Kiss1 mRNA Research Articles

OR16-01 The Expression of the Homeodomain Transcription Factor SIX3 within Kisspeptin Neurons Is Necessary for Reproduction in Mice

The homeodomain transcription factor SIX3 is necessary for normal reproductive processes in mice. The loss of a single allele results in haploinsufficient phenotypes that are reflective of individuals with Kallmann Syndrome, such as hypogonadism, reduced GnRH neurons, and anosmia. Six3 heterozygous mice have reproductive impairments including an increased time to first litter, increased estrous cycle lengths in females, and fewer litters in males compared to wildtype controls. Prior studies found that deleting SIX3 specifically from GnRH neurons did not recapitulate the same phenotypes produced from a global knockout of the Six3 allele and imposed no reproductive abnormalities. This suggests that the role of SIX3 in reproduction is due to SIX3 populations outside of GnRH neurons. Using qPCR and RNA-seq, we have verified that Six3 is expressed in arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV) populations of kisspeptin neurons and can regulate the kisspeptin promoter in vitro. This led us to hypothesize that SIX3 in kisspeptin neurons is required for Kiss1 regulation and maximal fertility. We selectively deleted Six3 from kisspeptin neurons by crossing a Six3-floxed mouse with a Kiss1-Cre mouse to produce Six3 flox/flox; Kiss1-Cre positive mice (cKO) and Six3 flox/flox, Kiss-Cre negative littermates (controls). In female mice, we found that the loss of SIX3 from kisspeptin neurons disrupted the estrous cycle and reduced fertility. Compared to controls, cKO mice had fewer cycles in a 16-day period (0.38 vs 1.44; p=0.004; n=8-9), and spent a greater percentage of time in diestrus (80.5 vs 61.8; p=0.002; n=8-9) and a smaller percentage of time in estrus (15.6 vs 29.2; p=0.030; n=8-9). The loss of kisspeptin-specific SIX3 also resulted in reduced fecundity, with the cKO female mice having fewer pups within 90 days (15.0 vs 34.4; p=0.048; n=5-6) compared to controls. In addition to its importance in female mice, kisspeptin-specific SIX3 also plays a role in male fertility. We found that male cKO mice had reduced motile sperm (52.2% vs 72.5%; p=0.012; n=7-9) compared to controls. We also assayed levels of Kiss1 mRNA in the male arcuate nucleus and observed decreased Kiss1 levels compared to controls (0.34 vs 1.01; p=0.042; n=3-4). These results support our hypothesis that SIX3 in kisspeptin neurons is necessary for critical reproductive processes in both females and males, including progression through the estrous cycle, fecundity, and sperm motility, potentially through Kiss1 regulation.

Effect of Sleeve Gastrectomy on Kisspeptin Expression in the Hypothalamus of Rats with Polycystic Ovary Syndrome

ObjectivesThe purpose of this study was to determine changes in the expression levels of kisspeptin‐1 (Kiss1) in the hypothalamus during the development of polycystic ovary syndrome (PCOS) and after treatment with sleeve gastrectomy (SG).MethodsThis study used chronic dehydroepiandrosterone (DHEA) alone and DHEA plus a high‐fat diet (HFD) to generate a PCOS rat model. Subsequently, SG was performed in the animals with PCOS and the effects on glucose tolerance, insulin sensitivity, sex hormones, estrous cyclicity, adiponectin, and Kiss1 expression in the hypothalamus were investigated.ResultsImpaired glucose tolerance, decreased insulin sensitivity, reduced adiponectin levels, disrupted estrous cyclicity, and elevated sex hormone levels associated with PCOS models were restored to normal following SG. In addition, SG was able to restore the increase in the expression of Kiss1 mRNA and Kiss1‐positive neurons in the arcuate nucleus of rats with PCOS. Interestingly, although SG did not result in a significant loss of body weight in rats administered DHEA under a chow diet, it resulted in comparable metabolic improvements and Kiss1 expression in rats that had been administered DHEA along with an HFD.ConclusionsThe recovery of normal levels of Kiss1 expression in the hypothalamus after SG in this study suggests that Kiss1 might play an important role in the development of PCOS and its improvement by SG.

Temporal expression of the KISS1/GPR54 system in goats’ testes and epididymides and its spatial expression in pubertal goats

Kisspeptin, encoded by the KISS1 gene, and its receptor GPR54 are essential in puberty onset and male fertility due to their central regulatory roles. However, the roles of KISS1/GPR54 in peripheral tissues remain unclear. This study aimed to investigate the temporal expression patterns of KISS1/GPR54 in goat testes and epididymides and its spatial expression patterns in pubertal goats. Immunohistochemical analysis revealed that kisspeptin/GPR54 were localized in Leydig, Sertoli, and germ cells of pubertal goats’ testis, as well as in principal and basal cells of the epididymis. RT-PCR revealed a marked variation in the KISS1/GPR54 expressions in the testes and epididymides from the age of first week to adulthood. KISS1 and GPR54 mRNA levels in testes decreased from the age of first week to two months and then increased from two months to puberty and adulthood. The KISS1 and GPR54 mRNA levels in Leydig cells decreased from the age of one week to two months and increased from two months to puberty, and then decreased from puberty to adulthood. Only GPR54 mRNA levels in the epididymides increased from the age of one week to two months and puberty, and then decreased from puberty to adulthood. RT-PCR analysis showed the different spatial expression patterns of KISS1/GPR54 in pubertal goat tissues. The KISS1 mRNA level was high in the hypothalamus, moderate in pancreas, liver, epididymis and testis; and low in the other tissues. The GPR54 expression was high in the pancreas and testis; moderate in pituitary, hypothalamus and mesenteric lymph node; and low in the other tissues. In conclusion, the KISS1/GPR54 system possessed distinct temporal expression profiles in goats’ testes and epididymides, as well as different spatial expression patterns in pubertal goat tissues, which implied the possible local role of this system in goats’ testes, epididymides, and other peripheral tissues.

Dysregulated Kisspeptin/Receptor Expression at the Maternal‐Fetal Interface in the Preeclamptic‐like BPH/5 Mouse

Preeclampsia (PE) is a hypertensive pregnancy syndrome estimated to cause the death of approximately 63,000 women worldwide annually. Insufficient invasion of extravillous trophoblast cells (EVT) into the maternal decidua and inadequate placentation are key adverse events in the pathogenesis of PE. Kisspeptins (KP), a family of small peptides derived from the kiss1 gene product, have been implicated as physiologic inhibitors of EVT invasion, a mechanism associated with upregulation of Tissue Inhibitor of Metalloproteinases (TIMP) and downregulation of Matrix Metalloproteinases (MMP). In PE, placental dysregulation of kiss1 and the KP receptor (kiss1r) have been proposed. Based on in vitro studies and evaluation of term PE placentas, it has been suggested that kiss1 and kiss1r mRNA are overexpressed at the maternal‐fetal interface during early gestation and excessively impair EVT invasion. However, the innate challenges involving human sample collection and early diagnosis of PE prevent a holistic understanding of KP role. Therefore, the objective of this study was to utilize the preeclamptic‐like BPH/5 mouse to investigate uteroplacental KP dysregulation throughout gestation. We have previously shown that pregnant BPH/5 females spontaneously develop poor placentation with decreased trophoblast invasion and inadequately remodeled decidual vessels. Hence, we hypothesized that uteroplacental expression of kiss1/kiss1r would be temporally dysregulated in the BPH/5 compared to the control C57 mice. Furthermore, we hypothesized that higher kiss1/kiss1r levels would be associated with upregulation of TIMP2, an important endogenous inhibitor of MMPs and cellular migration. To test our hypothesis, implantation sites and placentas of BPH/5 and C57 mice were collected at the embryonic days (e) 4.5, 7.5, 12.5, 14.5 and 18.5. The kiss1, kiss1r and TIMP2 mRNA levels were measured using RT‐PCR and comparisons were performed using unpaired t‐test. A similar expression pattern of kiss1 and kiss1r was seen throughout gestation in BPH/5 and control groups, with levels increasing from early (e4.5) to mid‐gestation (e14.5) and decreasing thereafter (e14.5 and e18.5). During the peak of decidualization (e7.5), BPH/5 mice presented 2‐fold higher kiss1 and kiss1r compared to C57 mice (n = 6; p ≤ 0. 05). In accordance with the proposed KP role, a 10‐fold increase in TIMP2 was also seen in BPH/5 mice at e7.5 (n = 6, p ≤ 0.05). In mid‐gestation (e14.5) kiss1r levels were 2‐fold lower in BPH/5 (n = 5) than in C57 (n = 6; p < 0.05), whereas kiss1 levels were not different among groups (n = 6; p > 0.05). In late gestation (e18.5), kiss1 and kiss1r were 3‐ and 2.5‐fold lower respectively in placentas of BPH/5 mice (n = 6; p ≤ 0.05). To the author’s knowledge, this is the first report of translational studies using an animal model of PE to investigate the role of KP in PE‐associated poor placental development. A kiss1/kiss1r dysregulation is reported, with higher expression levels in the preeclamptic‐like mouse in the peak of decidualization, and concurrent upregulation of TIMP2. Further studies are needed to investigate KP‐related upstream and downstream signaling pathways in the context of PE.

MicroRNA-124 and microRNA-378 inhibit the proliferation and invasion of colorectal cancer by upregulating KiSS1.

BackgroundThe KiSS1 gene is considered a tumor suppressor in various cancers. MicroRNAs are involved in many important life processes, and their regulation of gene expression may be as important as that of transcription factors. Here, we explore the roles of miR-124-3p and miR-378-3p in colorectal cancer and their relationships with the KiSS1 gene.MethodsThe effects of miR-124-3p and miR-378-3p on KiSS1 protein expression were observed by transfecting colorectal cancer cells (SW-480) with miR-124-3p and miR-378-3p mimics and inhibitors. Moreover, cell proliferation, migration and invasion were evaluated by ethynyl-20-deoxyuridine and Transwell experiments.ResultsThe KiSS1 mRNA and protein expression levels were significantly increased in mimic-transfected cells compared with those in untransfected cells, and the proliferation, migration and invasion abilities of the former were decreased; in addition, opposing results were obtained in the inhibitor and mimic groups.ConclusionsIn conclusion, our studies indicate that miR-124-3p and miR-378-3p upregulate the expression of KiSS1 and are associated with colorectal cancer metastasis and progression. miR-124-3p, miR-378-3p and KiSS1 may play important roles in colorectal cancer.

Identification of kisspeptin2 cDNA in the catfish Heteropneustes fossilis: Expression profile, in situ localization and steroid modulation

Kisspeptin (Kiss) is considered an upstream regulator of gonadotropin-releasing hormone in mammals but its role in non-mammalian vertebrates is not unequivocally established. In the catfish Heteropneustes fossilis, a 605 bp long cDNA was identified from the brain by cloning as well as by retrieving from the catfish transcriptome database. The open reading frame (ORF, 93–405 bp) codes for a 113 amino acids long precursor protein. Homology and phylogenetic analyses showed that the predicted protein belongs to the vertebrate Kiss2 type with a high degree of conservation in the Kiss2-10 region (FNFNPFGLRF). The kiss2 transcripts were expressed highly in the brain and gonads in a dimorphic manner with a female bias. In the brain, kiss2 transcripts showed regional differences with higher expression in the medulla oblongata and forebrain regions. The kiss2 transcripts showed significant seasonal variations with the highest expression in the brain in spawning phase and in the gonads in prespawning phase. The kiss2 transcripts were localized in the brain (nucleus preopticus, habenular nucleus, nucleus recessus posterioris, nucleus recessus lateralis) and stratum periventriculare (radial glial cells) of optic tectum, pituitary and ovary (follicular layer and germinal vesicle). Ovariectomy (1, 2, 3 and 4 weeks) decreased brain kiss2 mRNA levels and a single injection of estradiol-17β (E2; 0.5 μg/g body weight) in 3- week ovariectomized (OVX) and sham operated fish resulted in an increase in the transcript levels after 24 h. The E2 receptor antagonist Tamoxifen (TMX) produced biphasic effects on the kiss2 expression in the dose- response study. TMX inhibited the expression in the OVX fish, but elicited a stimulatory effect in the OVX + E2-treated fish. Testosterone (T) decreased, and progesterone (P4) inhibited (resting phase) or stimulated (prespawning phase) the transcript level in 3-week OVX fish. In the 3-week sham groups, E2 increased, and TMX, T and P4 inhibited the kiss2 transcript levels. The results suggest that Kiss2 is an important regulator of the brain- pituitary- gonadal- endocrine axis, and in habenular and optic tectum functions.

Effect of GABA-T on Reproductive Function in Female Rats.

Simple SummaryThis study evaluated the effect of γ-aminobutyric acid transaminase (GABA-T) on reproduction in female rats. Our results showed that GABA-T expressed in the reproductive axis of female rats at different developmental stages, and inhibiting GABA-T affected Rfrp-3, Gnrh and Kiss1 mRNA transcription levels in hypothalamic and reduced the concentration of GABA-T, LH, and P4 in serum. At the same time, the initiation of the puberty was delayed, and led to disordered estrous cycle and reduced breastfeeding performance in adult female rats. This study showed that GABA-T affects the reproductive function of female rats by regulating the levels of Gnrh, Kiss1 and Rfrp-3 in the hypothalamus as well as the concentrations of LH and P4.This study explored the role of γ-aminobutyric acid transaminase (GABA-T) in the puberty and reproductive performance of female rats. Immunofluorescence technique, quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the distribution of GABA-T and the expression of genes and hormones in female rats, respectively. The results showed that GABA-T was mainly distributed in the arcuate nucleus (ARC), paraventricular nucleus (PVN) and periventricular nucleus (PeN) of the hypothalamus, and in the adenohypophysis, ovarian granulosa cells and oocytes. Abat mRNA level at 28 d was lowest in the hypothalamus and the pituitary; at puberty, it was lowest in the ovary. Abat mRNA level was highest in adults in the hypothalamus; at infancy and puberty, it was highest in the pituitary; and at 21 d it was highest in the ovary. After vigabatrin (GABA-T irreversible inhibitor) was added to hypothalamus cells, the levels of Abat mRNA and Rfrp-3 mRNA were significantly reduced, but Gnrh mRNA increased at the dose of 25 and 50 μg/mL; Kiss1 mRNA was significantly increased but Gabbr1 mRNA was reduced at the 50 μg/mL dose. In prepubertal rats injected with vigabatrin, puberty onset was delayed. Abat mRNA, Kiss1 mRNA and Gnrh mRNA levels were significantly reduced, but Rfrp-3 mRNA level increased in the hypothalamus. Vigabatrin reduced the concentrations of GABA-T, luteinizing hormone (LH) and progesterone (P4), and the ovarian index. Lactation performance was reduced in adult rats with vigabatrin treatment. Four hours after vigabatrin injection, the concentrations of GABA-T and LH were significantly reduced in adult and 25 d rats, but follicle-stimulating hormone (FSH) increased in 25 d rats. In conclusion, GABA-T affects the reproductive function of female rats by regulating the levels of Gnrh, Kiss1 and Rfrp-3 in the hypothalamus as well as the concentrations of LH and P4.

Annual patterns of ocular melatonin level in the female grass puffer, Takifugu alboplumbeus: possible involvement in seasonal reproductive response.

The aim of this study was to investigate the expression patterns of ocular melatonin in the annual reproductive cycle of the female grass puffer. Spawning season of the female grass puffer is from June to July in Jeju, South Korea. Time-resolved fluoroimmunoassay revealed that levels of ocular melatonin, which show an annual change, peaked in May (spawning season). Additionally, expression of reproductive-related genes also showed annual patterns: GnRH1 peaked in August, GnRH2 peaked in February, GnRH3, Kiss2, and LPXRFa peaked in November. These results suggest that ocular melatonin may be related to the annual reproductive cycle in the grass puffer. To better understand the photic regulation of AANAT1a mRNA in the retina, we observed the nocturnal pattern of ocular melatonin levels daily, which shows a nocturnal pattern in both short photoperiod (SD) and long photoperiod (LD) conditions. In the brain, AANAT2 mRNA also shows a nocturnal pattern in both SD and LD; however, the time of peak expression of AANAT2 mRNA was unchanged in both conditions. Following intraperitoneal injection of melatonin for 2weeks, expression of GnRH2 and LPXRFa mRNA in the brain significantly increased, while that of Kiss2 mRNA was decreased, suggesting that melatonin has a reproduction-related effect. Furthermore, under SD and LD conditions for 14weeks, the gonadosomatic index more increased and the maturity of the ovary progressed under LD compared with those under SD, suggesting that the SD photoperiodic signal inactivated ovarian development. These results indicate that the ocular melatonin may have a possible role in the reproductive endocrinology of the grass puffer.

Estradiol Potentiates But Is Not Essential for Prolactin-Induced Suppression of Luteinizing Hormone Pulses in Female Rats.

Hyperprolactinemia causes infertility by suppressing gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion. Because effects of prolactin (PRL) on the hypothalamus usually require estradiol (E2), we investigated the role of E2 in PRL-induced suppression of LH pulses. Ovariectomized (OVX) rats treated with oil or E2 (OVX + E2) received a subcutaneous injection of ovine PRL (oPRL) 30 minutes before serial measurement of LH in the tail blood by enzyme-linked immunosorbent assay. E2 reduced pulsatile LH secretion. oPRL at 1.5 mg/kg further reduced LH pulse frequency in OVX + E2 but had no effect in OVX rats. The higher dose of 6-mg/kg oPRL decreased LH pulse frequency in both OVX and OVX + E2 rats, whereas pulse amplitude and mean LH levels were lowered only in OVX + E2 rats. Kisspeptin immunoreactivity and Kiss1 messenger ribonucleic acid (mRNA) levels were decreased in the arcuate nucleus (ARC) of OVX + E2 rats. oPRL decreased both kisspeptin peptide and gene expression in the ARC of OVX rats but did not alter the already low levels in OVX + E2 rats. In the anteroventral periventricular nucleus, oPRL did not change kisspeptin immunoreactivity and, paradoxically, increased Kiss1 mRNA only in OVX + E2 rats. Moreover, oPRL effectively reduced Gnrh expression regardless of E2 treatment. In this study we used tail-tip blood sampling to determine the acute effect of PRL on LH pulsatility in female rats. Our findings characterize the role of E2 in the PRL modulation of hypothalamic components of the gonadal axis and LH release, demonstrating that E2 potentiates but is not essential for the suppression of pulsatile LH secretion caused by hyperprolactinemia.

Sexually dimorphic gene expression and neurite sensitivity to estradiol in fetal arcuate Kiss1 cells.

Kiss1 neurons of the arcuate (ARC) nucleus form an interconnected network of cells that communicate via neurokinin B (encoded by Tac2) and its receptor (encoded by Tacr3) and play key roles in the control of the reproductive axis through sex hormone-regulated synthesis and release of kisspeptin peptides (Kp, encoded by Kiss1). The aim of this study was to determine whether the Kiss1 cell population of the ARC already displays sexually dimorphic features at embryonic age E16.5 in mice. At this time of development, Kiss1-GFP- and Kp-immunoreactive cell bodies were restricted to the ARC and not found in the pre-optic area (POA). The Kiss1-GFP cell population was identical in size between sexes but had significantly lower Kiss1, Tac2, and Tacr3 mRNA levels and lower Kp-ir fiber density in the POA in male compared to female fetuses. Receptors for androgen (Ar) and estrogen (Esr1, Esr2, Gpr30) and the Cyp19a1 gene (encoding the estradiol-producing enzyme aromatase) transcripts were also detected in fetal ARC Kiss1-GFP cells with significant sex differences for Ar (higher in males) and Esr1 (higher in females). Functional studies on primary cultures of sorted fetal Kiss1-GFP cells revealed a significant negative effect of estradiol treatment on neurite outgrowth on the fourth day of culture in the female group specifically. We conclude that the ARC Kiss1 cell population is already sexually differentiated at E16.5 and that its morphogenetic development may be particularly vulnerable to estradiol exposure at this early developmental time.

Effects of the Fertility Drugs Clomiphene Citrate and Letrozole on Kiss-1 Expression in Hypothalamic Kiss-1-Expressing Cell Models.

Clomiphene citrate (CC) and letrozole stimulate the hypothalamic-pituitary-ovarian axis and are used widely as oral fertility drugs to induce folliculogenesis. We examined whether these drugs increase Kiss-1 expression in hypothalamic cell models. We utilized two hypothalamic cell models, mHypoA-50 and mHypoA-55, which originated from Kiss-1 neurons in the anteroventral periventricular (AVPV) nucleus and arcuate (ARC) nucleus of the mouse hypothalamus, respectively. The cells were stimulated with CC or letrozole, after which Kiss-1 mRNA expression was determined. CC stimulated Kiss-1 gene expression in mHypoA-50 and mHypoA-55 cells. The basal expression of Kiss-1 was significantly increased in the presence of estradiol (E2) in mHypoA-50 cells, and the CC-induced increase in Kiss-1 expression was not observed in the presence of E2 in these cells. In contrast, E2 did not modify the basal expression of Kiss-1 in mHypoA-55 cells, and CC-induced Kiss-1 expression was still observed in the presence of E2. The significant increase in Kiss-1 gene expression in mHypoA-50 and mHypoA-55 cells was blunted in the presence of estrogen receptor antagonists. Aromatase was expressed in mHypoA-50 and mHypoA-55 cells. Letrozole, an aromatase inhibitor, increased Kiss-1 expression in mHypoA-55 ARC cells but not in mHypoA-50 AVPV cells. Although the basal expression of Kiss-1 was increased by E2, letrozole did not modulate Kiss-1 expression in mHypoA-50 cells. Letrozole-induced Kiss-1 gene expression in mHypoA-55 cells was not modulated in the presence of E2. The fertility drugs CC and letrozole modulated Kiss-1 expression in hypothalamic cell models.

Mating-induced increase in <i>Kiss1</i> mRNA expression in the anteroventral periventricular nucleus prior to an increase in LH and testosterone release in male rats

Kisspeptin has an indispensable role in gonadotropin-releasing hormone/gonadotropin secretion in mammals. In rodents, kisspeptin neurons are located in distinct brain regions, namely the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), arcuate nucleus (ARC), and medial amygdala (MeA). Among them, the physiological role of AVPV/PeN kisspeptin neurons in males has not been clarified yet. The present study aims to investigate the acute effects of the olfactory and/or mating stimulus with a female rat on hypothalamic and MeA Kiss1 mRNA expression, plasma luteinizing hormone (LH) and testosterone levels in male rats. Intact male rats were exposed to the following stimuli: exposure to clean bedding; exposure to female-soiled bedding as a female-olfactory stimulus; exposure to female-soiled bedding and mating stimulus with a female rat. The mating stimulus significantly increased the number of the AVPV/PeN Kiss1 mRNA-expressing cells in males within 5 minutes after the exposure, and significantly increased LH and testosterone levels, followed by an increase in male sexual behavior. Whereas, the males exposed to female-soiled bedding showed a moderate increase in LH levels and no significant change in testosterone levels and the number of the AVPV/PeN Kiss1 mRNA-expressing cells. Importantly, none of the stimuli affected the number of Kiss1 mRNA-expressing cells in the ARC and MeA. These results suggest that the mating-induced increase in AVPV/PeN Kiss1 mRNA expression may be, at least partly, involved in stimulating LH and testosterone release, and might consequently ensure male mating behavior. This study would be the first report suggesting that the AVPV/PeN kisspeptin neurons in males may play a physiological role in ensuring male reproductive performance.

Opposite effects of high- and low-dose di-(2-ethylhexyl) phthalate (DEHP) exposure on puberty onset, oestrous cycle regularity and hypothalamic kisspeptin expression in female rats.

Di-(2-ethylhexyl) phthalate (DEHP) is ubiquitous in the environment and has been proposed to lead to reproductive disruption. In this study, we systematically investigated the effects of different doses of DEHP exposure on female hypothalamic-pituitary-gonadal axis development. Female Sprague-Dawley rats were gavaged with vehicle (corn oil) or DEHP (5 or 500mgkg-1 day-1) during postnatal Days (PNDs) 22-28 or PNDs 22-70. Results demonstrated that the low and high doses of DEHP exerted opposite effects on puberty onset, circulating luteinising hormone, serum oestradiol and progesterone levels, with the low dose (5mgkg-1) promoting and the high dose (500mgkg-1) inhibiting these parameters. Significant dose-related differences were also found in the D500 group with longer oestrous cycle duration, lower ovarian/bodyweight ratio, fewer corpus lutea and more abnormal ovarian stromal tissue in comparison with the oil or D5 groups. Molecular data showed that the hypothalamic Kiss1 mRNA expression in the anteroventral periventricular but not in the arcuate nucleus significantly decreased in the D500 rats and increased in the D5 rats relative to the rats in the oil group. These findings suggested that the kisspeptin system is a potential target for DEHP to disrupt reproductive development and function.

Retinoblastoma binding protein 7 is involved in Kiss1 mRNA upregulation in rodents.

Kisspeptin, encoded by Kiss1, is essential for reproduction in mammals. Kiss1 expression is regulated by estrogen via histone acetylation in the Kiss1 promotor region. Thus, elucidation of histone modification factor(s) involved in the regulation of Kiss1 expression is required to gain further understanding of the mechanisms of its control. The RNA-seq analysis of isolated kisspeptin neurons, obtained from the arcuate nucleus (ARC) of female rats, revealed that Rbbp7, encoding retinoblastoma binding protein 7 (RBBP7), a member of histone modification and chromatin remodeling complexes, is highly expressed in the ARC kisspeptin neurons. Thus, the present study aimed to investigate whether RBBP7 is involved in Kiss1 expression. Histological analysis using in situ hybridization (ISH) revealed that Rbbp7 expression was located in several hypothalamic nuclei, including the ARC and the anteroventral periventricular nucleus (AVPV), where kisspeptin neurons are located. Double ISH for Rbbp7 and Kiss1 showed that a majority of kisspeptin neurons (more than 85%) expressed Rbbp7 mRNA in both the ARC and the AVPV of female rats. Further, Rbbp7 mRNA knockdown significantly decreased in vitro expression of Kiss1 in a mouse immortalized kisspeptin neuronal cell line (mHypoA-55). Estrogen treatment significantly decreased and increased Kiss1 mRNA levels in the ARC and AVPV of ovariectomized female rats, respectively, but failed to affect Rbbp7 mRNA levels in both the nuclei. Taken together, these findings suggest that RBBP7 is involved in the upregulation of Kiss1 expression in kisspeptin neurons of rodents in an estrogen-independent manner.

Immunization with oral KISS1 DNA vaccine inhibits testicular Leydig cell proliferation mainly via the hypothalamic–pituitary–testicular axis and apoptosis-related genes in goats

This study aimed to analyze the effect and mechanism of immunization of oral KISS1 DNA vaccine on the proliferation of goat testicular Leydig cells. Ten 8-week-old male goats were randomly divided into KISS1 DNA vaccine and control groups for immunization (five goats each group). These goats were sacrificed at 8 weeks after primary immunization, and the tissue samples of hypothalamus, pituitary, and testis and Leydig cell samples were collected for RT-PCR and CCK8 assay. Immunization with the oral KISS1 DNA vaccine effectively inhibited the proliferation of Leydig cells, the expression of hypothalamus KISS1, GPR54, and GnRH mRNA, pituitary GnRHR and LH mRNA, testicular LHR mRNA, and apoptosis-inhibitory gene Bcl-2 mRNA in Leydig cells. By contrast, the immunization enhanced the mRNA expression of apoptosis-promoting gene Bax and Clusterin in Leydig cells. These findings indicate that immunization with the oral KISS1 DNA vaccine can inhibit the proliferation of goat testicular Leydig cells mainly via the hypothalamic–pituitary–testicular axis and apoptosis-related genes.

Leptin actions through the nitrergic system to modulate the hypothalamic expression of the kiss1 mRNA in the female rat

Gonadotrophin-releasing hormone (GnRH) is the main controller of the reproductive axis and stimulates the synthesis and secretion of gonadotrophins. Estrogen is the main peripheral factor controlling GnRH secretion, and this action is mainly mediated by the transsynaptic pathway through nitric oxide, kisspeptin, leptin, among other factors. Kisspeptin is the most potent factor known to induce GnRH release. Nitric oxide and leptin also promote GnRH release; however, neurons expressing GnRH do not express the leptin receptor (OB-R). Leptin seems to modulate the expression of genes and proteins involved in the kisspeptin system. However, few kisspeptin-synthesizing cells in the arcuate nucleus (ARC) and few cells, if any, in the preoptic area (POA) express OB-R; this indicates an indirect mechanism of leptin action on kisspeptin. Nitric oxide is an important intermediate in the actions of leptin in the central nervous system. Thus, this work aimed to verify the numbers of nNOS cells were activated by leptin in different hypothalamic areas; the modulatory effects of the nitrergic system on the kisspeptin system; and the indirect regulatory effect of leptin on the kisspeptin system via nitric oxide. Ovariectomized rats were treated with estrogen or a vehicle and received an intracerebroventricular (i.c.v.) injection of a nitric oxide donor, leptin or neuronal nitric oxide synthase (nNOS) enzyme inhibitor. Thirty minutes after the injection, the animals were decapitated. Leptin acts directly on nitrergic neurons in different hypothalamic regions, and the effects on the ventral premammillary nucleus (PMV) and ventral dorsomedial hypothalamus (vDMH) are enhanced. The use of a nitric oxide donor or the administration of leptin stimulates the expression of the kisspeptin mRNA in the ARC of animals with or without estrogenic action; however, these changes are not observed in the POA. In addition, the action of leptin on the expression of the kisspeptin mRNA in the ARC is blocked by a nitric oxide synthesis inhibitor. We concluded that the effects of leptin on the central nervous system are at least partially mediated by the nitrergic system. Also, nitric oxide acts on the kisspeptin system by modulating the expression of the kisspeptin mRNA, and leptin at least partially modulates the kisspeptin system through the nitrergic system, particularly in the ARC.

Time‐of‐day‐dependent sensitivity of the reproductive axis to RFamide‐related peptide‐3 inhibition in female Syrian hamsters

In spontaneously ovulating rodent species, the timing of the luteinising hormone (LH) surge is controlled by the master circadian pacemaker in the suprachiasmatic nucleus (SCN). The SCN initiates the LH surge via the coordinated control of two opposing neuropeptidergic systems that lie upstream of the gonadotrophin-releasing hormone (GnRH) neuronal system: the stimulatory peptide, kisspeptin, and the inhibitory peptide, RFamide-related peptide-3 (RFRP-3; the mammalian orthologue of avian gonadotrophin-inhibitory hormone [GnIH]). We have previously shown that the GnRH system exhibits time-dependent sensitivity to kisspeptin stimulation, further contributing to the precise timing of the LH surge. To examine whether this time-dependent sensitivity of the GnRH system is unique to kisspeptin or a more common mechanism of regulatory control, we explored daily changes in the response of the GnRH system to RFRP-3 inhibition. Female Syrian hamsters were ovariectomised to eliminate oestradiol (E2 )-negative-feedback and RFRP-3 or saline was centrally administered in the morning or late afternoon. LH concentrations and Lhβ mRNA expression did not differ between morning RFRP-3-and saline-treated groups, although they were markedly suppressed by RFRP-3 administration in the afternoon. However, RFRP-3 inhibition of circulating LH at the time of the surge does not appear to act via the GnRH system because no differences in medial preoptic area Gnrh or RFRP-3 receptor Gpr147 mRNA expression were observed. Rather, RFRP-3 suppressed arcuate nucleus Kiss1 mRNA expression and potentially impacted pituitary gonadotrophs directly. Taken together, these findings reveal time-dependent responsiveness of the reproductive axis to RFRP-3 inhibition, possibly via variation in the sensitivity of arcuate nucleus kisspeptin neurones to this neuropeptide.

The effects of supraphysiological levels of testosterone on neural networks upstream of gonadotropin-releasing hormone neurons.

Objective(s):Several pathological conditions are associated with hyper-production of testosterone; however, its impacts are not well understood. Hence, we evaluated the effects of supraphysiological levels of testosterone on gonadotropin-releasing hormone (GnRH) system in the hypothalamus of male rats. Also, we assessed the expression of two excitatory (kisspeptin and neurokinin-B) and two inhibitory (dynorphin and RFamide-related-peptide) neuropeptides upstream of GnRH neurons as possible routes to relay androgen information. Materials and Methods:Gonadectomized (GDX) male rats received single injection of 100, 250 or 500 mg/kg testosterone undecanoate and three weeks later, posterior (PH) and anterior (AH) hypothalamus was dissected for evaluation of target genes using quantitative RT-PCR.Results:We found that GnRH mRNA in the PH was high in GDX rats and 500 mg/kg testosterone reduced GnRH level expression. Finding revealed extremely high level of Kiss1 mRNA in the PH of GDX rats. However, in GDX rats treated with different levels of testosterone, Kiss1 expression was not significantly different than control. We also found that testosterone replacement increased the Kiss1 mRNA level in the AH. Moreover, neurokinin-B mRNA level in PH of GDX rats was similar to control. However, excess testosterone levels were effective in significantly inducing the down-regulation of neurokinin-B expression. The basal level of dynorphin mRNA was increased following testosterone treatments in the AH, where we found no significant difference in the level of RFamide-related-peptide mRNA between the experimental groups. Conclusion:Excess levels of testosterone could act differently from its physiological concentration to regulate hypothalamic androgen sensitive neurons to control GnRH cell.

Exogenous kisspeptin (kp-10) resumes cyclicity in postpartum anestrus mithun cows

Present study was conducted to assess the effect of exogenous kisspeptin administration on estrus induction, Kiss1gene expression, endocrinological profiles and follicular development in post partum (PP) anestrus mithun cows. Animals who failed to resume cyclicity after day 90–100 of parturition were selected. Experimental cows were examined thoroughly through per-rectal as well as ultrasonograpic examination to confirm the anestrous status. PP anestrous healthy mithun cows (12), aged 5–6 years (body condition score: 5–6 of 10) were selected with uniform anestrus status and randomly divided into 2 groups namely treatment (Gr I) and control (Gr II), each group consist 6 cows. Anestrus cows in Gr 1 were injected with kisspeptin @ 1.30 μg per kg body weight at 3 day interval for 21 days after 90–100 days of post parturition whereas in control group, normal saline was injected as placebo. Blood samples were collected on the days of injection and at estrus in both the groups. Trans-rectal ultrasonography was done at 3 days interval till onset of estrus and ovulation to study the follicular development. Exogenous kisspeptin administration caused significantly early resumption of cyclicity in treatment as compared to control (24.64±10.43 vs 66.56±14.66 days) and significantly increased kiss1 and GPR54 mRNA expression in treatment as compared to control on the day of estrus (1.943±0.29 vs 0.424±0.062 and 1.84±0.31 vs 0.416±0.082, respectively). Similarly, circulating level of estradiol and follicle stimulating hormone (FSH) increased gradually after exogenous administration of kisspeptin, which reached peak on the day of estrus in treatment group (25.36±1.27 pg/ml and 15.65±1.22 ng/ml, respectively) whereas no significant difference was observed between days of treatment in control group except on the day of estrus (11.29±1.76 pg/ml and 9.86±1.06 ng/ml, respectively). Level of estradiol and FSH on the day of estrus was significantly higher in treatment as compared to control whereas non-significant difference was observed in plasma progesterone concentration. Number of medium and large follicles increased in treatment whereas only small follicles were observed in control group. Improved endocrinological profiles, follicle development and kiss gene profiles in post partum anestrous mithun cows following exogenous kisspeptin indicates that kisspeptin inducted or resumed the cyclicity in early.

Androgen signaling in male fishes: Examples of anti-androgenic chemicals that cause reproductive disorders

Similar to other vertebrates, androgens regulate spermatogenesis in fishes. In teleosts, the main androgen is 11-Ketotestosterone (11-KT), which is oxidized testosterone (T) at the C11 position. Compared to T, 11-KT is a nonaromatizable steroid, and does not convert to 17β-estradiol. However, circulatory levels of both T and 11-KT undergo seasonal variations along with testicular development. Physiological functions of androgens are mediated via androgen receptor (Ar). So far, nuclear Ar and membrane Ar have been identified in fishes. In the present study, we reviewed androgen biosynthesis in fishes, compared molecular structure of nuclear Ar in models of mammals and fishes, and investigated the mechanisms of action of environmental contaminants that differentially disrupt androgen signaling in fish reproduction. In the latter case, the adverse effects of vinclozolin (VZ) and bis(2-ethylhexyl) phthalate (DEHP) are compared. Both VZ and DEHP are capable of decreasing sperm quality in males. Vinclozolin causes an increase in 11-KT production associated with increases in kisspeptin (kiss-1) and salmon gonadotropin-releasing hormone (gnrh3) mRNA levels as well as circulatory levels of luteinizing hormone (Lh). In contrast, DEHP inhibits 11-KT production associated with a decrease in circulatory Lh levels. However, DEHP-inhibited 11-KT production is not associated with changes in kiss-1 and gnrh3 mRNA levels. Studies also show that VZ alters ar mRNA levels, while DEHP is without effect. These suggest that VZ and DEHP act differentially to cause androgen-dependent reproductive disorder in male fishes. Molecular analyses of the nuclear AR show that both DNA and ligand binding domains (DBD and LBD, respectively) are highly conserved within models of mammals and fishes. A phylogeny tree of the AR shows distinct clusters between mammals and fishes. In fishes, subtypes of Arα and Arβ are also separated in distinct clusters. Thus, further studies need to generate ar knockout fish model to better elucidate androgen regulation of reproduction in fishes via Ar.