Kinuyo Iwata

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.

Oestrogen-Dependent Suppression of Pulsatile Luteinising Hormone Secretion and Kiss1 mRNA Expression in the Arcuate Nucleus During Late Lactation in Rats

Follicular development and ovulation are strongly suppressed during lactation in mammals via a profound suppression of gonadotrophin secretion. The present study aimed to examine the role of oestrogen feedback action in suppressing luteinising hormone (LH) secretion and hypothalamic kisspeptin expression during the latter half of lactation. Plasma LH concentrations kept at low levels throughout the lactating period in intact and oestrogen‐replaced ovariectomised (OVX) lactating rats, whereas plasma LH concentrations gradually elevated from day 10 postpartum in lactating OVX rats. OVX lactating rats showed frequent LH pulses at late lactation, although the LH pulses were significantly inhibited by an oestrogen replacement, which is much less effective on LH release in nonlactating rats. Oestrogen replacement in lactating OVX rats significantly reduced the number of Kiss1 mRNA‐expressing cells in the arcuate nucleus (ARC) at late lactation, although the same oestrogen treatment did not affect the number of Kiss1‐expressing cells in nonlactating controls. Exogenous kisspeptin challenge (0.2 nmol) into the third cerebroventricle significantly increased LH secretion in lactating OVX, lactating OVX + subcutaneous 17β‐oestradiol and intact lactating rats at day 16 postpartum. These results suggest that LH pulse suppression during late lactation could be a result of the enhanced oestrogen‐dependent suppression of ARC kisspeptin expression.

Immunoelectron microscopic observation of the subcellular localization of kisspeptin, neurokinin B and dynorphin A in KNDy neurons in the arcuate nucleus of the female rat

KNDy neurons are named for their co-expression of three neuropeptides, kisspeptin, neurokinin B (NKB) and dynorphin A (DynA). These cells, located in the hypothalamic arcuate nucleus (ARC), are associated with generation of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulses to control follicular growth and steroidogenesis. However, subcellular sorting mechanisms for secretory vesicles containing these neuropeptides have not been elucidated. In this study, we analyzed the localization pattern of kisspeptin, NKB and DynA in the ARC of the ovariectomized rat immediately treated with estrogen using immunoelectron microscopy. First, we identified neuropeptides by dual-labeled fluorescence immunohistochemistry, with results indicating all three neuropeptides co-express within individual ARC cells in female rats. Next, we investigated the subcellular localization pattern of kisspeptin, NKB, and/or DynA using post-embedding double immunoelectron microscopy, indicating that each type of neuropeptide is contained within separate and individual neurosecretory vesicles. This suggests sorting and packaging of kisspeptin, NKB and DynA is differentially regulated within KNDy neurons. Our findings facilitate understanding of regulatory mechanisms underlying kisspeptin secretion in KNDy neurons, and generation of GnRH/LH pulses induced by kisspeptin in the ARC.

The effects of gonadal steroid manipulation on the expression of Kiss1 mRNA in rat arcuate nucleus during postnatal development.

Kisspeptins, encoded by Kiss1 gene, play pivotal roles in the regulation of reproduction. Recently, several studies reported a sex difference in Kiss1 expression in the arcuate nucleus (ARC) during the neonatal period. In this study, we investigated the effect of gonadal steroid manipulation on the sex difference in Kiss1 expression in ARC of rats. At neonatal and prepubertal stages, females had a greater number of Kiss1 neurons than the males. Gonadectomy at those stages resulted in significant increases in the Kiss1 neuron number and the sex differences disappeared. We also confirmed the expression of estrogen receptor α in kisspeptin neurons in neonates. Altogether, our results indicate that ARC Kiss1 expression is negatively regulated by gonadal steroids from early postnatal stages, and that the sex difference in ARC Kiss1 expression is attributed to the difference in circulating gonadal steroid levels. We also found that neonatal estrogenization inhibits Kiss1 expression and impairs negative feedback system.

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.

Effect of sex steroid hormones on the number of serotonergic neurons in rat dorsal raphe nucleus.

Disorders caused by the malfunction of the serotonergic system in the central nervous system show sex-specific prevalence. Many studies have reported a relationship between sex steroid hormones and the brain serotonergic system; however, the interaction between sex steroid hormones and the number of brain neurons expressing serotonin has not yet been elucidated. In the present study, we determined whether sex steroid hormones altered the number of serotonergic neurons in the dorsal raphe nucleus (DR) of adult rat brains. Animals were divided into five groups: ovariectomized (OVX), OVX+low estradiol (E2), OVX+high E2, castrated males, and intact males. Antibodies against 5-hydroxytryptamine (5-HT, serotonin) and tryptophan hydroxylase (Tph), an enzyme for 5-HT synthesis, were used as markers of 5-HT neurons, and the number of 5-HT-immunoreactive (ir) or Tph-ir cells was counted. We detected no significant differences in the number of 5-HT-ir or Tph-ir cells in the DR among the five groups. By contrast, the intensity of 5-HT-ir showed significant sex differences in specific subregions of the DR independent of sex steroid levels, suggesting that the manipulation of sex steroid hormones after maturation does not affect the number and intensive immunostaining of serotonergic neurons in rat brain. Our results suggest that, the sexual dimorphism observed in the serotonergic system is due to factors such as 5-HT synthesis, transportation, and degradation but not to the number of serotonergic neurons.

Effect of androgen on Kiss1 expression and luteinizing hormone release in female rats

Hyperandrogenic women have various grades of ovulatory dysfunction, which lead to infertility. The purpose of this study was to determine whether chronic exposure to androgen affects the expression of kisspeptin (ovulation and follicle development regulator) or release of luteinizing hormone (LH) in female rats. Weaned females were subcutaneously implanted with 90-day continuous-release pellets of 5α-dihydrotestosterone (DHT) and studied after 10 weeks of age. Number of Kiss1-expressing cells in both the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) was significantly decreased in ovary-intact DHT rats. Further, an estradiol-induced LH surge was not detected in DHT rats, even though significant differences were not observed between DHT and non-DHT rats with regard to number of AVPV Kiss1-expressing cells or gonadotrophin-releasing hormone (GnRH)-immunoreactive (ir) cells in the presence of high estradiol. Kiss1-expressing and neurokinin B-ir cells were significantly decreased in the ARC of ovariectomized (OVX) DHT rats compared with OVX non-DHT rats; pulsatile LH secretion was also suppressed in these animals. Central injection of kisspeptin-10 or intravenous injection of a GnRH agonist did not affect the LH release in DHT rats. Notably, ARC Kiss1-expressing cells expressed androgen receptors (ARs) in female rats, whereas only a few Kiss1-expressing cells expressed ARs in the AVPV. Collectively, our results suggest excessive androgen suppresses LH surge and pulsatile LH secretion by inhibiting kisspeptin expression in the ARC and disruption at the pituitary level, whereas AVPV kisspeptin neurons appear to be directly unaffected by androgen. Hence, hyperandrogenemia may adversely affect ARC kisspeptin neurons, resulting in anovulation and menstrual irregularities.

Distinct dynorphin expression patterns with low- and high-dose estrogen treatment in the arcuate nucleus of female rats†,‡

Abstract Kisspeptin (KISS1; encoded by Kiss1) neurons in the arcuate nucleus (ARC) coexpress tachykinin 3 (TAC3; also known as neurokinin B) and dynorphin A (PDYN). Accordingly, they are termed KNDy neurons and considered to be crucial in generating pulsatile release of gonadotropin-releasing hormone. Accumulating evidence suggests that Kiss1 and Tac3 are negatively regulated by estrogen. However, it has not been fully determined whether and how estrogen modulates Pdyn and PDYN. Here, we examined the expression of Pdyn mRNA and PDYN by in situ hybridization and immunohistochemistry, respectively, in the ARC of female rats after ovariectomy (OVX) and OVX plus low- or high-dose beta-estradiol (E2) replacement. We also investigated the effect of E2 on expression of Kiss1, KISS1, Tac3, and TAC3. Furthermore, colocalization of PDYN and estrogen receptor alpha (ESR1) was determined. Subsequently, we found that low-dose E2 treatment had no effect on Pdyn mRNA-expressing cells, but increased PDYN-immunoreactive (ir) cell numbers. In contrast, high-dose E2 treatment resulted in prominent reductions in both Pdyn mRNA-expressing and PDYN-ir cell numbers. Changes induced by low or high doses of E2 were similarly observed in the expression of Kiss1, KISS1, Tac3, and TAC3. The majority of PDYN-ir neurons coexpressed ESR1 in all groups. Our results indicate that E2 regulates the expression of PDYN, as well as KISS1 and TAC3, with regulation by E2 differing according to its levels. Summary Sentence Estrogen regulates dynorphin expression in the arcuate nucleus of female rats with low-dose estrogen suppressing dynorphin release and high-dose estrogen inhibiting dynorphin mRNA expression.