in DBH‐positive cells in the A1 field.

Tetrodotoxin‐resistant persistent Na+ current underlying pacemaker potentials of fish gonadotrophin‐releasing hormone neurones.

1. Gonadotrophin-releasing hormone (GnRH)-immunoreactive terminal nerve (TN) cells show endogenous regular beating discharges, which may be related to their putative neuromodulator functions. The ionic mechanism underlying the pacemaker potential was studied using intracellular and patch-pipette current clamp recordings from a whole brain in vitro preparation of a small fish brain. 2. The pacemaker potentials were resistant to 1.5-3 microM tetrodotoxin (TTX) and were not affected by Ca2+ channel blockers (amiloride, Ni2+, Co2+, Cd2+) or in Ca(2+)-free solution. In contrast, the pacemaker potentials were readily blocked by substituting tetramethylammonium or choline for Na+ in the perfusing solution, and the resting membrane potential became more hyperpolarized than the control level. 3. The present results suggest that the TTX-resistant persistent Na+ current, INa(slow), supplies the persistent depolarizing drive and plays an important role in the generation of pacemaker potentials in TN GnRH cells.

Studies on the neuronal systems involved in the oestrogen-negative feedback effect on gonadotrophin releasing hormone neurons in the ewe.

Oestrogen can act on the brain to exert negative and positive feedback effects on the secretion of gonadotrophin releasing hormone (GnRH), but this cannot be effected through the GnRH cells themselves because they have no oestrogen receptors. We have used intraventricular injection of agonists and antagonists of various transmitters to determine which systems are involved in the control of GnRH by steroid hormones. We have also developed a model for the injection of drugs directly into the medial pre-optic area of the sheep brain to study the action on the GnRH cell bodies. Opioid systems seem to be involved in the mediation of the feedback effects of progesterone, but not oestrogen. Noradrenaline appears to be involved in the negative feedback regulation of GnRH secretion during the anoestrous period. Thus, under the strong negative influence of oestrogen at this time GnRH/luteinizing hormone (LH) secretion is reduced but can be restored by the preoptic micro-injection of noradrenaline. Gamma-amino-butyric acid (GABA) agonists and antagonists inhibit LH secretion when injected into the medial pre-optic region of the hypothalamus of ovariectomized ewes with or without oestrogen treatment. We have no evidence that GABA is involved in the negative feedback regulation of GnRH, since neither agonists nor antagonists will reverse the effects of steroid feedback. There appears to be a shift in the GABA receptor subtype function with season; in the breeding season only GABAA ligands are effective, whereas during the non-breeding season both GABAA and GABAB type ligands will affect LH secretion. This suggests that the seasonal shift in responsiveness to the negative feedback effects of oestrogen involves a shift in the function of GABA receptor subtypes.

Neurophysiological control of the secretion of gonadotrophin-releasing hormone and luteinizing hormone in the sheep--a review

The anterior pituitary gland secretes pulses of luteinizing hormone (LH) in response to pulses of gonadotrophin-releasing hormone (GnRH) released into the hypophysial portal blood by the hypothalamus. The pulsatile nature of the secretions is very important because the frequency of the pulses is directly related to the activity of the GnRH neurons. We can therefore take advantage of this phenomenon to develop mechanistic interpretations of responses to experimental treatments designed to unravel the neural pathways that influence what is, arguably, the most important individual signal controlling the activity of the reproductive system. We might also resolve the disagreements in the literature covering the neuropharmacology of gonadotrophin secretion. In this review, we describe work towards this end in the sheep. Most (95%) of the 2500 GnRH cell bodies in the sheep brain are located in a region covering the anterior hypothalamus, the medial preoptic area, the diagonal band of Broca, and the septum. The axons of up to 50% of these cells terminate in the organum vasculosum of the lamina terminalis. The remainder terminate in the median eminence and form the final common pathway for the many factors that affect gonadotrophin secretion. Among the factors known to affect the frequency of the pulses (or the activity of the GnRH neurons) are nutrition, pheromones, photoperiod and gonadal steroids (negative and positive feedback). Factors that affect GnRH pulse amplitude are more difficult to determine because variations in pituitary responsiveness prevent the use of LH patterns as a 'bioassay'. Techniques developed recently have allowed the direct measurement of GnRH pulse amplitude and revealed inhibitory effects of oestradiol, but we do not know whether this effect is due to a reduction in the amount of GnRH released by each neurone or a reduction in the number of neurones releasing a pulse. It is unlikely that the factors that alter pulse frequency do so by directly affecting the GnRH cells. For example, it is obvious that other cells, with specific receptors for pheromonal or nutritional stimuli, formulate a signal that is transferred to the GnRH cells via interneurones. Similarly, it is likely that a hypothalamic clock intervenes between photoperiodic inputs and GnRH output. Opioidergic neurons have been proposed as a link in this system, but the complexity of their action makes it unlikely that they directly affect the GnRH neurons. The responses to steroids are simple and rapid, but steroid receptors have not been found in GnRH cells, so at least one other set of interneurones is involved.(ABSTRACT TRUNCATED AT 400 WORDS)