Central injection of neuropeptide B induces luteinizing hormone release in male and female rats.

Neuropeptide B (NPB) has been identified as an endogenous peptide ligand for the orphan receptor NPBWR1. However, the effect of NPB on the central regulatory mechanisms of reproductive functions remains unclear. Our findings indicated the presence of Npb, Npw (which is another ligand for NPBWR1), and Npbwr1 mRNA in the hypothalamus of male and female rats at each stage of the estrous cycle. Npb mRNA expression was found to be significantly higher in diestrus compared to estrus. The expression of Npw mRNA was one order of magnitude lower than that of Npb mRNA, and Npw mRNA expression in diestrus was significantly higher than that in the other stages of the estrous cycle. Furthermore, Npbwr1 mRNA expression was found to be significantly higher in diestrus compared to the other stages of the estrous cycle and intact males. Notably, estrogen did not alter the expression of Npb, Npw, and Npbwr1 mRNAs in the hypothalamus of females. Central injection of NPB increased plasma luteinizing hormone (LH) levels in both intact males and estrogen-primed ovariectomized females but not in ovariectomized females. These results suggest that NPB-NPBWR1 signaling would be a facilitatory regulatory mechanism in the reproductive function of male and female rats. To the best of our knowledge, this study is the first report to describe the central role of NPB-NPBWR1 signaling in LH regulation in mammals.

Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression.

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2-3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

Effect of a neurokinin 3 receptor-selective agonist administration on the embryos recovered from superovulated cows.

Superovulation (SOV) treatment of cows results in unovulated follicles and inconsistent quality of the recovered embryos. It has been demonstrated that luteinizing hormone (LH) secretion is suppressed during SOV treatment of cows, which may cause insufficient follicle development and variation in the development of recovered embryos and unovulated follicles. Pulsatile gonadotropin-releasing hormone/LH secretion is controlled by the activity of kisspeptin, neurokinin B and dynorphin (KNDy) neurons in the arcuate nucleus in many mammals. As neurokinin B promotes the activity of KNDy neurons, we hypothesized that senktide, a neurokinin B receptor agonist, has the potential as a therapeutic drug to improve the ovulation rate and quality of recovered embryos in SOV-treated cows via stimulation of LH secretion. Senktide was administered intravenously (30 or 300 nmol/min) for 2 h, beginning from 72 h after the start of SOV treatment. LH secretion was examined before and after administration, and embryos were collected 7 d after estrus. Senktide administration increased LH secretion in SOV-treated cows. The ratios of code 1, code 1 and 2, and blastocyst stage embryos to recovered embryos were increased by senktide (300 nmol/min) administration. Moreover, the mRNA levels of MTCO1, COX7C, and MTATP6 were upregulated in recovered embryos of senktide (300 nmol/min)-administered animals. These results indicate that the administration of senktide to SOV-treated cows enhances LH secretion and upregulates the expression of genes involved in mitochondrial metabolism in embryos, thereby improving embryo development and embryo quality.

Deterioration of mitochondrial biogenesis and degradation in the endometrium is a cause of subfertility in cows.

To investigate possible causes of reproductive failure, we conducted global endometrial gene expression analyses in fertile and subfertile cows. Ingenuity pathway analysis showed that RICTOR and SIRT3 are significant upstream regulators for highly expressed genes in fertile cows, and are predicted to be activated upstream regulators of normal mitochondrial respiration. Canonical pathway analysis revealed that these highly expressed genes are involved in the activation of mitochondrial oxidative phosphorylation. Therefore, in subfertile cows, the inactivation of RICTOR and SIRT3 may correlate with decreased capacity of mitochondrial respiration. Furthermore, the expression levels of most mitochondrial DNA genes and nuclear genes encoding mitochondrial proteins were higher in subfertile cows. The mitochondrial DNA copy number was significantly higher in the endometrium of subfertile cows, whereas the ATP content did not differ between fertile and subfertile cows. Quantitative reverse transcription-PCR analysis demonstrated that the expression of PGC1a, TFAM, MFN1, FIS1, and BCL2L13 were significantly lower in subfertile cows. In addition, transmission electron microscopy images showed mitochondrial swelling in the endometrial cells of the subfertile cow. These results suggest that poor-quality mitochondria accumulate in the endometrium owing to a reduced capacity for mitochondrial biogenesis, fusion, fission, and degradation in subfertile cows, and may contribute to infertility.

Kiss1-dependent and independent release of luteinizing hormone and testosterone in perinatal male rats.

Prenatal and postnatal biphasic increases in plasma testosterone levels derived from perinatal testes are considered critical for defeminizing/masculinizing the brain mechanism that regulates sexual behavior in male rats. Hypothalamic kisspeptin neurons are indispensable for stimulating GnRH and downstream gonadotropin, as well as the consequent testicular testosterone production/release in adult male rats. However, it is unclear whether kisspeptin is responsible for the increase in plasma testosterone levels in perinatal male rats. The present study aimed to investigate the role of Kiss1/kisspeptin in generating perinatal plasma LH and the consequent testosterone increase in male rats by comparing the plasma testosterone and LH profiles of wild-type (Kiss1+/+) and Kiss1 knockout (Kiss1-/-) male rats. A biphasic pattern of plasma testosterone levels, with peaks in the prenatal and postnatal periods, was found in both Kiss1+/+ and Kiss1-/- male rats. Postnatal plasma testosterone and LH levels were significantly lower in Kiss1-/- male rats than in Kiss1+/+ male rats, whereas the levels in the prenatal embryonic period were comparable between the genotypes. Exogenous kisspeptin challenge significantly increased plasma testosterone and LH levels and the number of c-Fos-immunoreactive GnRH neurons in neonatal Kiss1-/- and Kiss1+/+ male rats. Kiss1 and Gpr54 (kisspeptin receptor gene) were found in the testes of neonatal rats, but kisspeptin treatment failed to stimulate testosterone release in the cultured testes of both genotypes. These findings suggest that postnatal, but not prenatal, testosterone increase in male rats is mainly induced by central kisspeptin-dependent stimulation of GnRH and consequent LH release.

Effect of diet-induced obesity on kisspeptin-neurokinin B-dynorphin A neurons in the arcuate nucleus and luteinizing hormone secretion in sex hormone-primed male and female rats.

Metabolic stress resulting from either lack or excess of nutrients often causes infertility in both sexes. Kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC) has been suggested to be a key players in reproduction via direct stimulation of the pulsatile gonadotropin-releasing hormone (GnRH) and subsequent gonadotropin release in mammalian species. In this study, we investigated the effect of high-fat diet (HFD) on hypothalamic KNDy gene expression to examine the pathogenic mechanism underlying obesity-induced infertility in male and female rats. Male and female rats at 7 weeks of age were fed with either a standard or HFD for 4 months. In the male rats, the HFD caused a significant suppression of ARC Kiss1 and Pdyn gene expressions, but did not affect the plasma luteinizing hormone (LH) levels and sizes of the morphology of the testis and epididymis. In the female rats, 58% of the HFD-fed female rats exhibited irregular estrous cycles, whereas the remaining rats showed regular cycles. Two of the 10 rats that showed HFD-induced irregular estrous cycles showed profound suppression of LH pulse frequency and the number of ARC Kiss1-expressing cells, whereas the other females showed normal LH pulses and ARC Kiss1 expression. Our finding shows that suppression of ARC Kiss1 expression might be the initial pathological change of hypogonadotropic hypogonadism in HFD-fed male rats, while the obese-related infertility in the female rats may be mainly induced by KNDy-independent pathways. Taken together, ARC kisspeptin neurons in male rats may be susceptible to HFD-induced obesity compared with those in female rats.

Gene-expression profile and postpartum transition of bovine endometrial side population cells†.

The mechanism of bovine endometrial regeneration after parturition remains unclear. Here, we hypothesized that bovine endometrial stem/progenitor cells participate in the postpartum regeneration of the endometrium. Flow cytometry analysis identified the presence of side population (SP) cells among endometrial stromal cells. Endometrial SP cells were shown to differentiate into osteoblasts and adipocytes. RNA-seq data showed that the gene expression pattern was different between bovine endometrial SP cells and main population cells. Gene Set Enrichment Analysis identified the enrichment of stemness genes in SP cells. Significantly (false discovery rate < 0.01) upregulated genes in SP cells contained several stem cell marker genes. Gene ontology (GO) analysis of the upregulated genes in SP cells showed enrichment of terms related to RNA metabolic process and transcription. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of upregulated genes in SP cells revealed enrichment of signaling pathways associated with maintenance and differentiation of stem/progenitor cells. The terms involved in TCA cycles were enriched in GO and KEGG pathway analysis of downregulated genes in SP cells. These results support the assumption that bovine endometrial SP cells exhibit characteristics of somatic stem/progenitor cells. The ratio of SP cells to endometrial cells was lowest on days 9-11 after parturition, which gradually increased thereafter. SP cells were shown to differentiate into epithelial cells. Collectively, these results suggest that bovine endometrial SP cells were temporarily reduced immediately after calving possibly due to their differentiation to provide new endometrial cells.

Inducible Kiss1 knockdown in the hypothalamic arcuate nucleus suppressed pulsatile secretion of luteinizing hormone in male mice.

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.

Colocalization of GPR120 and anterior pituitary hormone-producing cells in female Japanese Black cattle.

Negative energy balance in domestic animals suppresses their reproductive function. These animals commonly use long-chain fatty acids (LCFAs) from adipocytes as an energy source under states of malnutrition. The G-protein coupled receptor, GPR120, is a specific receptor for LCFAs, but its role in reproductive function remains unknown in domestic animals. The purpose of this study was to examine whether GPR120 is involved in the reproductive system of cattle. GPR120 mRNA expression was evaluated in brain, pituitary, and ovarian tissue samples by RT-PCR. GPR120 gene expression was detected with high intensity only in the anterior pituitary sample, and GPR120-immunoreactive cells were found in the anterior pituitary gland. Double immunohistochemistry of GPR120 in the anterior pituitary hormone-producing cells, such as gonadotropes, thyrotropes, lactotropes, somatotropes, and corticotropes, was performed to clarify the distribution of GPR120 in the anterior pituitary gland of ovariectomized heifers. Luteinizing hormone ß subunit (LHß)- and follicle-stimulating hormone ß subunit (FSHß)-immunoreactive cells demonstrated GPR120 immunoreactivity at 80.7% and 85.9%, respectively. Thyrotropes, lactotropes, somatotropes, and corticotropes coexpressed GPR120 at 21.1%, 5.4%, 13.6%, and 14.5%, respectively. In conclusion, the present study suggests that GPR120 in the anterior pituitary gland might mediate LCFA signaling to regulate gonadotrope functions, such as hormone secretion or production, in cattle.

GnRH(1-5), a metabolite of gonadotropin-releasing hormone, enhances luteinizing hormone release via activation of kisspeptin neurons in female rats.

Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC), which coexpress neurokinin B and dynorphin, are involved in gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulse generation, while the anteroventral periventricular nucleus (AVPV) kisspeptin neurons are responsible for GnRH/LH surge generation. The present study aims to examine whether GnRH(1-5), a GnRH metabolite, regulates LH release via kisspeptin neurons. GnRH(1-5) was intracerebroventricularly injected to ovariectomized and estrogen-treated Wistar-Imamichi female rats. Immediately after the central GnRH(1-5) administration at 2 nmol, plasma LH concentration increased, resulting in significantly higher levels of the area under the curve and baseline of plasma LH concentrations compared to vehicle-injected controls. On the other hand, in Kiss1 knockout rats, GnRH(1-5) administration failed to affect LH secretion, suggesting that the facilitatory effect of GnRH(1-5) on LH release is mediated by kisspeptin neurons. Double in situ hybridization (ISH) for Kiss1 and Gpr101, a GnRH(1-5) receptor gene, revealed that few Kiss1-expressing cells coexpress Gpr101 in both ARC and AVPV. On the other hand, double ISH for Gpr101 and Slc17a6, a glutamatergic marker gene, revealed that 29.2% of ARC Gpr101-expressing cells coexpress Slc17a6. Further, most of the AVPV and ARC Kiss1-expressing cells coexpress Grin1, a gene encoding a subunit of NMDA receptor. Taken together, these results suggest that the GnRH(1-5)-GPR101 signaling facilitates LH release via indirect activation of kisspeptin neurons and that glutamatergic neurons may mediate the signaling. This provides a new aspect of kisspeptin- and GnRH-neuronal communication with the presence of stimulation from GnRH to kisspeptin neurons in female rats.

Neonatal Estrogen Causes Irreversible Male Infertility via Specific Suppressive Action on Hypothalamic Kiss1 Neurons.

Aberrant exposure to estrogen-like compounds during the critical developmental period may cause improper hypothalamic programming, thus resulting in reproductive dysfunction in adulthood in male mammals. Kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC) have been suggested to govern tonic GnRH/gonadotropin release to control reproduction in male mammals. In this study, we report that chronic exposure to supraphysiological levels of estrogen during the neonatal period caused an irreversible suppression of KNDy genes in the ARC, resulting in reproductive dysfunction in male rats. Daily estradiol benzoate (EB) administration from days 0 to 10 postpartum caused smaller seminiferous tubules, abnormal spermatogenesis, and a decrease in plasma testosterone in adult male rats. The neonatal EB treatment profoundly suppressed LH pulse and ARC KNDy gene expression at adulthood, but it failed to affect the number of GnRH gene-expressing cells in male rats. The EB treatment failed to affect gene expression of other neuropeptides, such as GHRH, proopiomelanocortin, and agouti-related protein in the ARC, suggesting that ARC KNDy neurons would be a specific target of neonatal estrogen to cause male reproductive dysfunction. Because LH secretory responses to kisspeptin challenge and GnRH expression were spared in male rats with the EB treatment, LH pulse suppression is most probably due to ARC KNDy deficiency. Taken together, the current study indicates that chronic exposure to estrogenic chemicals in the developing brain causes a defect of ARC KNDy neurons, resulting in an inhibition of pulsatile GnRH/LH release and the failure of spermatogenesis and steroidogenesis.

Morphological analysis for neuronal pathway from the hindbrain ependymocytes to the hypothalamic kisspeptin neurons.

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 ß-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.

Long-Term Neonatal Estrogen Exposure Causes Irreversible Inhibition of LH Pulses by Suppressing Arcuate Kisspeptin Expression via Estrogen Receptors α and ß in Female Rodents.

Exposure to estrogen during the developmental period causes reproductive dysfunction in mammals, because the developing brain is highly sensitive to estrogens. In the present study, we report that long-term exposure to supraphysiological doses of estrogen during the neonatal critical period causes irreversible suppression of Kiss1/kisspeptin expression in the arcuate nucleus (ARC) via estrogen receptor-alpha (ERα) and ERß, resulting in reproductive dysfunction in female rats. Daily estradiol-benzoate (EB) administration from days 0 to 10 postpartum caused persistent vaginal diestrus in female rats. The female rats showed profound suppression of pulsatile luteinizing hormone (LH) release and ARC Kiss1/kisspeptin expression even after ovariectomy at adulthood. In contrast, female rats treated with a single EB injection at day 5 postpartum exhibited persistent vaginal estrus and showed comparable LH pulses and numbers of ARC Kiss1-expressing cells to vehicle-treated controls after ovariectomy at adulthood. Because the LH secretory response to exogenous kisspeptin was spared in female rats with neonatal long-term estrogen exposure, the LH pulse suppression was most probably due to ARC kisspeptin deficiency. Furthermore, neonatal estrogen might act through both ERα and ERß, because EB exposure significantly reduced the number of ARC Kiss1-expressing cells in wild-type mice but not in ERα or ERß knockout mice. Taken together, long-term exposure to supraphysiological doses of estrogen in the developing brain might cause defects in ARC kisspeptin neurons via ERα and ERß, resulting in inhibition of pulsatile LH release and lack of estrous cyclicity.

Erratum: J Reprod Dev, Vol. 59, No. 3, p. 266-272, Dynorphin-Kappa Opioid Receptor Signaling Partly Mediates Estrogen Negative Feedback Effect on LH Pulses in Female Rats.

Figure 3(a) have been corrected.

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.

Identification of hypothalamic arcuate nucleus-specific enhancer region of Kiss1 gene in mice.

Pulsatile secretion of GnRH plays a pivotal role in follicular development via stimulating tonic gonadotropin secretion in mammals. Kisspeptin neurons, located in the arcuate nucleus (ARC), are considered to be an intrinsic source of the GnRH pulse generator. The present study aimed to determine ARC-specific enhancer(s) of the Kiss1 gene by an in vivo reporter assay. Three green fluorescent protein (GFP) reporter constructs (long, medium length, and short) were generated by insertion of GFP cDNA at the Kiss1 locus. Transgenic female mice bearing the long and medium-length constructs showed apparent GFP signals in kisspeptin-immunoreactive cells in both the ARC and anteroventral periventricular nucleus, in which another population of kisspeptin neurons are located. On the other hand, transgenic mice bearing 5'-truncated short construct showed few GFP signals in the ARC kisspeptin-immunoreactive cells, whereas they showed colocalization of GFP- and kisspeptin-immunoreactivities in the anteroventral periventricular nucleus. In addition, chromatin immunoprecipitation and chromosome conformation capture assays revealed recruitment of unoccupied estrogen receptor-α in the 5'-upstream region and intricate chromatin loop formation between the 5'-upstream and promoter regions of Kiss1 locus in the ARC. Taken together, the present results indicate that 5'-upstream region of Kiss1 locus plays a critical role in Kiss1 gene expression in an ARC-specific manner and that the recruitment of estrogen receptor-α and formation of a chromatin loop between the Kiss1 promoter and the 5' enhancer region may be required for the induction of ARC-specific Kiss1 gene expression. These results suggest that the 5'-upstream region of Kiss1 locus functions as an enhancer for ARC Kiss1 gene expression in mice.

KISS1 gene expression in the developing brain of female pigs in pre- and peripubertal periods.

Puberty is associated with an increase in gonadotropin secretion as a result of an increase in gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin is considered to play a key role in puberty onset in many mammalian species, including rodents, ruminants and primates. The present study aimed to determine if changes in hypothalamic expression of the KISS1 gene, encoding kisspeptin, are associated with the onset of puberty in pigs. The animals (n=4 in each group) were perfused with 4% paraformaldehyde at 0, 1, 2, 3 and 4 months old, as prepubertal stages, and at 5 months old, as the peripubertal stage, following each blood sampling. KISS1 gene expressions in coronal sections of brains were visualized by in situ hybridization. Plasma luteinizing hormone (LH) was measured by radioimmunoassay. KISS1 mRNA signals were observed in the arcuate nucleus (ARC) at all ages examined without any significant difference in the number of KISS1-expressing cells, indicating that the KISS1 gene is constantly expressed in the ARC throughout pubertal development in pigs. The plasma LH concentration was the highest in 0-month-old piglets and significantly decreased in the 1- and 2 month-old groups (P<0.05), suggesting a developing negative feedback mechanism affecting gonadotropin release during the prepubertal period. Considering the potent stimulating effect of kisspeptin on gonadotropin release in prepubertal pigs, kisspeptin secretion rather than kisspeptin synthesis may be responsible for the onset of puberty in pigs.

Microarray analysis of perinatal-estrogen-induced changes in gene expression related to brain sexual differentiation in mice.

Sexual dimorphism of the behaviors or physiological functions in mammals is mainly due to the sex difference of the brain. A number of studies have suggested that the brain is masculinized or defeminized by estradiol converted from testicular androgens in perinatal period in rodents. However, the mechanisms of estrogen action resulting in masculinization/defeminization of the brain have not been clarified yet. The large-scale analysis with microarray in the present study is an attempt to obtain the candidate gene(s) mediating the perinatal estrogen effect causing the brain sexual differentiation. Female mice were injected with estradiol benzoate (EB) or vehicle on the day of birth, and the hypothalamus was collected at either 1, 3, 6, 12, or 24 h after the EB injection. More than one hundred genes down-regulated by the EB treatment in a biphasic manner peaked at 3 h and 12-24 h after the EB treatment, while forty to seventy genes were constantly up-regulated after it. Twelve genes, including Ptgds, Hcrt, Tmed2, Klc1, and Nedd4, whose mRNA expressions were down-regulated by the neonatal EB treatment, were chosen for further examination by semiquantitative RT-PCR in the hypothalamus of perinatal intact male and female mice. We selected the genes based on the known profiles of their potential roles in brain development. mRNA expression levels of Ptgds, Hcrt, Tmed2, and Nedd4 were significantly lower in male mice than females at the day of birth, suggesting that the genes are down-regulated by estrogen converted from testicular androgen in perinatal male mice. Some genes, such as Ptgds encoding prostaglandin D2 production enzyme and Hcrt encording orexin, have been reported to have a role in neuroprotection. Thus, Ptgds and Hcrt could be possible candidate genes, which may mediate the effect of perinatal estrogen responsible for brain sexual differentiation.

dynorphin-kappa opioid receptor signaling partly mediates estrogen negative feedback effect on LH pulses in female rats.

Accumulating evidence suggests that the arcuate nucleus (ARC) kisspeptin/neurokinin B (NKB)/dynorphin (KNDy) neurons play a role in estrogen negative feedback action on pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release. The present study aimed to determine if dynorphin (Dyn) is involved in estrogen negative feedback on pulsatile GnRH/LH release. The effect of the injection of nor-binaltorphimine (nor-BNI), a kappa-opioid receptor (KOR) antagonist, into the third cerebroventricle (3V) on LH pulses was determined in ovariectomized (OVX) adult female rats with/without replacement of negative feedback levels of estradiol (low E2). The mean LH concentrations and baseline levels of LH secretion in nor-BNI-injected, low E2-treated rats were significantly higher compared with vehicle-treated controls. On the other hand, the nor-BNI treatment failed to affect any LH pulse parameters in OVX rats without low E2 treatment. These results suggest that Dyn is involved in the estrogen negative feedback regulation of pulsatile GnRH/LH release. The low E2 treatment had no significant effect on the numbers of ARC Pdyn (Dyn gene)-,Kiss1- and Tac2 (NKB gene)-expressing cells. The treatment also did not affect mRNA levels of Pdyn and Oprk1 (KOR gene) in the ARC-median eminence region, but significantly increased the ARC kisspeptin immunoreactivity. These findings suggest that the negative feedback level of estrogen suppresses kisspeptin release from the ARC KNDy neurons through an unknown mechanism without affecting the Dyn and KOR expressions in the ARC. Taken together, the present result suggests that Dyn-KOR signaling is a part of estrogen negative feedback action on GnRH/LH pulses by reducing the kisspeptin release in female rats.

Epigenetic regulation of Kiss1 gene expression mediating estrogen-positive feedback action in the mouse brain.

This study aims to determine the epigenetic mechanism regulating Kiss1 gene expression in the anteroventral periventricular nucleus (AVPV) to understand the mechanism underlying estrogen-positive feedback action on gonadotropin-releasing hormone/gonadotropin surge. We investigated estrogen regulation of the epigenetic status of the mouse AVPV Kiss1 gene locus in comparison with the arcuate nucleus (ARC), in which Kiss1 expression is down-regulated by estrogen. Histone of AVPV Kiss1 promoter region was highly acetylated, and estrogen receptor α was highly recruited at the region by estrogen. In contrast, the histone of ARC Kiss1 promoter region was deacetylated by estrogen. Inhibition of histone deacetylation up-regulated in vitro Kiss1 expression in a hypothalamic non-Kiss1-expressing cell line. Gene conformation analysis indicated that estrogen induced formation of a chromatin loop between Kiss1 promoter and the 3' intergenic region, suggesting that the intergenic region serves to enhance estrogen-dependent Kiss1 expression in the AVPV. This notion was proved, because transgenic reporter mice with a complete Kiss1 locus sequence showed kisspeptin neuron-specific GFP expression in both the AVPV and ARC, but the deletion of the 3' region resulted in greatly reduced GFP expression only in the AVPV. Taken together, these results demonstrate that estrogen induces recruitment of estrogen receptor α and histone acetylation in the Kiss1 promoter region of the AVPV and consequently enhances chromatin loop formation of Kiss1 promoter and Kiss1 gene enhancer, resulting in an increase in AVPV-specific Kiss1 gene expression. These results indicate that epigenetic regulation of the Kiss1 gene is involved in estrogen-positive feedback to generate the gonadotropin-releasing hormone/gonadotropin surge.