Accumulating evidence suggests that kisspeptin-GPR54 signaling is indispensable for gonadotropin-releasing hormone (GnRH)/gonadotropin secretion and consequent reproductive functions in mammals. Conventional Kiss1 knockout (KO) mice and rats are reported to be infertile. To date, however, no study has investigated the effect of inducible central Kiss1 KO/knockdown on pulsatile gonadotropin release in male mammals. Here we report an in vivo analysis of inducible conditional Kiss1 knockdown male mice. The mice were generated by a bilateral injections of either adeno-associated virus (AAV) vectors driving Cre recombinase (AAV-Cre) or AAV vectors driving GFP (AAV-GFP, control) into the hypothalamic arcuate nucleus (ARC) of Kiss1-floxed male mice, in which exon 3 of the Kiss1 gene were floxed with loxP sites. Four weeks after the AAV-Cre injection, the mice showed a profound decrease in the both number of ARC Kiss1-expressing cells and the luteinizing hormone (LH) pulse frequency. Interestingly, pulsatile LH secretion was apparent 8 weeks after the AAV-Cre injection despite the suppression of ARC Kiss1 expression. The control Kiss1-floxed mice infected with AAV-GFP showed apparent LH pulses and Kiss1 expression in the ARC at both 4 and 8 weeks after the AAV-GFP injection. These results with an inducible conditional Kiss1 knockdown in the ARC of male mice suggest that ARC kisspeptin neurons are responsible for pulsatile LH secretion in male mice, and indicate the possibility of a compensatory mechanism that restores GnRH/LH pulse generation.

The present study aimed to evaluate whether novel conditional kisspeptin neuron-specific Kiss1 knockout (KO) mice utilizing the Cre-loxP system could recapitulate the infertility of global Kiss1 KO models, thereby providing further evidence for the fundamental role of hypothalamic kisspeptin neurons in regulating mammalian reproduction. We generated Kiss1-floxed mice and hypothalamic kisspeptin neuron-specific Cre-expressing transgenic mice and then crossed these two lines. The conditional Kiss1 KO mice showed pubertal failure along with a suppression of gonadotropin secretion and ovarian atrophy. These results indicate that newly-created hypothalamic Kiss1 KO mice obtained by the Cre-loxP system recapitulated the infertility of global Kiss1 KO models, suggesting that hypothalamic kisspeptin, but not peripheral kisspeptin, is critical for reproduction. Importantly, these Kiss1-floxed mice are now available and will be a valuable tool for detailed analyses of roles of each population of kisspeptin neurons in the brain and peripheral kisspeptin-producing cells by the spatiotemporal-specific manipulation of Cre expression.

Follicular development and ovulation are profoundly suppressed during lactation in mammals. This suppression is suggested to be mainly due to the suckling-induced inhibition of kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and consequent inhibition of pulsatile GnRH/LH release. We examined whether central somatostatin (SST) signaling mediates the suckling-induced suppression of pulsatile LH secretion. SST has been reported to be expressed in the posterior intralaminar thalamic nucleus (PIL), where the suckling stimulus is postulated to be relayed to the hypothalamus during lactation. SST inhibitory receptors (SSTRs) are abundantly expressed in the ARC, where kisspeptin/neurokinin B/dynorphin A (KNDy) neurons are located. Histological and quantitative studies revealed that the suckling stimulus increased the number of SST-expressing cells in the PIL, and Sstr2 expression in the ARC. Furthermore, a central injection of an SSTR2 antagonist caused a significant increase in pulsatile LH release in lactating rats. Double labeling of Sstr2 and the neurokinin B gene, as a marker for ARC KNDy neurons, showed Sstr2 expression was abundantly detected in the ARC, but few KNDy neurons coexpressed Sstr2 in lactating rats. Taken together, these findings suggest the suckling-induced activation of SST-SSTR2 signaling mediates, at least in part, the suppression of pulsatile LH secretion during lactation in rats, probably via the indirect effects of SST on KNDy neurons. These results provide a new aspect on the role of central SST-SSTR signaling in understanding the mechanism underlying lactational anestrus.

Hindbrain ependymocytes are postulated to have a glucose-sensing role in regulating gonadal functions. Previous studies have suggested that malnutrition-induced suppression of gonadotropin secretion is mediated by noradrenergic inputs from the A2 region in the solitary tract nucleus to the paraventricular nucleus (PVN), and by corticotropin-releasing hormone (CRH) release in the hypothalamus. However, no morphological evidence to indicate the neural pathway from the hindbrain ependymocytes to hypothalamic kisspeptin neurons, a center for reproductive function in mammals, currently exists. The present study aimed to examine the existence of a neuronal pathway from the hindbrain ependymocytes to kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). To determine this, wheat-germ agglutinin (WGA), a trans-synaptic tracer, was injected into the fourth ventricle (4V) in heterozygous Kiss1-tandem dimer Tomato (tdTomato) rats, where kisspeptin neurons were visualized by tdTomato fluorescence. 48 h after the WGA injection, brain sections were taken from the forebrain, midbrain and hindbrain and subjected to double immunohistochemistry for WGA and dopamine β-hydroxylase (DBH) or CRH. WGA immunoreactivities were found in vimentin-immunopositive ependymocytes of the 4V and the central canal (CC), but not in the third ventricle. The WGA immunoreactivities were detected in some tdTomato-expressing cells in the ARC and AVPV, DBH-immunopositive cells in the A1-A7 noradrenergic nuclei, and CRH-immunopositive cells in the PVN. These results suggest that the hindbrain ependymocytes have neuronal connections with the kisspeptin neurons, most probably via hindbrain noradrenergic and CRH neurons to relay low energetic signals for regulation of reproduction.

Ependymocytes are one of the energy-sensing cells that regulate animal reproduction through their responsiveness to changes in extracellular glucose levels and the expression of pancreatic-type glucokinase and glucose transporter 2, which play a critical role in sensing blood glucose levels in pancreatic β-cells. Molecular mechanisms underlying glucose sensing in the ependymocytes remain poorly understood. The AMP-activated protein kinase (AMPK), a serine/threonine kinase highly conserved in all eukaryotic cells, has been suggested to be an intracellular fuel gauge that detects cellular energy status. The present study aims to clarify the role AMPK of the lower brainstem ependymocytes has in sensing glucose levels to regulate reproductive functions. First, we will show that administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, into the 4th ventricle suppressed pulsatile LH release in female rats. Second, we will demonstrate the presence of AMPK catalytic subunit immunoreactivities in the rat lower brainstem ependymocytes. Third, transgenic mice were generated to visualize the ependymocytes with Venus, a green fluorescent protein, expressed under the control of the mouse vimentin promoter for further in vitro study. The Venus-labeled ependymocytes taken from the lower brainstem of transgenic mice revealed that AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, increased in vitro intracellular calcium concentrations. Taken together, malnutrition-induced AMPK activation of ependymocytes of the lower brainstem might be involved in suppression of GnRH/LH release and then gonadal activities.

The reproductive function of vertebrates is regulated by the hypothalamic-pituitary-gonadal axis. In sexually mature females, gonadotropin-releasing hormone (GnRH) neurons in the preoptic area (POA) are assumed to be responsible for a cyclic large increase in GnRH release, the GnRH surge, triggering a luteinizing hormone (LH) surge, which leads to ovulation. Precise temporal regulation of the preovulatory GnRH/LH surge is important for successful reproduction because ovulation should occur after follicular development. The time course of the circulating level of estrogen is correlated with the ovulatory cycle throughout vertebrates. However, the neural mechanisms underlying estrogen-induced preovulatory GnRH surge after folliculogenesis still remain unclear, especially in non-mammals. Here, we used a versatile non-mammalian model medaka for the analysis of the involvement of estrogen in the regulation of POA-GnRH (GnRH1) neurons. Electrophysiological analysis using a whole brain-pituitary in vitro preparation, which maintains the hypophysiotropic function of GnRH1 neurons intact, revealed that 17β-estradiol (E2 ) administration recovers the ovariectomy-induced lowered GnRH1 neuronal activity in the evening, indicating the importance of E2 for upregulation of GnRH1 neuronal activity. The importance of E2 was also confirmed by the fact that GnRH1 neuronal activity was low in short-day photoperiod-conditioned females (low E2 model). However, E2 failed to upregulate the firing activity of GnRH1 neurons in the morning, suggesting the involvement of additional time-of-day signal(s) for triggering GnRH/LH surges at an appropriate timing. We also provide morphological evidence for the localization of estrogen receptor subtypes in GnRH1 neurons. In conclusion, we propose a working hypothesis in which both estrogenic and time-of-day signals act in concert to timely upregulate the firing activity of GnRH1 neurons that trigger the GnRH surge at an appropriate timing in a female-specific manner. This neuroendocrinological mechanism is suggested to be responsible for the generation of ovulatory cycles in female teleosts in general.