Mika Kinoshita

Significance of Neonatal Testicular Sex Steroids to Defeminize Anteroventral Periventricular Kisspeptin Neurons and the GnRH/LH Surge System in Male Rats

The brain mechanism regulating gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release is sexually differentiated in rodents. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) have been suggested to be sexually dimorphic and involved in the GnRH/LH surge generation. The present study aimed to determine the significance of neonatal testicular androgen to defeminize AVPV kisspeptin expression and the GnRH/LH surge-generating system. To this end, we tested whether neonatal castration feminizes AVPV kisspeptin neurons and the LH surge-generating system in male rats and whether neonatal estradiol benzoate (EB) treatment suppresses the kisspeptin expression and the LH surge in female rats. Immunohistochemistry, in situ hybridization, and quantitative real-time RT-PCR were performed to investigate kisspeptin and Kiss1 mRNA expressions. Male rats were castrated immediately after birth, and females were treated with EB on postnatal Day 5. Neonatal castration caused an increase in AVPV kisspeptin expression at peptide and mRNA levels in the genetically male rats, and the animals showed surge-like LH release in the presence of the preovulatory level of estradiol (E2) at adulthood. On the other hand, neonatal EB treatment decreased the number of AVPV kisspeptin neurons and caused an absence of E2-induced LH surge in female rats. Semiquantitative RT-PCR analysis showed that neonatal steroidal manipulation affects Kiss1 expression but does not significantly affect gene expressions of neuropeptides (neurotensin and galanin) and enzymes or transporter for neurotransmitters (gamma-aminobutyric acid, glutamate, and dopamine) in the AVPV, suggesting that the manipulation specifically affects Kiss1 expressions. Taken together, our present results provide physiological evidence that neonatal testicular androgen causes the reduction of AVPV kisspeptin expression and failure of LH surge in genetically male rats. Thus, it is plausible that perinatal testicular androgen causes defeminization of the AVPV kisspeptin system, resulting in the loss of the surge system in male rats.

Central Lipoprivation-Induced Suppression of Luteinizing Hormone Pulses Is Mediated by Paraventricular Catecholaminergic Inputs in Female Rats

The present study aims to clarify the role of fatty acids in regulating pulsatile LH secretion in rats. To produce an acute central lipoprivic condition, mercaptoacetate (MA), an inhibitor of fatty acids oxidation, was administered into the fourth cerebroventricle (4V) in ad libitum fed ovariectomized (OVX) rats (0.4, 2, and 10 micromol/rat) with or without an estradiol (E2) implant producing diestrus plasma E2 levels. Pulsatile LH secretion was suppressed by 4V MA administration in a dose-dependent manner in both OVX and OVX plus E2 rats. Mean LH levels and LH pulse frequency and amplitude were significantly reduced by the highest dose of MA in OVX rats, and by the middle and highest dose of MA in E2-treated rats, suggesting that estrogen enhanced LH suppression. Blood glucose levels increased immediately after the highest dose of MA in both groups. Fourth ventricular injection of trimetazidine (2 and 3 micromol/rat), another inhibitor of fatty acids oxidation, also inhibited pulsatile LH release, resulting in significant and dose-dependent suppression of LH pulse frequency and an increase in blood glucose levels in OVX plus E2 rats. In contrast, peripheral injection of the highest 4V dose of MA (10 micromol/rat) did not alter LH release or blood glucose levels. Microdialysis of the hypothalamic paraventricular nucleus (PVN) revealed that norepinephrine release in the region was increased by 4V MA administration. Preinjection of alpha-methyl-p-tyrosine, a catecholamine synthesis inhibitor, into the PVN completely blocked the lipoprivic inhibition of LH and the counter-regulatory increase in blood glucose levels in OVX plus E2 rats. Together, these studies indicate that fatty acid availability may be sensed by a central detector, located in the lower brainstem to maintain reproduction, and that noradrenergic inputs to the PVN mediate this lipoprivic-induced suppression of LH release.

A rat model for the energetic regulation of gonadotropin secretion: role of the glucose-sensing mechanism in the brain

Energy availability has been considered to regulate gonadal activity by modulating the release of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) at various reproductive phases, such as lactation and puberty in domestic as well as wild animals. Experimental models with rats and sheep have demonstrated that fasting or glucoprivation suppresses pulsatile LH release. From those experiments, the information on energy deficiency is considered to be detected by specific central sensors and conveyed to the hypothalamus to regulate LH release as well as food intake. Noradrenergic neurons, originating in the medulla oblongata and projecting to the hypothalamic paraventricular nucleus (PVN), is reported to be one of the pathways mediating the response of LH release to energy deficiency. The other component is considered to be an energy-sensing mechanism in the brain. Glucose or other oxidizable fuels may function as a metabolic signal to regulate LH release. Previous studies suggest the presence of a glucose-sensing mechanism in the rat hindbrain. From our previous results in the rat, the ependymocytes lining the wall of the cerebroventricle could possibly serve as a glucose sensor to regulate GnRH/LH release. Greater understanding of the nature of the energy-sensing mechanism in the brain will contribute to the nutritional manipulation of reproductive performance in domestic animals in various conditions.

Oestrogen-dependent stimulation of luteinising hormone release by galanin-like peptide in female rats.

Galanin‐like peptide (GALP), a ligand for three types of galanin receptor, is reported to have a role in regulating luteinising hormone (LH) release in male rodents and primates, but its role in LH release in female rodents remains controversial. The present study was conducted to test whether GALP has a stimulatory role in regulating LH secretion in female rats. The effect of i.c.v. infusion of GALP (5 nmol) on pulsatile LH release was investigated in Wistar‐Imamichi strain female rats, or lean and obese Zucker rats. In oestradiol‐17β (oestradiol)‐primed ovariectomised (OVX) Wistar‐Imamichi female rats, i.c.v. infusion of GALP caused a gradual increase in LH release for the first 1.5 h after the infusion followed by an increased LH pulse frequency during the next 1.5 h, resulting in a significant increase in the mean LH concentrations and baseline levels of LH pulses throughout the sampling period and in the frequency of LH pulses at the last half of the period compared to vehicle‐treated controls. The stimulatory effect of GALP was oestrogen‐dependent because the same GALP treatment did not affect LH release in OVX rats in the absence of oestradiol. In lean Zucker rats, LH pulses were found in oestradiol‐primed OVX individuals and central GALP infusion increased mean LH concentrations in the last half of the period. By contrast, few LH pulses were found in oestradiol‐primed OVX obese Zucker rats reportedly with lower hypothalamic GALP expression. Central GALP infusion caused an apparent but transient increase in LH release, resulting in the significant increase in all pulse parameters of LH pulses compared to vehicle‐treated controls in the first half of the sampling period. These results suggest that hypothalamic GALP is likely involved in stimulating GnRH/LH release, and that the stimulatory effect of GALP on LH release is oestrogen‐dependent in female rats.

Involvement of brain ketone bodies and the noradrenergic pathway in diabetic hyperphagia in rats

Uncontrolled type 1 diabetes leads to hyperphagia and severe ketosis. This study was conducted to test the hypothesis that ketone bodies act on the hindbrain as a starvation signal to induce diabetic hyperphagia. Injection of an inhibitor of monocarboxylate transporter 1, a ketone body transporter, into the fourth ventricle normalized the increase in food intake in streptozotocin (STZ)-induced diabetic rats. Blockade of catecholamine synthesis in the hypothalamic paraventricular nucleus (PVN) also restored food intake to normal levels in diabetic animals. On the other hand, hindbrain injection of the ketone body induced feeding, hyperglycemia, and fatty acid mobilization via increased sympathetic activity and also norepinephrine release in the PVN. This result provides evidence that hyperphagia in STZ-induced type 1 diabetes is signaled by a ketone body sensed in the hindbrain, and mediated by noradrenergic inputs to the PVN.