Kiss1r mRNA Expression Research Articles

Investigation of subfertility in the female Nsmf knockout mouse

To study if a pituitary or ovarian defect contributes to subfertility of the female Nsmf knockout (KO) mouse, an animal model of the hypogonadotropic hypogonadism gene NSMF. Analysis of hypothalamic, pituitary and ovarian gene expression at baseline, serum gonadotropin levels before and after gonadotropin-releasing hormone (GnRH) stimulation, ovarian response and implantation after superovulation, gonadotropin effects after ovariectomy, and ovarian NSMF protein expression. University research laboratory. None; mice were used. Gonadotropin-releasing hormone stimulation, superovulation, and ovariectomy in separate experiments. Gene expression in the hypothalamus, pituitary, and ovary; ovarian response and implantation after superovulation; serum gonadotropins after GnRH stimulation and ovariectomy; Western blot to measure ovarian NSMF expression. We found increased hypothalamic Kiss1, Gnrh1, and Jak2 mRNA expression in female Nsmf KO vs. wild type (WT) mice. However, pituitary gonadotropin, and GnRH receptor gene expression was not affected, and serum gonadotropin levels were normal. Gonadotropins increased after ovariectomy for both groups. Baseline Kiss1, Fshr, Prkaca, Prkar1a, and Gdf9 ovarian mRNA expression was increased and Cyp19a1 expression was decreased in Nsmf KO mice, while superovulated Nsmf KO mice had reduced ovarian Kiss1r, Prkar1a, and Fshr mRNA expression, 50% less oocytes, and normal implantation. Western blot demonstrated NSMF protein expression in the ovary of WT mice. Altered hypothalamic and ovarian gene expression was demonstrated in female Nsmf KO mice. It is possible that increased hypothalamic Gnrh1 and Kiss1 mRNA expression could compensate for reduced NSMF enabling a normal pituitary gonadotropin response. Impaired superovulation response, altered ovarian gene expression, and decreased number of oocytes indicate ovarian dysfunction, but a uterine factor cannot be excluded. These findings provide an anatomic basis for future mechanistic studies of subfertility in female Nsmf KO mice.

Hesperidin Mitigates Cyclophosphamide-Induced Testicular Dysfunction via Altering the Hypothalamic Pituitary Gonadal Axis and Testicular Steroidogenesis, Inflammation, and Apoptosis in Male Rats.

Cyclophosphamide (CP) is a cytotoxic, cell cycle, non-specific, and antiproliferative drug. This study aimed to address the toxic effects of CP on male fertility and the possible ameliorative role of hesperidin (HSP). Thirty-two adult albino rats were randomly divided into four groups, namely, the negative control, HSP, CP-treated, and CP+HSP-treated groups. The CP-treated rats showed a significant reduction in the levels of serum LH, FSH, testosterone, prolactin, testicular glutathione peroxidase (GPx), and total antioxidant capacity (TAC) with an elevation in levels of malondialdehyde (MDA), and p53, and iNOS immune expression, compared to the control group. A significant downregulation in hypothalamic KISS-1, KISS-1r, and GnRH, hypophyseal GnRHr, and testicular mRNA expression of steroidogenesis enzymes, PGC-1α, PPAR-1, IL10, and GLP-1, as well as a significant upregulation in testicular mRNA of P53 and IL1β mRNA expression, were detected in the CP-treated group in comparison to that in the control group. The administration of HSP in CP-treated rats significantly improved the levels of serum LH, FSH, testosterone, prolactin, testicular GPx, and TAC, with a reduction in levels of MDA, and p53, and iNOS immune expression compared to the CP-treated group. A significant upregulation in hypophyseal GnRHr, and testicular mRNA expression of CYP19A1 enzymes, PPAR-1, IL10, and GLP-1, as well as a significant downregulation in testicular mRNA of P53 and IL1β mRNA expression, were detected in the CP+HSP-treated group in comparison to that in the CP-treated group. In conclusion, HSP could be a potential auxiliary agent for protection from the development of male infertility.

Effects of intracerebroventricular and intravenous administration of Kisspeptin-54 and Gonadotropin-releasing hormone agonist in rats with ovarian hyperstimulation

PurposeWe aim to determine the effect of local and systemic administration of kisspeptin-54 on ovarian hyperstimulation. MethodsImmature female rats were used. In order to generate the ovarian hyperstimulation model, 50 IU PMSG was administered for 4 consecutive days and a single dose of 25 IU hCG was administered to all groups except for the sham group. To synchronize the sham group, a single dose of 10 IU PMSG followed by 10 IU hCG (48 h later) was applied. Kisspeptin-54 and gonadotropin-releasing hormone (GnRH) agonists were administered 48 h after hCG injection. While intracerebroventricular injection is performed with stereotaxic surgery, Intravenous administration was from the tail vein. Ovarian weights were measured. FSH, LH, estrogen and progesterone hormones were detected in serum by ELISA. VEGFa, IL-1β, TNF-α, MCP-1 immunohistochemical staining was performed on the ovaries and hypothalamus and their optical densities were determined with Image J. Kiss1R mRNA expression was determined by qRT-PCR. ResultsOvarian weights increased significantly in the OHSS group and the systemic GnRH agonist group. The optical densities of VEGFa, IL-1β, TNF- α and MCP-1 immunoreactivity showed us that both local and systemic applied kisspeptin-54 attenuates the level of investigated inflammation parameters in the ovaries. Moreover, local administration of kisspeptin-54 has been shown to enhance the level of Kiss1R mRNA in both the ovaries and the hypothalamus. Conclusion(s)Local and systemic administration of Kisspeptin-54 as a post-treatment reduces inflammation parameters in the ovaries. These findings promote the potential use of kisspeptin-54 on OHSS.

Comprehensive RNA-seq reveals molecular changes in kidney malignancy among people living with HIV

To heighten the awareness of kidney malignancy in patients with HIV infection to facilitate the early diagnosis of kidney cancer, the differentially expressed mRNAs were analyzed in this malignant tumor using RNA sequencing. We identified 2,962 protein-coding transcripts in HIV-associated kidney cancer. KISS1R, CAIX, and NPTX2 mRNA expression levels were specifically increased in HIV-associated kidney cancer while UMOD and TMEM213 mRNA were decreased in most cases based on real-time PCR analyses. These findings were similar to those noted for the general population with renal cell carcinoma. Immunohistochemical staining analysis also showed that a total of 18 malignant kidney cases among the people living with HIV (PLWH) exhibited positive staining for KISS1R and CAIX. Pathway analysis of the differentially expressed mRNAs in HIV-associated kidney cancer revealed that several key pathways were involved, including vascular endothelial growth factor-activated receptor activity, IgG binding, and lipopolysaccharide receptor activity. Altogether, our findings reveal the identified molecular changes in kidney malignancy, which may offer a helpful explanation for cancer progression and open up new therapeutic avenues that may decrease mortality after a cancer diagnosis among PLWH.

Comprehensive RNA-Seq Reveals Molecular Changes in Kidney Malignancy Among People Living With HIV

Background: Malignancy of the kidney is a rapidly progressive kidney disease and a major source of morbidity and mortality among people living with HIV (PLWH). Patients with HIV-associated kidey cancer experience higher cancer-specific mortality than the general population,and its mechanism remains poorly understood. Methods: To heighten the awareness of kidney malignancy in patients with HIV infection to facilitate the early diagnosis of kidney cancer, we identified 2460 protein-coding transcripts in HIV-associated kidney cancer using comprehensive RNA sequencing (RNA-seq). Results:KISS1R, CAIX,and NPTX2 mRNA expression levels were specifically increased in HIV-associated kidney cancer, and UMOD and TMEM213 mRNA were decreased in most cases based on real-time PCR analyses. These findings were similar to those noted for the general population with renal cell carcinoma. Immunohistochemical staining analysis also showed that a total of 16 of 18 kidney malignant cases among PLWH exhibited positive staining for KISS1R and CAIX. Conclusion: The identified molecular changes in kidney malignancy may offer a helpful explanation for cancer progression and open up new therapeutic avenues that may decrease mortality after a cancer diagnosis among PLWH.

Role of the Kisspeptin/KISS1 Receptor System in the Testicular Development of Mice.

Kisspeptin and its receptor KISS1R have been found to be essential regulators of reproductive function. Previous data have revealed the presence of Kiss1 and Kiss1r mRNAs in the hypothalamus and the testis of humans and rodents. However, the precise location and possible physiological role of the kisspeptin/KISS1R system in the testis remain ambiguous. We first produced an anti-KISS1R immunoglobulin Y antibody for KISS1R identification. To detect the exact sites of KISS1R and kisspeptin expression in the testis, we conducted immunohistochemistry assays on sections of testes. We used real-time polymerase chain reactions (PCR) to identify Kiss1r in mice and to determine the expression levels of testicular genes. Finally, to verify the upstream regulation on the Kisspeptin/KISS1 receptor system, we treated primary mouse Leydig cells and MA-10 cells with luteinizing hormone (LH) and Br-cAMP, respectively and examined Kiss1 and Kiss1r mRNA expression. Immunohistochemistry assays revealed that kisspeptin was expressed in Leydig cells and KISS1R was localized in the seminiferous tubules. With real-time PCR, we found Kiss1r mRNA was constitutively expressed in the mouse testis from birth until the postnatal fourth week. Furthermore, mRNA expression of Kiss1 was synchronized with that of Insl3 and Cyp19a. However, the expression of the LH receptor-encoding gene increased 1 week earlier than did Kiss1 expression. This indicated that the kisspeptin/KISS1R system in the testis may be controlled by LH and cAMP signaling pathways. Finally, we confirmed that Kiss1 mRNA expression was increased in both LH-treated primary Leydig cells and Br-cAMP-treated MA-10 cells (p < 0.05). On the other hand, cotreatment of both cell lines with Br-cAMP and a protein kinase A inhibitor RP-cAMP significantly suppressed 50% of Br-cAMP-induced Kiss1 expression (p < 0.05). We discovered that Kiss1 expression in mouse Leydig cells was induced by LH through the cAMP/PKA pathway. Based on the presence of kisspeptin receptors on spermatids, we inferred that kisspeptin and development-related factors have synergistic effects on spermatogenesis. Nevertheless, more studies are required to elaborate the role of the kisspeptin/KISS1R system in testicular development.

Ghrelin antagonist overrides the mRNA expression of NPY in hypothalamus in feed restricted ewes.

A negative energy balance (NEB) is detrimental to reproduction in animals. A suggested link between NEB and reproductive failure is the gastrointestinal hormone ghrelin, because of the association between ghrelin and the hypothalamo-pituitary-gonadal axis. The [D-Lys3]-Growth Hormone Releasing Peptide-6 ([D-Lys3]-GHRP-6) is a ghrelin antagonist that acts on ghrelin receptors (GHS-R1). The objective of this study was to evaluate the effect of [D-Lys3]-GHRP-6 on reproduction variables in feed restricted ewes. Two experiments were conducted. Experiment I was conducted for 30 days; and Experiment II for 13 days. In both experiments the ewes (n = 18) were randomly assigned to: Control (CO): fed to meet maintenance requirements; Feed restriction (FR): 80% of maintenance restriction; or Ghrelin antagonist (GA): feed restricted and daily subcutaneous of 7.5μg/kg of [D-Lys3]-GHRP-6. Plasma was collected to measure hormones and metabolite concentration. In Experiment II, the hypothalamus and ovaries were collected on day 13. In both Experiments, sheep allocated to the FR and GA treatments decreased their body weight compared with sheep in the CO group (P < 0.06); progesterone however, did not differ between treatments (P > 0.10). Experiment I: Plasma ghrelin concentration was greater (P < 0.01) in FR and GA compared with CO ewes. Plasma non-esterified fatty acids concentration was greater (P < 0.01) in GA and FR than CO. Experiment II: Kisspeptin1-Receptor (Kiss1-R) mRNA expression was greater in FR (P < 0.01) and tended to be greater in GA (P = 0.10) compared with CO ewes. The neuro peptide-Y (NPY) mRNA expression was greater (P = 0.03) in FR than CO; and tended to be greater (P = 0.06) compared with GA ewes. Growth hormone releasing hormone (GhRH) mRNA expression was greater in GA (P = 0.04) and tended to be greater in FR (P = 0.07) compared with CO ewes. Feed restriction increased GhRH, NPY, and Kiss-R mRNA expression in hypothalamus without affecting reproductive variables.Ghrelin antagonist may prevent an increase inNPY expression in ewes.

Role of adiponectin in ovarian follicular development and ovarian reserve.

Adiponectin levels are associated with anti-Müllerian hormone (AMH) and kisspeptin levels in non-ovarian tissues. The objective of the present study was to investigate the association between adiponectin and the genes important for ovarian follicular development and ovarian reserve, specifically AMH and kisspeptin, and their corresponding receptors. In the first experiment, the mRNA levels of anti-Müllerian hormone (amh) and its receptor (Amhr2), as well as those of kisspeptin (Kiss1) and its receptor (Kiss1r), were quantified by reverse transcription-polymerase chain reaction analysis in the ovaries of two groups of mice [adiponectin-knockout (KO) vs. control] that underwent oophorectomy. The second experiment measured follicular phase serum AMH and follicular fluid adiponectin levels in 25 women who underwent controlled ovarian hyperstimulation for in vitro fertilization. Compared with the control mice, adiponectin-KO mice had 6.5 times lower Kiss1 mRNA levels (P=0.009) and a tendency for lower ovarian Kiss1r mRNA expression levels (P=0.06). However, adiponectin-KO mice had significantly higher Amhr2 mRNA levels (P=0.01). In all women participants, there was a positive correlation between serum AMH and follicular fluid adiponectin concentrations (r=0.54, P=0.006). The findings of the present study indicate that adiponectin may play a role in ovarian physiology through its impact on genes crucial for ovarian follicular development and ovarian reserve, such as kisspeptin and AMH. Understanding the role of adiponectin in ovarian function may improve our knowledge of the pathophysiology underlying ovulatory dysfunction in obese women, who usually have low adiponectin levels, and overcome reproductive barriers.

Region-specific changes in brain kisspeptin receptor expression during estrogen depletion and the estrous cycle.

Kisspeptin acts as a potent neuropeptide regulator of reproduction through modulation of the hypothalamic-pituitary-gonadal axis. Previous studies revealed sex differences in brain expression patterns as well as regulation of expression by estrogen. Alternatively, sex differences and estrogen regulation of the kisspeptin receptor (encoded by Kiss1r) have not been examined at cellular resolution. In the current study, we examined whether Kiss1r mRNA expression also exhibits estrogen sensitivity and sex-dependent differences using in situ hybridization. We compared Kiss1r mRNA expression between ovariectomized (OVX) rats and estradiol (E2)-replenished OVX rats to examine estrogen sensitivity, and compared expression between gonadally intact male rats and female rats in diestrus or proestrus to examine sex differences. In OVX rats, E2 replenishment significantly reduced Kiss1r expression specifically in the hypothalamic arcuate nucleus (ARC). A difference in Kiss1r expression was also observed between diestrus and proestrus rats in the hypothalamic paraventricular nucleus (PVN), but not in the ARC. Thus, estrogen appears to have region- and context-specific effects on Kiss1r expression. However, immunostaining revealed minimal colocalization of estrogen receptor alpha (ERα) in Kiss1r-expressing neuronal populations of ARC and PVN, indicating indirect or ERα-independent regulation of Kiss1r expression. Surprisingly, unlike the kisspeptin ligand, no sexual dimorphisms were observed in either the brain distribution of Kiss1r expression or in the number of Kiss1r-expressing neurons within enriched brain nuclei. The current study reveals marked differences in regulation between kisspeptin and kisspeptin receptor, and provides an essential foundation for further study of kisspeptin signaling and function in reproduction.

Role of kisspeptin and Kiss1R in the regulation of prolactin gene expression in rat somatolactotroph GH3 cells.

Hypothalamic kisspeptin is a known principal activator of gonadotropin-releasing hormone neurons and governs the hypothalamic-pituitary-gonadal axis. Previous reports have shown that kisspeptin is also released into the hypophyseal portal circulation and directly affects the anterior pituitary. In this study, we examined the direct effect of kisspeptin on pituitary prolactin-producing cells. The rat pituitary somatolactotroph cell line GH3 expresses the kisspeptin receptor (Kiss1R); however, in these cells, kisspeptin failed to stimulate prolactin-promoter activity. When GH3 cells overexpressed Kiss1R, kisspeptin clearly increased prolactin-promoter activity, with a concomitant increase in extracellular signal-regulated kinase (ERK) and cAMP/protein kinase A (PKA) signaling pathways. In the experiments using GH3 cells overexpressing Kiss1R, kisspeptin did not potentiate thyrotropin-releasing hormone (TRH)-induced prolactin-promoter activity, but it potentiated the pituitary adenylate cyclase-activating polypeptide-induced prolactin-promoter activity, with a concomitant enhancement of ERK and PKA signaling pathways. Although the basal and TRH-induced prolactin-promoter activities were not modulated by increasing amounts of Kiss1R expression in GH3 cells, kisspeptin-stimulated prolactin-promoter activity was increased by the amount of Kiss1R overexpression. Endogenous Kiss1r mRNA expression in GH3 cells was significantly increased by treatment with estradiol (E2) but not by TRH. In addition, kisspeptin's ability to stimulate prolactin-promoter activity was restored after E2 treatment in non-transfected GH3 cells. Our current observations suggest that kisspeptin might have a direct effect on prolactin expression in the anterior pituitary prolactin-producing cells under the influence of E2, which may regulate Kiss1R expression and function.

Development of an excitatory kisspeptin projection to the oxytocin system in late pregnancy.

Oxytocin release from the posterior pituitary gland stimulates uterine contraction during birth but the central mechanisms that activate oxytocin neurones for birth are not well characterized. We found that that kisspeptin fibre density around oxytocin neurones increases in late-pregnant rats. These kisspeptin fibres originated from hypothalamic periventricular nucleus neurones that upregulated kisspeptin expression in late pregnancy. Oxytocin neurones were excited by central kisspeptin administration in late-pregnant rats but not in non-pregnant rats or early- to mid-pregnant rats. Our results reveal the emergence of a new excitatory kisspeptin projection to the oxytocin system in late pregnancy that might contribute to oxytocin neurone activation for birth. The hormone oxytocin promotes uterine contraction during parturition. Oxytocin is synthesized by magnocellular neurones in the hypothalamic supraoptic and paraventricular nuclei and is released into the circulation from the posterior pituitary gland in response to action potential firing. Systemic kisspeptin administration increases oxytocin neurone activity to elevate plasma oxytocin levels. Here, immunohistochemistry revealed that rats on the expected day of parturition (day21 of gestation) had a higher density of kisspeptin-positive fibres in the perinuclear zone surrounding the supraoptic nucleus (which provides dense glutamatergic and GABAergic innervation to the supraoptic nucleus) than was evident in non-pregnant rats. Retrograde tracing showed the kisspeptin projections to the perinuclear zone originated from the hypothalamic periventricular nucleus. Quantitative RT-PCR showed that kisspeptin receptor mRNA, Kiss1R mRNA, was expressed in the perinuclear zone-supraoptic nucleus and that the relative Kiss1R mRNA expression does not change over the course of pregnancy. Finally, intracerebroventricular administration of kisspeptin increased the firing rate of oxytocin neurones in anaesthetized late-pregnant rats (days 18-21 of gestation) but not in non-pregnant rats, or in early- or mid-pregnant rats. Taken together, these results suggest that kisspeptin expression is upregulated in the periventricular nucleus projection to the perinuclear zone of the supraoptic nucleus towards the end of pregnancy. Hence, this input might activate oxytocin neurones during parturition.

Diurnal regulation of hypothalamic kisspeptin is disrupted during mouse pregnancy.

Kisspeptin, the neuropeptide product of the Kiss1 gene, is critical in driving the hypothalamic-pituitary-gonadal (HPG) axis. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (Arc) of the hypothalamus mediate differential effects, with the Arc regulating negative feedback of sex steroids and the AVPV regulating positive feedback, vital for the preovulatory surge and gated under circadian control. We aimed to characterize hypothalamic Kiss1 and Kiss1r mRNA expression in nonpregnant and pregnant mice, and investigate potential circadian regulation. Anterior and posterior hypothalami were collected from C57BL/6J mice at diestrus, proestrus, and days 6, 10, 14, and 18 of pregnancy, at six time points across 24h, for real-time PCR analysis of gene expression. Analysis confirmed that Kiss1 mRNA expression in the AVPV increased at ZT13 during proestrus, with a luteinizing hormone surge observed thereafter. No diurnal regulation was seen at diestrus or at any stage of pregnancy. Anterior hypothalamic Avp mRNA expression exhibited no diurnal variation, but Avpr1a peaked at 12:00h during proestrus, possibly reflecting the circadian input from the suprachiasmatic nucleus to AVPV Kiss1 neurons. Rfrp (Npvf) expression in the posterior hypothalamus did not demonstrate diurnal variation at any stage. Clock genes Bmal1 and Rev-erbα were strongly diurnal, but there was little change between diestrus/proestrus and pregnancy. Our data indicate the absence of the circadian input to Kiss1 in pregnancy, despite high gestational estradiol levels and normal clock gene expression, and may suggest a disruption of a kisspeptin-specific diurnal rhythm that operates in the nonpregnant state.

The suppressive effect of immune stress on LH secretion is absent in the early neonatal period in rats

Some physiological functions display weak responses to stress in the early neonatal period; i.e., they exhibit stress hyporesponse periods. In this study, we evaluated whether gonadotropin regulatory factors exhibit stress hyporesponsive periods in male and female rats. Rats were intraperitoneally injected with lipopolysaccharide (100μg/kg) (LPS group) or saline (control group) on postnatal day (PND) 5, 10, 15, or 25. Then, their serum luteinizing hormone (LH) concentrations and hypothalamic mRNA levels of gonadotropin regulatory factors; i.e., kisspeptin (Kiss1), the kisspeptin receptor (Kiss1r), and gonadotropin-releasing hormone (GnRH), were measured at 2h after the injection. The hypothalamic mRNA levels of pro-inflammatory cytokines were also measured because they suppress gonadotropin secretion. The serum LH concentration of the LPS group was lower than that of the control group at PND25 in both sexes, but no such difference was seen at PND5, 10, or 15 in either sex. In both sexes, the hypothalamic tumor necrosis factor (TNF)α and interleukin (IL)-6 mRNA expression levels of the LPS group were higher than those of the control group at PND25, but not at PND5 or 10. The hypothalamic IL-1β mRNA expression level of the LPS group was higher than that of the control group at all time points. The hypothalamic Kiss1, Kiss1r, and GnRH mRNA expression levels of the LPS and control groups did not differ at any time point in either sex. These findings suggest that gonadotropin regulatory factors exhibit stress hyporesponse periods. The hypothalamic–pituitary–gonadal axis (HPG) might become responsive to immune stress between PND15 and 25, which could be related to enhanced hypothalamic cytokine expression. The avoidance of infectious stress during the early neonatal period might be important for normal development of the HPG axis.

Fasting induced kisspeptin signaling suppression is regulated by glutamate mediated cues in adult male rhesus macaque (Macaca mulatta)

Kisspeptin signaling is suppressed by short term fasting. It has been reported that hypothalamic Kiss1 and Kiss1r mRNA expression decreased after 48h of fasting in male rhesus monkey. But the mechanism involved in the reduction of kisspeptin signaling after 48h of fasting is unknown. Recent studies have suggested the role of afferent excitatory and inhibitory pathways in the regulation of kisspeptin neurons. Therefore, this study was designed to observe the changes in the glutamate and GABA signaling during fed and 48h fasting states by performing immunofluorescence to examine the interaction of kisspeptin neurons with NR1 subunit of NMDA receptors and by performing SYBR green qRT-PCR to measure and quantify the levels of Kiss1, Kiss1r, NR1 and GAD67 mRNA in the POA and MBH of adult male rhesus macaque (Macaca mulatta) during 48h of fasting (n=2) and fed ad libitum (n=2). Plasma testosterone (p<0.05) and blood glucose levels were significantly (p<0.001) decreased after short term fasting. Our results clearly showed that expression of hypothalamic Kiss1, Kiss1r and NR1 mRNA was significantly (p<0.05) reduced in adult male rhesus monkeys which were fasted for 48h as compared to those which were fed ad libitum. There was no clear difference in the GAD67 mRNA contents between the two groups. Number of kisspeptin neurons and the interactions of kisspeptin neurons with NR1 were significantly (p<0.05) reduced after 48h fasting. These observations suggest that decreased kisspeptin signaling during fasting may occur due to reduction in glutamatergic inputs to kisspeptin neurons. Our results also suggest that fasting induced suppression of kisspeptin signaling is not mediated through GABAergic neurons.

Effects of kisspeptin-10 on in vitro proliferation and kisspeptin receptor expression in primary epithelial cell cultures isolated from bovine placental cotyledons of fetuses at the first trimester of pregnancy

Kisspeptin (Kp) and Kiss-1 receptor (Kiss-1R) expressions have been reported to be in the placenta, and a possible involvement of the Kiss-1R/Kps system in regulating trophoblast invasion and proliferation has been hypothesized. The aim of the present study was to investigate whether Kiss-1R activation by kisspeptin-10 (Kp-10) could modulate in vitro proliferation and progesterone (P4) secretion of bovine primary placental cell lines isolated from cotyledons of fetuses in the first trimester of pregnancy. The involvement of Kiss-1R in the cell responses observed was also analyzed. Uteri from cows at the first trimester of pregnancy were obtained from local abattoirs. Fetal cotyledon fragments were digested with collagenase in low glucose Dulbecco's Modified Eagle's Medium and cell lines were isolated. After being characterized for epithelial polygonal morphology, the presence of binucleate cells, male gender, and the expression of cytokeratin and zona occludens 2, cell lines were cultured in a low glucose Dulbecco's Modified Eagle's Medium–based expansion medium in the presence of 0.01, 0.1, 1, and 10 μM Kp-10. Control cells were cultured in the absence of Kp-10. Cell population doubling time was evaluated for each culture passage (P) from P1 to P10. Cells were tested for Kiss-1R mRNA expression analysis by real-time reverse transcription–polymerase chain reaction, and culture media were analyzed for P4 concentration by radioimmunoassay. Kisspeptin-10 modulated in vitro proliferation of epithelial cell lines isolated from cotyledons recovered from bovine fetuses in the first trimester of pregnancy. Inhibitory (line A) or stimulatory (line B) effects of Kp-10 on cell proliferation were found in different cell lines and observed cell responses were found to be related to Kiss-1R mRNA levels. Inhibition of cell proliferation matched with not significant variation of Kiss-1R expression, whereas stimulation of cell proliferation was found to be related to Kiss-1R upregulation. In both cell lines, no effect of Kp-10 on P4 secretion was found at any tested concentration. These results lead to the conclusion that the Kiss-1R/Kps system is involved in the regulation of cell proliferation of bovine placental cotyledon cell lines isolated at the first trimester of pregnancy but, at this gestational stage, it may not be involved in modulating placental P4 secretion.

Kisspeptin receptor haplo-insufficiency causes premature ovarian failure despite preserved gonadotropin secretion.

Premature ovarian failure (POF) affects 1% of women in reproductive age, but its etiology remains uncertain. Whereas kisspeptins, the products of Kiss1 that act via Kiss1r (aka, Gpr54), are known to operate at the hypothalamus to control GnRH/gonadotropin secretion, additional actions at other reproductive organs, including the ovary, have been proposed. Yet, their physiological relevance is still unclear. We present here a series of studies in Kiss1r haplo-insufficient and null mice suggesting a direct role of kisspeptin signaling in the ovary, the defect of which precipitates a state of primary POF. Kiss1r hypomorph mice displayed a premature decline in ovulatory rate, followed by progressive loss of antral follicles, oocyte loss, and a reduction in all categories of preantral follicles. These alterations were accompanied by reduced fertility. Because of this precocious ovarian ageing, mice more than 48 weeks of age showed atrophic ovaries, lacking growing follicles and corpora lutea. This phenomenon was associated with a drop in ovarian Kiss1r mRNA expression, but took place in the absence of a decrease in circulating gonadotropins. In fact, FSH levels increased in aged hypomorph animals, reflecting loss of follicular function. In turn, Kiss1r-null mice, which do not spontaneously ovulate and have arrested follicular development, failed to show normal ovulatory responses to standard gonadotropin priming and required GnRH prestimulation during 1 week in order to display gonadotropin-induced ovulation. Yet, the magnitude of such ovulatory responses was approximately half of that seen in control immature wild-type animals. Altogether, our data are the first to demonstrate that Kiss1r haplo-insufficiency induces a state of POF, which is not attributable to defective gonadotropin secretion. We also show that the failure of follicular development and ovulation linked to the absence of Kiss1r cannot be fully rescued by (even extended) gonadotropin replacement. These findings suggest a direct ovarian role of kisspeptin signaling, the perturbation of which may contribute to the pathogenesis of POF.

Kiss1 and Kiss1r mRNA expression in the rat placenta: Changes with gestational age and regulation by glucocorticoids

IntroductionKisspeptin, the neuropeptide product of the KISS1 gene, is synthesized by neurons within the hypothalamus and is critical for fertility. Human placenta also expresses KISS1 and kisspeptin receptor (KISS1R) mRNA within the trophoblast compartment, where it is thought to act as a physiological invasion inhibitor. MethodsWe determined the expression of Kiss1 mRNA in rat placenta and examined the effect of gestational age and feto-placental growth restriction, achieved through excess maternal glucocorticoid exposure. ResultsDexamethasone induced fetal growth restriction at both day 16 and day 22 of gestation, but placental growth restriction only at day 22. Real-time quantitative RT-PCR revealed an increase in Kiss1 and Kiss1r mRNA from day 16–22 in the labyrinth and junctional zones of the rat placenta. Immunolocalization confirmed kisspeptin expression in the placenta and was prominent in trophoblast tissue. Dexamethasone exposure elevated the expression of Kiss1 mRNA in the labyrinth and junctional zones of day 16 placentas. In contrast, Kiss1 mRNA in the labyrinth zone was reduced following dexamethasone-treatment at day 22. Kiss1r expression was increased in both placental zones at day 16 and 22 in response to dexamethasone-treatment. ConclusionsWe confirm the presence of Kiss1 and Kiss1r mRNA in the rat placenta with expression increasing over the final third of pregnancy, suggestive of a role in restricting placental growth. Furthermore, the effects of dexamethasone on placental Kiss1/Kiss1r suggest glucocorticoid-induced placental growth retardation could be mediated, in part, via early stimulation of Kiss1 and the subsequent inhibition of trophoblast proliferation and invasion.

Kisspeptin Is Essential for the Full Preovulatory LH Surge and Stimulates GnRH Release from the Isolated Ovine Median Eminence

Kisspeptins are the product of the Kiss1 gene and potently stimulate GnRH secretion. In sheep, Kiss1 mRNA-expressing cells are found in the arcuate nucleus (ARC) and dorsal-lateral preoptic area and both appear to mediate the positive feedback effect of estradiol to generate the preovulatory GnRH/LH surge. To determine the role of kisspeptin in transmitting estrogen-positive feedback in the hypothalamus, we administered the kisspeptin antagonist p-271 to ewes subjected to an estradiol benzoate-induced LH surge. Kisspeptin antagonist treatment significantly attenuated these LH surges. We further examined the response to kisspeptin treatment prior to the LH surge. Kisspeptin significantly stimulated GnRH secretion into the hypophysial portal system, but the response to kisspeptin was similar in luteal and late-follicular phase ewes. Kiss1r mRNA expression in GnRH neurons was also similar across the estrous cycle. To examine alternative pathways for kisspeptin stimulation of GnRH neurons, we examined the origin of kisspeptin neuronal fibers in the external zone of the median eminence (ME) using neuronal tracing and immunohistochemical techniques. ARC populations of kisspeptin neurons project fibers to the ME. Finally, we showed kisspeptin stimulates GnRH release from ovine ME-cultured explants. This suggests direct kisspeptin to GnRH terminal-to-terminal communication within the ME. Overall, these data indicate an essential role for kisspeptin in receiving stimulatory estrogen signals and generating the full positive feedback GnRH/LH surge. Kisspeptin neurons of the ARC project to the external zone of the ME and kisspeptin acts upon the GnRH fibers at this level.

Fasting reduces the kiss1 mRNA levels in the caudal hypothalamus of gonadally intact adult female rats

Kisspeptin, which is the product of the kiss1 gene and its receptor kiss1r, have emerged as the essential gatekeepers of reproduction. The present study used gonadally intact female rats to evaluate fasting-induced suppression of the KiSS-1 system of anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) under normal physiological conditions. Starting on the day of estrous, one group of rats was subjected to 72 h of food deprivation, while the other group of rats was able to continue feeding ad libitum. The length of the estrous cycle was significantly longer in the food-deprived rats as compared to the feeding rats. At the end of the 72-h food deprivation period, all of the food-deprived rats were at the diestrous phase, with their serum concentrations of LH and leptin significantly lower than that observed in the feeding rats. In addition, as compared to the feeding rats, the expression levels of kiss1 mRNA were significantly lower in the food-deprived rats in the posterior hypothalamic block, which contained the ARC, but not in the anterior hypothalamic block, which contain the AVPV. However, both the kiss1r mRNA expression levels in the anterior and posterior hypothalamic blocks and the neurokinin B and neurokinin 3 receptor mRNA expression levels in the posterior hypothalamic block were not significantly different between the feeding and food-deprived rats. Thus, lower kiss1 mRNA levels in the ARC appear to be responsible for the fasting-induced inhibition of gonadotrophin secretion and subsequent prolongation of the estrous cycle.

Sensitivities of mRNA expression levels of Kiss1 and its receptor, Kiss1r, to nutritional status are changed during the developmental period in female rats

Decreased activity of kisspeptin, the product of the hypothalamic Kiss1 gene, is the major cause of the suppression of reproductive function in subnutritional conditions. The sensitivities of the endocrine and the hypothalamic neuronal systems to nutritional status develop during the neonatal period. We examined the developmental changes in the sensitivity of hypothalamic mRNA expression of Kiss1 and its receptor, Kiss1r, to nutritional status in female rats. Kiss1 mRNA expression was reduced by 24 h food deprivation (24 h FD) at postnatal day 25, but not at postnatal day 5 or 15. Kiss1r mRNA expression was reduced by the 12 or 24 h FD at postnatal days 5 and 25, but not at postnatal day 15. Kiss1r mRNA level was found to be correlated with the plasma leptin level, and the administration of leptin, which increased the serum leptin concentration above the physiological range, restored the acute FD-induced suppression of Kiss1r mRNA expression. These data suggest that the hypothalamic Kiss1 and Kiss1r mRNA expression is differentially affected by the nutritional condition at different age points. It is speculated that the sensitivity of Kiss1 mRNA, which is expressed in kisspeptin neuron, to nutritional status develops during the neonatal period. On the other hand, it seems that the sensitivity of Kiss1r mRNA, which is expressed in GnRH neuron, to nutritional status has been already established during the early neonatal period. These data also show that hypoleptinemia plays a role in the reduction of hypothalamic Kiss1r mRNA expression under subnutritional conditions.