Gonadotrophin-Releasing Hormone Pulse Generator Activity in the Hypothalamus of the Goat

Pulsatile release of gonadotrophin-releasing hormone (GnRH) is indispensable to maintain normal gonadotrophin secretion. The pulsatile secretion of GnRH is associated with synchronised electrical activity in the mediobasal hypothalamus (i.e. multiple unit activity; MUA), which is considered to reflect the rhythmic oscillations in the activity of the neuronal network that drives pulsatile GnRH secretion. However, the cellular source of this ultradian rhythm in GnRH activity is unknown. Direct input from kisspeptin neurones in the arcuate nucleus (ARC) to GnRH cell bodies in the medial preoptic area or their terminals in the median eminence could be the intrinsic source for driving the GnRH pulse generator. To determine whether kisspeptin signalling could be responsible for producing pulsatile GnRH secretion, we studied goats, measured plasma levels of luteinising hormone (LH) and recorded MUA in the posterior ARC, where the majority of kisspeptin neuronal cell bodies are located. Rhythmic volleys of MUA were found to be accompanied by LH pulses with regular intervals in the ARC, where kisspeptin neuronal cell bodies were found. Exogenous administration of kisspeptin stimulated a sustained increase in LH secretion, without influencing MUA, suggesting that the GnRH pulse generator, as reflected by MUA, originated from outside of the network of GnRH neurones, and could plausibly reflect the pacemaker activity of kisspeptin neurones, whose projections reach the median eminence where GnRH fibres project. These observations suggest that the kisspeptin neurones in the ARC may be the intrinsic source of the GnRH pulse generator.     

Octopus Gonadotrophin-Releasing Hormone: A Multifunctional Peptide in the Endocrine and Nervous Systems of the Cephalopod

The optic gland, which is analogous to the anterior pituitary in the context of gonadal maturation, is found on the upper posterior edge of the optic tract of the octopus Octopus vulgaris . In mature octopus, the optic glands enlarge and secrete a gonadotrophic hormone. A peptide with structural features similar to that of vertebrate gonadotophin-releasing hormone (GnRH) was isolated from the brain of octopus and was named oct-GnRH. Oct-GnRH showed luteinising hormone-releasing activity in the anterior pituitary cells of the Japanese quail Coturnix coturnix . Oct-GnRH immunoreactive signals were observed in the glandular cells of the mature optic gland. Oct-GnRH stimulated the synthesis and release of sex steroids from the ovary and testis, and elicited contractions of the oviduct. Oct-GnRH receptor was expressed in the gonads and accessory organs, such as the oviduct and oviducal gland. These results suggest that oct-GnRH induces the gonadal maturation and oviposition by regulating sex steroidogenesis and a series of egg-laying behaviours via the oct-GnRH receptor. The distribution and expression of oct-GnRH in the central and peripheral nervous systems suggest that oct-GnRH acts as a multifunctional modulatory factor in feeding, memory processing, sensory, movement and autonomic functions.     

Modulation of Gonadotrophin-Releasing Hormone Secretion by an Endogenous Circadian Clock

The mechanisms mediating positive feedback effects of oestradiol on pre-ovulatory gonadotrophin releasing-hormone (GnRH) surge generation in female mammals, although well-explored, are still incompletely understood. In addition to binding to and signalling through classical nuclear receptor-mediated pathways in afferent hypothalamic neurones, recent evidence suggests that ovarian steroids may use membrane-bound receptors or nonclassical signalling pathways to directly influence cell function leading to the generation of GnRH surge secretion. We review recent investigations into the role of the endogenous molecular circadian clock on modulation of GnRH gene expression and neuropeptide secretion, and will explore potential molecular mechanisms by which ovarian steroids may directly induce secretory changes at the level of the GnRH neurone, examining closely whether circadian clock gene oscillations may be involved.     

Effects of Bovine Follicular Fluid on Pulsatile Secretion of Gonadotrophin-Releasing Hormone and Gonadotrophins in Ovariectomized Ewes

Ovariectomized ewes were injected with charcoal-extracted bovine follicular fluid (n = 5) or with hypophysectomized ewe serum (n = 5) after which hypophyseal portal blood samples were taken to monitor the release of gonadotrophin-releasing hormone (GnRH). Peripheral blood samples were also taken to monitor plasma luteinizing hormone and follicle-stimulating hormone (FSH) concentrations. Bovine follicular fluid treatment caused a 50% decrease in plasma FSH concentrations whereas hypophysectomized ewe serum did not significantly alter plasma FSH levels. The frequency and amplitude of luteinizing hormone and GnRH pulses was similar in both experimental groups. It is concluded that inhibin activity in bovine follicular fluid selectively suppresses the release of FSH by direct action on the pituitary gland, with no effect on GnRH secretion.     

Coexistence of Gonadotrophin-Releasing Hormone and Galanin: Immunohisto-chemical and Functional Studies

Our observation of galanin-immunoreactive perikarya with morphological characteristics strikingly similar to those containing gorvadotrophin-releasmg hormone (GnRH) led us to undertake double-label immunohistochemical studies on the distribution of these peptides. A considerable proportion of GnRH-containing perikarya in the rat preoptic area were found to be immunoreactive for galanin. In order to establish whether this coexistence contributes to the control of luteinizing hormone (LH) release, the two peptides have been perfused alone and in combination through anterior pituitary cell columns derived from female rats in various endocrine conditions. Galanin at 0.1 μ M had a small stimulatory effect on LH release from cells obtained on the day of pro-oestrus; this effect was less than that obtained with GnRH at 4 nM. Cells from oestrous rats showed no LH release in response to galanin. The studies undertaken thusfar have not indicated that galanin has a priming effect on the release of LH induced by subsequent treatment with galanin or a potentiating effect when administered together with GnRH. Delta sleep-inducing peptide, another neuropeptide recently reported to be coexistent with GnRH, was also found to be capable of inducing a small discharge of LH from anterior pituitary cells obtained on the day of pro-oestrus. Further studies will be required to establish the full consequences of the coexistence of these peptides and GnRH.     

Modulation of Gonadotrophin-Releasing Hormone Pulse Generator Activity by the Pheromone in Small Ruminants

In small ruminants, such as goats and sheep, a primer pheromone produced by males induces an out-of-seasonal ovulation in anoestrous females, a phenomenon known as the male effect. The male effect is unique in that an external chemical stimulus can immediately modulate the activity of the hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator. We have established a monitoring method of the GnRH pulse generator activity in Shiba goat. Using this method as a sensitive bioassay to assess the male effect pheromone activity, we have shown that the male effect pheromone is synthesised in an androgen-dependent manner in the sebaceous glands or their vicinity in specific body regions in goats. Although chemical identity of the pheromone is yet to be determined, analyses of male goat hair extracts by gas chromatography fractionation suggest that the male effect pheromone is a volatile substance with relatively small molecular weight. From morphological and molecular biological studies in goats, it is suggested that the pheromone molecule is detected by a member of the V1R family located on both the olfactory neurones and the vomeronasal sensory neurones, and the pheromone signal is conveyed to the medial nucleus of amygdala via the main olfactory and vomeronasal pathways and, subsequently, to the hypothalamic GnRH pulse generator to enhance its activity.     

Effects of Gonadotrophin-Releasing Hormone Variants on Reproductive Organs and Plasma Testosterone in the Male Lizard, Podarcis s. sicula

The effects of various gonadotrophin-releasing hormone (GnRH) forms (mammalian GnRH (mGnRH), chicken I GnRH (cGnRH-I), chicken II GnRH (cGnRH-II) and salmon GnRH (sGnRH)) on the genital apparatus and plasma testosterone level in the male lizard, Podarcis s. sicula , have been investigated.
In short duration experiments (20 min to 76 h) GnRH forms did not affect testicular and epididymal morphology. A single dose (0.05 μg) of mGnRH, cGnRH-II and sGnRH, however, induced a rise in plasma testosterone after 20 to 40 min. Variable results were obtained in the animals given GnRH variants every 12 h for 3 days since mGnRH and cGnRH-I caused a decrease of circulating hormone; cGnRH-II and sGnRH a slight increase.
Daily peptide administration, for 15 to 30 days, caused severe inhibition of both testicular and epididymal activity and a significant drop of circulating testosterone.
In Podarcis s. sicula , species specificity of pituitary sensitivity to GnRH variants appeared to be low. On the other hand, this gland seemed to show some desensitization after chronic peptide administration.     

Neurobiological Mechanisms Underlying Oestradiol Negative and Positive Feedback Regulation of Gonadotrophin-Releasing Hormone Neurones

The feedback actions of ovarian oestradiol during the female reproductive cycle are among the most unique in physiology. During most of the cycle, oestradiol exerts homeostatic, negative feedback upon the release of gonadotrophin-releasing hormone (GnRH). Upon exposure to sustained elevated oestradiol levels, however, there is a switch in the feedback effects of this hormone to positive, resulting in induction of a surge in the release of GnRH that serves as a neuroendocrine signal to initiate the ovulatory cascade. We review recent developments stemming from studies in an animal model exhibiting daily switches between positive and negative feedback that have probed the neurobiological mechanisms, including changes in neural networks and intrinsic properties of GnRH neurones, underlying this switch in oestradiol action.     

Spatiotemporal expression patterns of slit and robo genes in the rat brain.

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a repulsive guidance system that prevents inappropriate axons from crossing the central nervous system midline; this repulsive system is mediated by the secreted extracellular matrix protein Slit and its receptors Roundabout (Robo). Three distinct slit genes (slit1, slit2, and slit3) and three distinct robo genes (robo1, robo2, rig-1) have been cloned in mammals. However, to date, only Robo1 and Robo2 have been shown to be receptors for Slits. In rodents, Slits have been shown to function as chemorepellents for several classes of axons and migrating neurons. In addition, Slit can also stimulate the formation of axonal branches by some sensory axons. To identify Slit-responsive neurons and to help analyze Slit function, we have studied, by in situ hybridization, the expression pattern of slits and their receptors robo1 and robo2, in the rat central nervous system from embryonic stages to adult age. We found that their expression patterns are very dynamic: in most regions, slit and robo are expressed in a complementary pattern, and their expression is up-regulated postnatally. Our study confirms the potential role of these molecules in axonal pathfinding and neuronal migration. However, the persistence of robo and slit expression suggests that the couple slit/robo may also have an important function in the adult brain.     

Lesions of the Suprachiasmatic Nucleus Indicate the Presence of a Direct Vasoactive Intestinal Polypeptide-Containing Projection to Gonadotrophin-Releasing Hormone Neurons in the Female Rat

In non-seasonal breeders like the rat, the influence of the suprachiasmatic nucleus (SCN) on reproduction is most clearly expressed in the female. Complete lesions of the SCN induce persistent oestrus (anovulation) in intact female rats, whereas oestrogen implantation in ovariectomized rats results in daily luteinizing hormone surges. Vasoactive intestinal polypeptide (VIP), a peptide synthesized in cell bodies of the SCN, inhibits the increase in pulsatile luteinizing hormone release observed in ovariectomized female rats. In search of the anatomical basis for these observations, the present study employs an immunocytochemical double staining for VIP and gonadotrophin-releasing hormone (GnRH) at the light microscopical level. It was demonstrated that approximately 45% of the GnRH positive neurons in the diagonal band of Broca, the preoptic and anterior hypothalamic area of female rats are innervated by VIP-containing processes. To investigate whether these VIP-containing fibres represent a direct projection of the SCN to the GnRH system, unilateral thermic SCN lesions were made. Lesions that unilaterally destroyed the majority of the VIP synthesizing cells in the SCN resulted in at least a 50% decrease of the VIP innervation of GnRH cell bodies at the lesioned side compared to the intact side. Lesions not affecting the VIP synthesizing cell population in the SCN did not change the percentage of GnRH neurons innervated by VIP-containing fibres, while partial lesions resulted in intermediate effects. These results indicate that the majority of the light microscopical VIP-containing input on GnRH neurons in the hypothalamus is derived from the SCN. It is suggested that the reported effects of VIP on luteinizing hormone release may, at least in part, be induced via a direct effect of VIP on GnRH cell bodies. This direct SCN-GnRH pathway provides an anatomical basis for diurnal influences on the regulation of the female reproductive cycle.     

An olfactory input to the hippocampus of the cat: Field potential analysis

Hippocampal responses to electrical stimulation of the prepyriform cortex in the cat were studied both in acute experiments under halothane anesthesia and in awake cats with chronically indwelling electrodes. Analysis of field potentials and unit activity indicated the extent to which different hippocampal subareas were activated, the laminar level at which the synaptic action took place and the dynamics of the evoked responses. It was found that: (1) the main generator of evoked responses in the hippocampus upon prepyriform cortex stimulation is localized in the fascia dentata and CA3 (CA1 pyramidal cells, and probably also subiculum cells, are activated but in a lesser degree); (2) the initial synaptic activity takes place at the most distal part of the dendrites of fascia dentata granule cells and CA3 pyramidal cells; and (3) this synaptic activity corresponds to an EPSP that leads to a transient increase in the firing rate of the hippocampal units, which is often followed by a long-lasting decrease in firing rate.We conclude that the pathway from the prepyriform cortex via lateral entorhinal cortex to hippocampal neurons may enable olfactory inputs to effectively excite hippocampal neurons.     

Stimulation of Luteinizing Hormone-Beta Messenger Ribonucleic Acid and Post-Translational Modification of Luteinizing Hormone Isoforms by Second Messengers Mediating the Action of Gonadotrophin-Releasing Hormone

Several second messenger systems have been implicated in mediating the action of gonadotrophin-releasing hormone on the pituitary gonadotrophs and numerous studies have shown that activation of these systems induces luteinizing hormone (LH) secretion. However, it is not known how gonadotrophin-releasing hormone or the second messenger systems induce de novo LH biosynthesis and post-translational modification of the hormone. In these experiments hemipituitary glands have been perifused with drugs which activate second messengers or stimulate protein kinase C directly. The LH secretory responses have been correlated with measurements of common a and LHβ mRNA and the molecular species of LH which were present in the pituitary perifusate after exposure to the drugs. Gonadotrophin-releasing hormone (50 ng/ml, 42 nM), with and without the presence of extracellular Ca²⁺, the Ca²⁺ ionophore, A23187 (10 μM), and phorbol 12-myristate (1 μM) all stimulated an increase in LHβ mRNA compared with controls and the appearance of a different isoform of LH to that found stored in and released from the unstimulated pituitary gland. Phospholipase C was without effect on LHβ mRNA levels and showed minimal efficacy in inducing the appearance of the different LH isoform.     

Kisspeptin/Metastin: A Key Molecule Controlling Two Modes of Gonadotrophin-Releasing Hormone/Luteinising Hormone Release in Female Rats

Kisspeptin (also known as metastin), a hypothalamic peptide, has attracted attention as a key molecule in the release of gonadotrophin-releasing hormone (GnRH) in various mammalian species, such as rodents, sheep and primates. Two populations of kisspeptin neurones in the brain may control two modes of GnRH release to time the onset of puberty and regulate oestrous cyclicity in rats and mice. One population of kisspeptin neurones, located in the anteroventral periventricular nucleus, appears to be responsible for the induction of the GnRH surge that leads to the luteinising hormone surge and ovulation. The other, located in the hypothalamic arcuate nucleus, appears to be involved in generating GnRH pulses, resulting in luteinising hormone pulses followed by follicular development and steroidogenesis in the ovary. The present review focuses on the physiological role of the two populations of kisspeptin neurones in controlling gonadal functions by generating the two modes of GnRH release in a female rat model.     

A Biological Role for the Gonadotrophin-Releasing Hormone (GnRH) Metabolite, GnRH-(1-5)

Gonadotrophin-releasing hormone (GnRH) was first isolated in the mammal and shown to be the primary regulator of the reproductive system through its initiation of pituitary gonadotrophin release. Subsequent to its discovery, this form of GnRH has been shown to be one of many structural variants found in the brain and peripheral tissues. Accordingly, the original form first discovered and cloned in the mammal is commonly referred to as GnRH-I. In addition to the complex regulation of GnRH-I synthesis, release and function, further evidence suggests that the processing of GnRH-I produces yet another layer of complexity in its activity. GnRH-I is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15), which cleaves the hormone at the covalent bond between the fifth and sixth residue of the decapeptide (Tyr₅-Gly₆) to form GnRH-(1-5). It was previously thought that the cleavage of GnRH-I by EP24.15 represents the initiation of its degradation. Here, we review the evidence for the involvement of GnRH-(1-5), the metabolite of GnRH-I, in the regulation of GnRH-I synthesis, secretion and facilitation of reproductive behaviour.     

Spatial distribution of neural activity in the anterior olfactory nucleus evoked by odor and electrical stimulation

Several lines of evidence indicate that complex odorant stimuli are parsed into separate data streams in the glomeruli of the olfactory bulb, yielding a combinatorial "odotopic map." However, this pattern does not appear to be maintained in the piriform cortex, where stimuli appear to be coded in a distributed fashion. The anterior olfactory nucleus (AON) is intermediate and reciprocally interconnected between these two structures, and also provides a route for the interhemispheric transfer of olfactory information. The present study examined potential coding strategies used by the AON. Rats were exposed to either caproic acid, butyric acid, limonene, or purified air and the spatial distribution of Fos-immunolabeled cells was quantified. The two major subregions of the AON exhibited different results. Distinct odor-specific spatial patterns of activity were observed in pars externa, suggesting that it employs a topographic strategy for odor representation similar to the olfactory bulb. A spatially distributed pattern that did not appear to depend on odor identity was observed in pars principalis, suggesting that it employs a distributed representation of odors more similar to that seen in the piriform cortex.     

Differences in chemo- and cytoarchitectural features within pars principalis of the rat anterior olfactory nucleus suggest functional specialization

The anterior olfactory nucleus (AON) lies between the olfactory bulb and piriform cortex and is the first bilaterally innervated structure in the olfactory system. It is typically divided into two subregions: pars externa and pars principalis. We examined the cytoarchitecture of pars principalis, the largest cellular area of the region, to determine whether it is homogeneously organized. Quantitative Nissl studies indicated that large cells (cell body area >2 standard deviations (SD) larger than the mean cell size) are densest in lateral and dorsolateral regions, while small cells (>1 SD smaller than the mean) are more numerous in medial and ventral areas. Further evidence for regional differences in the organization of the AON were obtained with immunohistochemistry for calbindin (CALB), parvalbumin (PARV), glutamic acid decarboxylase (GAD), and choline transporter (CHT). Cells immunopositive for CALB (CALB+) were denser in the deep portion of Layer II, although homogeneously dispersed throughout the circumference of the AON. PARV+ cells were located in the superficial half of Layer II and were sparse in ventral and medial regions. CHT+ and GAD+ fibers were denser in lateral versus medial regions. No regional differences were found in GAD+ somata, or in norepinephrine transporter or serotonin transporter immunoreactivity. The observed regional differences in cyto- and chemoarchitectural features may reflect functional heterogeneity within the AON.     

Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat

The immunohistochemical localization of calcium-binding protein (CaBP) in the cerebellum, hippocampal formation and olfactory bulb of the rat was examined using rabbit anti-human or sheep anti-chick antisera purified by affinity chromatography. CaBP-like immunoreactivity was observed within the somata and dendrites of: (1) cerebellar Purkinje cells; (2) dentate granule cells, CA1 pyramidal cells and scattered interneurons in the stratum radiatum of the hippocampus; (3) periglomerular cells in the olfactory bulb. Staining was conspicuously absent in certain major cell types in each of these structures including cerebellar granule cells, hippocampal pyramidal cells in the CA3 region and both mitral and granule cells in the olfactory bulb. Immunoreactive fibers in the cerebellum presumably corresponding to climbing fiber inputs from the inferior olive and efferent projections to the deep cerebellar nuclei, were also observed. In the hippocampus intense staining was present in the mossy fiber projection to the CA3 region and in the terminal regions of the perforant path projection from entorhinal cortex.