Satoshi Ohkura

Introduction: Pulsatile GnRH secretion is a key determinant for normal gonadal activities. Recent emerging evidence suggests that neurokinin B (NKB) plays a pivotal role in generating GnRH pulse. Here, we aimed to develop novel compounds with potent NKB receptor (NK3R) agonistic activity for future therapeutic applications to improve gonadal functions. Materials and methods: On the basis of the structure-activity relationship study of NKB and tachykinin peptides, we designed and synthesized 72 compounds. Their binding affinity and agonistic activity for G_q-coupled NK3 receptor were evaluated by an in vitro-binding inhibition assay and an intracellular Ca²⁺ flux assay respectively. Actions of the peptides on the GnRH pulse generator were evaluated by the multiple-unit activity (MUA) recording method in ovariectomized goats, in which the electrophysiological manifestations of the GnRH pulse generator activity can be represented as characteristic increases in MUA (MUA volleys). Thirty nanomoles of each peptide was i.v. administered at the mid-point of two successive endogenous MUA volleys occurring with a constant interval of ~25 min. The given peptide was assessed to stimulate the GnRH pulse generator when the next MUA volley occurred within 240 s after administration. Results and discussion: Eight compounds were found to exhibit superior in vitro activity than senktide, a classical NK3R agonist, and induce an MUA volley after administration. Because those compounds were capable of stimulating the GnRH pulse generator by peripheral administration, they might be potential candidates for novel agents to improve reproduction acting centrally.

Neurons essential for reproductive functions are distributed in hypothalamus. The precise functions of these neurons are not fully analyzed at a cellular level, especially in domestic animals. To analyze cellular mechanisms that control ruminant reproduction, we generated neuronal cell lines by immortalizing fetal goat hypothalamus. The hypothalamic arcuate nucleus was collected from a female Shiba goat at E118. The tissue was dispersed and plated. Twenty-four hours after the primary culture, lentiviral vector containing SV40 large T antigen gene (T-Ag) was added, then cells were selected by G418. We performed cell cloning from the obtained cell population, and analyzed gene expressions of each clone by RT-PCR. The expression of neuronal marker (neuron specific enolase (NSE)), glial marker (glial fibrillary acidic protein (GFAP)), T-Ag, and GAPDH were examined. The primary-cultured cells acquired strong proliferative activity after the viral infection. All clones expressed T-Ag, suggesting that these cells were immortalized by the insertion of T-Ag. We obtained 61 clones from the immortalized population. Thirty-six out of 61 clones were NSE-positive and GFAP-negative, which were defined to be neuron-derived cell lines. In summary, we established a number of neuronal cell lines derived from goat hypothalamus. Further analysis of expression of neuropeptides or receptors related to reproduction is required to determine neuronal cell lines appropriate for examining central mechanism regulating reproduction in vitro. This work was supported in part by the Research Program on Innovative Technologies for Animal Breeding, Reproduction, and Vaccine Development from the Agriculture, Forestry and Fisheries Research Council, Japan (REP2005).

The aim of the present study was to evaluate ovarian and hormonal responses to follicular phase administration of a metastin/kisspeptin analog, TAK-683, with a low dose, determined as the minimally effective dose for LH stimulation in ovariectomized goats. At 7–10 days after ovulation, goats received a prostaglandin F₂α (PGF₂α) injection (day 0) followed by 10 days of CIDR-G^® treatment. A PGF₂α was injected again at CIDR removal. Then, they received 5 μg of TAK-683 (0 h) intravenously (n=5, I.V.) or subcutaneously (n=3, S.C.) at 12 h after CIDR removal. Vehicle was given intravenously in the same manner (n=4, Cont). Blood samples were collected at 10-min (2–6 h), 2-h (6–24 h) or 6-h (24–48 h) I.V. for analysis of endocrine profiles. Ovarian ultrasonographic images were assessed daily to confirm ovulation after the treatment. A surge-like release of LH was immediately observed after the injection in all animals in I.V. (peak time: hours 4.2±0.6, peak concentration: 73.3±27.5 ng/ml) and S.C. (peak time: hours 4.6±0.4, peak concentration: 62.6±23.2 ng/ml) groups. Estradiol concentrations at hours 24 and 30 in I.V. and S.C. groups were significantly lower than those in the Cont group. Ovulation was detected within 3 days after TAK-683 injection in all animals in I.V. and S.C. groups. There was no significant difference in the changes in the luteal diameter and blood progesterone levels after ovulation between I.V. and S.C. groups. These results show that follicular phase administration of the minimally effective dose of TAK-683 to ovariectomized goats induces a surge-like release of LH followed by ovulation in ovary-intact goats.

Metastin/Kisspeptin is a potent secretagogue of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) secretion. The present study aims to determine if a metastin/kisspeptin analogue, TAK-683, has a stimulatory effects on LH secretion in peripheral plasma and GnRH release in the pituitary portal circulation in castrated male or ovariectomized female goats. In experiment 1, castrated (n=5) or ovariectomized (n=5) goats were received a bolus intravenous (iv) injection of TAK-683 (3.5 pmol/head - 3.5 nmol/head). A single injection of TAK-683 stimulated LH secretion in a dose-dependent manner in both castrated and ovariectomized goats. The effective dose (3.5 nmol) of TAK-683 was much lower than that (380 nmol) of the metastin/kisspeptin C-terminal active decapeptide, kisspeptin-10 (1), indicating that TAK-683 is a potent analogue to induce LH secretion. In experiment 2, portal blood sampling techniques were applied to monitor change in GnRH release in pituitary portal circulation. A bolus iv injection of TAK-683 (35 nmol/head) strongly stimulated GnRH release in castrated male goats (n=6). The duration of elevated GnRH/LH secretion lasted for more than 4 hours after the injection. This observation provides direct evidence that TAK-683 induces LH secretion by stimulating hypothalamic GnRH release. In experiment 3, a chronic application of TAK-683 was achieved by subcutaneous implantation of an osmotic pump containing TAK-683 solution (50 nmol/kg BW/week) and pulsatile GnRH/LH secretion was examined in castrated male goats (n=5). By contrast to a bolus iv injection of TAK-683, a chronic treatment of this analogue for 5 to 6 days caused severe suppression of GnRH and LH pulses. This “paradoxical effect” on GnRH/LH secretion was achieved by 3 days after the initiation of TAK-683 application. The present findings demonstrate that TAK-683 is a potent secretagogue of GnRH/LH secretion and that continuous exposure to this metastin/kisspeptin analogue attenuates the responsiveness of GnRH neurons, which in turn decreases LH secretion. These series of experiments using a metastin/kisspeptin analogue suggests that endogenous metastin/kisspeptin is a potent stimulator of LH secretion via GnRH release from the hypothalamus and would be critical for the control of reproductive functions in goats as in other mammalian species. (1) Ohkura S et al., J Neuroendocrinol 2009; 21:813 Nothing to Disclose: SO, TT, TK, YW, TO, MK, HO

Metastin/kisspeptin plays critical roles in the control of gonadotropin-releasing hormone (GnRH) secretion. It has been suggested that metastin neurons in the arcuate nucleus (ARC) participate in the generation of the GnRH pulse generator activity. TAK-683 is a metastin analogue, and chronic administration of the compound potently suppresses testosterone secretion in male rats or pulsatile luteinizing hormone (LH) secretion in goats. To clarify the role of ARC metastin neurons, effects of chronic administration of TAK-683 on the GnRH pulse generator activity was examined in goats. Four ovariectomized goats were implanted with recording electrodes aimed at close proximity of ARC metastin neurons, and multiple unit activity (MUA) was recorded. Characteristic bursts in MUA (MUA volleys) were considered to the electrophysiological manifestation of the GnRH pulse generator. An osmotic pump containing TAK-683 (500 nmol/kgBW/week) was subcutaneously implanted. On one day before and 5 days after the implantation, blood samples were taken for 4 hrs, while monitoring MUA. In addition, the goats received a bolus intracerebroventricular injection of human metastin (10 nmol) at the midpoint of the experimental period on each day. Plasma LH concentrations were determined by RIA. Prior to the TAK-683 treatment, periodic fluctuations of LH secretion (LH pulses) were observed with a constant interpulse interval of approximately 25 min. In each goat, LH pulses were invariably associated with MUA volleys, confirming that the MUA volley represents the GnRH pulse generator activity. The injection of human metastin markedly increased LH secretion. After the TAK-683 treatment for 5 days, LH secretion was completely suppressed and no LH pulse was detected, whereas periodic MUA volleys were observed with similar frequency and magnitude as prior to the treatment. Although the metastin injection resulted in a slight rise in LH secretion in the TAK-683 treated goat as well, the stimulatory effect of metastin on LH secretion was remarkably reduced. The present results suggest that chronic administration of TAK-683 potently attenuates the responsiveness of GnRH neurons to metastin by acting at the downstream of the GnRH pulse generator. The apparent dissociation between the MUA volley and LH pulse indicates that the MUA volley is not a consequence of activity of GnRH neurons, and suggests that ARC metastin neurons may be the intrinsic source of the GnRH pulse generator activity. Nothing to Disclose: YW, TY, SO, TT, MK, HO

Estrous cyclicity is controlled by a cascade of neuroendocrine events, involving the activation of the hypothalamo-pituitary-gonadal axis. Two modes of gonadotropin-releasing hormone (GnRH) are well established to regulate the estrous cycle: one is a tonic or pulse mode of secretion which is responsible for the stimulation of follicular development and steroidogenesis; the other is a surge mode, which is solely responsible for the induction of luteinizing hormone (LH) surges, eventually leading to ovulation. Metastin/kisspeptin-GPR54 signaling has been suggested to control ovarian cyclicity through regulating the two modes of GnRH release. A population of metastin/kisspeptin neurons located in the anteroventral periventricular nucleus (AVPV) is considered to trigger GnRH surge and thus to mediate the estrogen positive feedback action on GnRH release. The other hypothalamic population of metastin/kisspeptin neurons is located in the arcuate nucleus (ARC) and could be involved in generating GnRH pulses and mediating negative feedback action of estrogen on GnRH release. GnRH neurons express mRNA for GPR54, a metastin/kisspeptin receptor, and have a close association with metastin/kisspeptin neurons at the cell body and terminal level, but the precise mechanism by which this peptide regulates the two modes of GnRH release needs to be determined. Metastin/kisspeptin, therefore, is a key hypothalamic neuropeptide, which is placed immediately upstream of GnRH neurons and relays the peripheral steroidal information to GnRH neurons to control estrous cyclicity.

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.