Facilitatory and inhibitory role of central amylin administration in the regulation of the gonadotropin-releasing hormone pulse generator activity in goats.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is essential for regulating reproductive functions in mammals. GnRH pulses are governed by a neural mechanism that is termed the GnRH pulse generator. In the present study, we investigated the role of central calcitonin receptor (CTR) signaling in the regulation of the GnRH pulse generator activity in ovariectomized goats by administering amylin, an endogenous ligand for CTR, into the lateral ventricle. GnRH pulse generator activity was measured using multiple unit activity (MUA) recordings in the mediobasal hypothalamus. We analyzed changes in the interval of characteristic increases in MUA (MUA volleys). The MUA volley interval shortened immediately after amylin administration, followed by prolonged intervals. Double in situ hybridization for KISS1 (kisspeptin gene) and CALCR (CTR gene) revealed that low expression levels of CALCR were found in the arcuate kisspeptin neurons, which is suggested as the main population of neurons, involved in GnRH pulse generator activity. These results suggest that central amylin-CTR signaling has a biphasic role in the regulation of GnRH pulse generator activity by acting on cells other than the arcuate kisspeptin neurons in goats.

Immunohistochemical characterization of the arcuate kisspeptin/neurokinin B/dynorphin (KNDy) and preoptic kisspeptin neuronal populations in the hypothalamus during the estrous cycle in heifers.

Elucidating the physiological mechanisms that control reproduction is an obvious strategy for improving the fertility of cattle and developing new agents to control reproductive functions. The present study aimed to identify kisspeptin neurons in the bovine hypothalamus, clarifying that a central mechanism is also present in the cattle brain, as kisspeptin is known to play an important role in the stimulation of gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in other mammals. To characterize kisspeptin neurons in the bovine hypothalamus, the co-localizations of kisspeptin and neurokinin B (NKB) or kisspeptin and dynorphin A (Dyn) were examined. Hypothalamic tissue was collected from Japanese Black or Japanese Black × Holstein crossbred cows during the follicular and luteal phases. Brain sections, including the arcuate nucleus (ARC) and the preoptic area (POA), were dual immunostained with kisspeptin and either NKB or Dyn. In the ARC, both NKB and Dyn were co-localized in kisspeptin neurons during both the follicular and luteal phases, demonstrating the presence of kisspeptin/NKB/Dyn-containing neurons, referred to as KNDy neurons, in cows. In the POA, no co-localization of kisspeptin with either NKB or Dyn was detected. Kisspeptin expression in the follicular phase was higher than that in the luteal phase, suggesting that kisspeptin expression in the POA is positively controlled by estrogen in cows. The kisspeptin neuronal populations in the ARC and POA likely play important roles in regulating the GnRH pulse and surge, respectively, in cows.

Molecular and Epigenetic Mechanism Regulating Hypothalamic Kiss1 Gene Expression in Mammals.

After the discovery of hypothalamic kisspeptin encoded by the Kiss1 gene, the central mechanism regulating gonadotropin-releasing hormone (GnRH) secretion, and hence gonadotropin secretion, is gradually being unraveled. This has increased our understanding of the central mechanism regulating puberty and subsequent reproductive performance in mammals. Recently, emerging evidence has indicated the molecular and epigenetic mechanism regulating hypothalamic Kiss1 gene expression. Here we compile data regarding DNA and histone modifications in the Kiss1 promoter region and provide a hypothetic scheme of the molecular and epigenetic mechanism regulating Kiss1 gene expression in two populations of hypothalamic kisspeptin neurons, which govern puberty and subsequent reproductive performance via GnRH/gonadotropin secretion.

The effects of chronic subcutaneous administration of an investigational kisspeptin analog, TAK-683, on gonadotropin-releasing hormone pulse generator activity in goats.

The continuous activation of the kisspeptin receptor by its agonists causes the abrogation of kisspeptin signaling, leading to decreased pulsatile luteinizing hormone (LH) secretion. Employing this phenomenon as a tool for probing kisspeptin action, this study aimed to clarify the role of kisspeptin in gonadotropin-releasing hormone (GnRH) pulse generation in goats. We examined the effects of chronic administration of TAK-683, an investigational kisspeptin analog, on LH secretion, GnRH immunostaining, pituitary responses to exogenous GnRH, and GnRH pulse generator activity, reflected by a characteristic increase in multiple-unit activity (MUA volley). An osmotic pump containing TAK-683 was subcutaneously implanted on day 0. TAK-683 treatment dose-dependently suppressed pulsatile LH secretion on day 1. Higher doses of chronic TAK-683 profoundly suppressed pulsatile LH secretion but had little effect on GnRH immunostaining patterns and pituitary responses to GnRH on day 5. In ovariectomized goats, MUA volleys occurred at approximately every 30 min on day -1. On day 5 of chronic TAK-683 administration, pulsatile LH secretion was markedly suppressed, whereas MUA volleys were similar to those observed on day -1. Male pheromones and senktide (neurokinin B receptor agonist) induced an MUA volley but had no effect on LH secretion during chronic TAK-683 administration. The results indicate that the chronic administration of a kisspeptin analog profoundly suppresses pulsatile LH secretion without affecting GnRH content, pituitary function or GnRH pulse generator activity, and they suggest an indispensable role for kisspeptin signaling in the cascade driving GnRH/LH pulses by the GnRH pulse generator.

Development of novel neurokinin 3 receptor (NK3R) selective agonists with resistance to proteolytic degradation.

Neurokinin B (NKB) regulates the release of gonadotropin-releasing hormone (GnRH) via activation of the neurokinin-3 receptor (NK3R). We evaluated the biological stability of NK3R selective agonists to develop novel NK3R agonists to regulate reproductive functions. On the basis of degradation profiles, several peptidomimetic derivatives were designed. The modification of senktide with (E)-alkene dipeptide isostere generated a novel potent NK3R agonist with high stability and prolonged bioactivity.

Differential effects of continuous exposure to the investigational metastin/kisspeptin analog TAK-683 on pulsatile and surge mode secretion of luteinizing hormone in ovariectomized goats.

The aim of the present study was to determine if the estradiol-induced luteinizing hormone (LH) surge is influenced by the constant exposure to TAK-683, an investigational metastin/kisspeptin analog, that had been established to depress the pulsatile gonadotropin-releasing hormone (GnRH) and LH secretion in goats. Ovariectomized goats subcutaneously received TAK-683 (TAK-683 group, n=6) or vehicle (control group, n=6) constantly via subcutaneous implantation of an osmotic pump. Five days after the start of the treatment, estradiol was infused intravenously in both groups to evaluate the effects on the LH surge. Blood samples were collected at 6-min intervals for 4 h prior to the initiation of either the TAK-683 treatment or the estradiol infusion, to determine the profiles of pulsatile LH secretion. They were also collected at 2-h intervals from -4 h to 32 h after the start of estradiol infusion for analysis of LH surges. The frequency and mean concentrations of LH pulses in the TAK-683 group were remarkably suppressed 5 days after the start of TAK-683 treatment compared with those of the control group (P<0.05). On the other hand, a clear LH surge was observed in all animals of both groups. There were no significant differences in the LH concentrations for surge peak and the peak time of the LH surge between the TAK-683 and control groups. These findings suggest that the effects of continuous exposure to kisspeptin or its analog on the mechanism(s) that regulates the pulsatile and surge mode secretion of GnRH/LH are different in goats.

Central distribution of kiss2 neurons and peri-pubertal changes in their expression in the brain of male and female red seabream Pagrus major.

kisspeptins that are encoded by kiss1 gene are now considered the key regulator of reproduction from a number of studies in mammals. In most vertebrates, a paralogue of kiss1, called kiss2, is also present, and the functional significance of kisspeptins is not known precisely. In the present study, we have cloned kiss2 from a perciform teleost, the red seabream Pagrus major. The amino acid sequence deduced from the red seabream kiss2 contained a highly conserved 10-amino-acid residue, Kiss2(10) or kp-10. A kiss1-like transcript was also identified, but it appears to be non-functional due to the presence of a "premature" stop codon. Neurons that express kiss2 mRNA were distributed in the dorsal (NRLd) and ventral (NRLv) parts of nucleus recessi lateralis in the hypothalamus. In some fish a few kiss2-expressing neurons were detected in the preoptic area and nucleus ventralis tuberis. The number of kiss2-expressing neurons in the NRLd was larger during the first spawning season in both males and females compared with fish in the post-spawning periods. In males the number of kiss2 neurons in the NRLd of maturing fish was also larger than those in the post-spawning periods. In males the number of kiss2 neurons in the NRLv showed a similar pattern of changes to that of NRLd, while significant changes were not detected for females. The numbers of gonadotropin-releasing hormone 1 (GnRH1)-immunoreactive neurons in the preoptic area showed a similar pattern of change as those of kiss2 cells of the NRLd in both males and females (and also the NRLv in males). These results are in good agreement with the hypothesis that kiss2 neurons are involved in pubertal processes via regulatory influences on GnRH1 neurons in red seabream.

Morphological evidence for direct interaction between kisspeptin and gonadotropin-releasing hormone neurons at the median eminence of the male goat: an immunoelectron microscopic study.

Kisspeptin has been thought to play pivotal roles in the control of both pulse and surge modes of gonadotropin-releasing hormone (GnRH) secretion. To clarify loci of kisspeptin action on GnRH neurons, the present study examined the morphology of the kisspeptin system and the associations between kisspeptin and GnRH systems in gonadally intact and castrated male goats. Kisspeptin-immunoreactive (ir) and Kiss1-positive neurons were found in the medial preoptic area of intact but not castrated goats. Kisspeptin-ir cell bodies and fibers in the arcuate nucleus (ARC) and median eminence (ME) were fewer in intact male goats compared with castrated animals. Apposition of kisspeptin-ir fibers on GnRH-ir cell bodies was very rare in both intact and castrated goats, whereas the intimate association of kisspeptin-ir fibers with GnRH-ir nerve terminals was observed in the ME of castrated animals. Neurokinin B immunoreactivity colocalized not only in kisspeptin-ir cell bodies in the ARC but also in kisspeptin-ir fibers in the ME, suggesting that a majority of kisspeptin-ir fibers projecting to the ME originates from the ARC. A dual immunoelectron microscopic examination revealed that nerve terminals containing kisspeptin-ir vesicles made direct contact with GnRH-ir nerve terminals at the ME of castrated goats. There was no evidence for the existence of the typical synaptic structure between kisspeptin- and GnRH-ir fibers. The present results suggest that the ARC kisspeptin neurons act on GnRH neurons at the ME to control (possibly the pulse mode of) GnRH secretion in males.

Neurobiological mechanisms underlying GnRH pulse generation by the hypothalamus.

Gonadotropin-releasing hormone (GnRH) secretion has two modes of release in mammalian species; the surge mode and the pulse mode. The surge mode, which is required for the induction of the preovulatory gonadotropin discharge in most species, is induced by the positive feedback of estrogen secreted by the mature ovarian follicle. The pulse mode of GnRH secretion stimulates tonic luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion which drives folliculogenesis, spermatogenesis and steroidogenesis and is negatively fine-tuned by estrogen or androgen. The GnRH pulse-generating mechanism is sensitive to environmental cues, such as photoperiod, nutrition and stress surge-generating mechanism is relatively emancipated from these environmental cues. The present article first provides a brief historical background to the work that led to the concept of the GnRH pulse generator: a hypothalamic network that is central to our understanding of the regulation of reproduction. We then discuss possible neurobiological mechanisms underlying GnRH pulse generation, and conclude by proposing that kisspeptin neurons in the arcuate nucleus are key players in this regard.

Molecular characterization and estrogen regulation of hypothalamic KISS1 gene in the pig.

Kisspeptin-GPR54 signaling plays an essential role in normal reproduction in mammals via stimulation of gonadotropin secretion. Here, we cloned the porcine KISS1 cDNA from the hypothalamic tissue and investigated the effect of estrogen on the distribution and numbers of KISS1 mRNA-expressing cells in the porcine hypothalamus. The full length of the cDNA was 857 bp encoding the kisspeptin of 54 amino acids, with the C-terminal active motif designated kisspeptin-10 being identical to that of mouse, rat, cattle, and sheep. In situ hybridization analysis revealed that KISS1-positive cell populations were mainly distributed in the hypothalamic periventricular nucleus (PeN) and arcuate nucleus (ARC). KISS1 expression in the PeN of ovariectomized (OVX) pigs was significantly upregulated by estradiol benzoate (EB) treatment. On the other hand, KISS1-expressing cells were abundantly distributed throughout the ARC in both OVX and OVX with EB animals. The number of KISS1-expressing neurons was significantly lowered by EB treatment only in the most caudal part of the ARC, but other ARC populations were not affected. The present study thus suggests that the PeN kisspeptin neurons could be responsible for the estrogen positive feedback regulation to induce gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) surge in the pig. In addition, the caudal ARC kisspeptin neurons could be involved in the estrogen negative feedback regulation of GnRH/LH release. This is the first report of identification of porcine KISS1 gene and of estrogen regulation of KISS1 expression in the porcine brain, which may be helpful for better understanding of the role of kisspeptin in reproduction of the pig.

Further evidence for the role of glucose as a metabolic regulator of hypothalamic gonadotropin-releasing hormone pulse generator activity in goats.

The present study examined the relative importance of blood glucose vs. free fatty acids as a metabolic signal regulating GnRH release as measured electrophysiologically by multiple-unit activity (MUA) in the arcuate nucleus/median eminence region in ovariectomized, estradiol-treated goats. MUA was recorded before, during, and after: 1) cellular glucoprivation by peripheral infusion of 2-deoxy-d-glucose (2DG; 25, 50, and 75 mg/kg.h, iv); 2) peripheral hypoglycemia in response to various doses (15-195 mU/kg.h, iv) of insulin infusion; and 3) cellular lipoprivation induced by peripheral infusion of sodium mercaptoacetate (MA; 2.4 mg/kg.h alone or combined with 25 mg/kg.h of 2DG, iv), and effects on the interval of characteristic increases in MUA (MUA volleys) were examined. Infusion of the highest dose of 2DG increased the mean interval between MUA volleys, whereas the lower doses of 2DG had no effect on volley interval. The MUA volley intervals lengthened as insulin-induced hypoglycemia became profound. There was a negative correlation between MUA volley intervals and blood glucose concentrations during insulin infusion, and coinfusion of glucose with insulin returned the MUA volley interval to a normal frequency. Infusion of MA alone or MA with 2DG did not increase MUA volley intervals. These findings demonstrate that glucose availability, but not fatty acids, regulates the GnRH pulse generator activity in the ruminant. Glucose is considered a key metabolic regulator that fine-tunes pulsatile GnRH release.

Simultaneous observation of the GnRH pulse generator activity and plasma concentrations of metabolites and insulin during fasting and subsequent refeeding periods in Shiba goats.

The time course of GnRH pulse generator activity and plasma concentrations of energy substrates and insulin were simultaneously observed in female goats during 4-day fasting and subsequent refeeding in the presence or absence of estrogen for a better understanding of the mechanism of energetic control of gonadotropin secretion in ruminants. The GnRH pulse generator activity was electrophysiologically assessed with the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus. In estradiol-treated ovariectomized (OVX+E2) goats, the MUA volley intervals increased as fasting progressed. Plasma concentrations of non-esterified fatty acid and ketone body increased, while those of acetic acid and insulin decreased during fasting. The MUA volley intervals and plasma concentrations of those metabolites and insulin were restored to pre-fasting levels after subsequent refeeding. In ovariectomized (OVX) goats, changes in plasma metabolites and insulin concentrations were similar to those in OVX+E2 goats, but the MUA volley intervals were not altered. The present results demonstrated that fasting suppressed GnRH pulse generator activity in an estrogen-dependent manner. Changes in plasma concentrations of energy substrates and insulin during fasting were associated with the GnRH pulse generator activity in the presence of estrogen, but not in the absence of the steroid in female goats.