Hypothalamic Kiss1 mRNA Research Articles

Effect of nourishing and purging fire Chinese herbal mixture on delaying light-induced premature puberty in rats.

To elucidate the mechanism of the nourishing Yin and purging fire Chinese herbal mixture (NYPF) in delaying light-induced premature puberty in rats. Twenty-one days old female Sprague-Dawley rats were randomly assigned to normal group (N), long light exposure group (L), NYPF and normal saline group (NS). Rats in the L, NYPF and NS groups were exposed to 16 h: 350 lux light/8 h: dark, while rats in the N group were exposed to 12 h: 50 lux light/12 h: dark. NYPF and normal saline was administered to the rats in the NYPF group or NS group, respectively, from day 21. Five rats in every group were sacrificed at 9 p.m. on day 28 (P28), on the day when rat's vulva opened in the L group (L-VO), on the day when the first estrous interphase occurred in rats of L group (L-E1), and on the day when the second estrous interphase occurred in rats of L group (L-E2), respectively. On day 34, all rats in the L group, 80% of rats in the NS group, 40% of rats in the N group, and 20% of rats in the NYPF group showed complete opening of the vulva. At P28, mRNA level of hypothalamic kisspeptin (Kiss-1) in the L group was significantly higher than that in the N group (P < 0.05). The rats in the L and NS groups had significantly lower hypothalamic arginine-phenylalanine-amide (RFamide)-related peptide 3 (RFRP-3) mRNA levels than those in the N group (P < 0.05), whereas RFRP-3 mRNA level was significantly higher in the NYPF group than that in the L group (P < 0.05). At L-VO, the ovarian index of the L and NS groups was significantly higher than that of the N group (P < 0.05) and estradiol (E2) level of the NYPF group was significantly lower than that of the N and NS groups (P < 0.05); hypothalamic Kiss-1 mRNA level in the L and NS groups was significantly higher than that in the N and NYPF groups (P < 0.05), whereas hypothalamic RFRP-3 mRNA level in the L, NYPF, and NS groups was significantly lower than that in the N group (P < 0.05). At L-E1, E2 level of the L and NS groups was significantly higher than that of the N group (P < 0.01), whereas it was significantly lower in the NYPF group than that of the N, L, and NS groups (P < 0.01), and serum luteinizing hormone level of the L and NS groups was significantly higher than that of the N group (P < 0.05); levels of serum melatonin and ovarian melatonin receptor 1 (MT-1) mRNA in the L, NYPF, and NS groups were significantly lower than those in the N group (P < 0.05). At L-E2, the uterine organ index of the NYPF group was significantly lower than that of the L group (P < 0.05); and ovarian MT-1 mRNA level of the L and NS groups was significantly lower than that in the N group (P < 0.05). NYPF can delay puberty onset in rats exposed to strong light for a prolonged duration, and regulation of the gene expression of Kiss-1 and RFRP-3 in the hypothalamus has been suggested as one of the mechanisms.

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.

Socs3 ablation in kisspeptin cells partially prevents lipopolysaccharide-induced body weight loss

Many cytokines have been proposed to regulate reproduction due to their actions on hypothalamic kisspeptin cells, the main modulators of gonadotropin-releasing hormone (GnRH) neurons. Hormones such as leptin, prolactin and growth hormone are good examples of cytokines that lead to Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation, consequently exerting effects in kisspeptin neurons. Different studies have investigated how specific components of the JAK/STAT signaling pathway affect the functions of kisspeptin cells, but the role of the suppressor of cytokine signaling 3 (SOCS3) in mediating cytokine actions in kisspeptin cells remains unknown. Cre-Loxp technology was used in the present study to ablate Socs3 expression in kisspeptin cells (Kiss1/Socs3-KO). Then, male and female control and Kiss1/Socs3-KO mice were evaluated for sexual maturation, energy homeostasis features, and fertility. It was found that hypothalamic Kiss1 mRNA expression is significantly downregulated in Kiss1/Socs3-KO mice. Despite reduced hypothalamic Kiss1 mRNA content, these mice did not present any sexual maturation or fertility impairments. Additionally, body weight gain, leptin sensitivity and glucose homeostasis were similar to control mice. Interestingly, Kiss1/Socs3-KO mice were partially protected against lipopolysaccharide (LPS)-induced body weight loss. Our results suggest that Socs3 ablation in kisspeptin cells partially prevents the sickness behavior induced by LPS, suggesting that kisspeptin cells can modulate energy metabolism in mice in certain situations.

Active kisspeptin DNA vaccines oral immunization disrupt mRNA hormone receptors expression in ram lambs

To evaluate the efficacy of oral immunization with active kisspeptin DNA vaccine on the expression of hormone receptor mRNA. For this study, ten 56-day-old Hu breed ram lambs were randomly assigned to the treatment and control groups (n = 5). Treatment Experimental group received C500/pKS-asd and the control group received C500/pVAX-asd (aspartate-β semialdehyde dehydrogenase orally on days 0, 28, and 56, and blood samples were taken at each immunization interval (14-day) and tissues samples were collected at the end of the experimental period (day 98). The collected samples were stored in the refrigerator at −20 °C and liquid nitrogen, respectively, for laboratory examination. Total RNA was extracted from samples using TRIzol reagent and quantitative real-time polymerase chain reaction (QPCR) was used to quantify the levels of KISS1, G protein-coupled receptor-54 (Kiss1r), and gonadotrophin-releasing hormone (GnRH) mRNA in the hypothalamus. Levels of luteinizing hormone receptor (LHR) and luteinizing hormone beta (LHβ) mRNA, and follicle-stimulating hormone receptor (FSHR) and follicle-stimulating hormone beta (FSHβ) mRNA in the testes and pituitary were analyzed, respectively. Further, gonadotropin-releasing hormone receptor (GnRHR) mRNA expression level in the pituitary was measured. Moreover, the Kiss1r concentration level in the blood was measured using an indirect ELISA. The concentration of Kiss1r in the blood was lower in the treatment group than in the control group (p < 0.05). The levels of testicular FSHR and LHR mRNA were significantly lower in the treatment group (p < 0.05) when compared to the control group. Furthermore, the treatment group's levels of hypothalamic KISS1, Kiss1r, and GnRH mRNA were significantly lower (p < 0.05) than the controls. LH, FSH, and GnRHR mRNA expression in the pituitary were also significantly lower in the treatment group (p < 0.01 and p < 0.05, respectively). These findings imply that oral immunization with active kisspeptin DNA vaccine suppresses hormone receptor mRNA expression in the ram lambs.

Effects of Feed Restriction and Fermented Potato Protein Supplementation on IGF1, GHR and Kiss1 mRNA Expression in Intact Nursery Gilts

Background: Decreased feed intake is a common stress response presented after weaning that leads to a negative effect on performances. Health management and feed supplementation were commonly used to solve such problems. The objectives of this research were to study the effects of nutritional stress and the benefit of fermented potato protein supplementation in intact nursery gilts. Methods: Prepubertal nursery gilts were assigned into the following feed treatments for 30 days: 1) normal feed, 2) normal feed with fermented potato protein (FPP) supplementation, 3) restricted feed which received 60% amount of normal feed and 4) restricted feed with FPP supplementation. Result: Serum IGF-1 and IGF1 mRNA expression of semitendinosus muscle were significantly increased by FPP supplementation (P less than 0.05). All pig groups showed no different on serum cortisol and hypothalamic Kiss1 mRNA expression levels. The degree of feed restriction was suggested to be an important determinant of response of serum IGF-1, cortisol and hypothalamic Kiss1 mRNA expression. In this study, an elevated serum IGF-1 concentration correlated with IGF1 mRNA upregulation in a muscle rather than in a liver.

Increased testicular insulin-like growth factor 1 is associated with gonadal activation by recombinant growth hormone in immature rats

BackgroundIn children, recombinant human growth hormone (rhGH) therapy for treatment of short stature has raised concerns of the early onset of puberty. Puberty is initiated by the activation of the hypothalamus-pituitary-gonad axis. Insulin-like growth factor-1 (IGF1) has been known to mediate physiologic effects of GH. To understand the mechanism of precocious sexual maturation following prepubertal GH therapy, the effects of rhGH on the hypothalamus-pituitary-gonad axis were examined in the immature male rats.MethodsImmature male rats were given by daily injection of rhGH (1 or 2 IU/kg) from postnatal day (PND) 21 to PND 23 or 30. The effects of rhGH on kisspeptin-GnRH-LH system in the hypothalamus-pituitary axis, systemic and testicular IGF1, spermatogenesis, steroidogenesis, and circulating testosterone levels were examined. The effects of rhGH on the IGF1 expression and steroidogenesis were examined in progenitor LCs in vitro.ResultsTesticular steroidogenic pathway and spermatogenesis marker mRNA levels, number and size of 17β-hydroxysteroid dehydrogenase (+) LCs, and blood testosterone levels of rhGH rats were significantly higher than those of controls on PNDs 24 and 31. Hypothalamic Kiss1 and Gnrh1 mRNA of rhGH rats were significantly higher than those of controls on PND 24, indicating early activation of hypothalamic kisspeptin-GnRH neurons by rhGH. Hypothalamic Igf1 mRNA levels of rhGH rats were significantly higher than those of controls on PND 24 but significantly lower than those of controls on PND 31. Testicular Igf1 mRNA levels were significantly higher in rhGH rats than in the controls on PNDs 24 and 31 whereas circulating IGF1 levels were not. In progenitor LCs, rhGH significantly increased Igf1 and steroidogenic pathway mRNA levels and testosterone production.ConclusionsLocal increases in testicular IGF1 might be an important mediator of gonadal maturation via activation of LCs steroidogenesis in immature rats given rhGH.

Male subfertility effects of sub-chronic ethanol exposure during stress in a rat model.

Stressful conditions increase alcohol consumption in men. Clinical studies link disruption of the neuroendocrine stress system with alcoholism, but the effect of alcohol in a stress condition on male fertility is still relatively poorly understood. This project was undertaken to evaluate the effect of sub-chronic alcohol in a stress condition on male fertility in a rat model. Male Sprague-Dawley rats were randomly divided into a control group, a stress group that was exposed to restraint stress, an ethanol group that was injected with ethanol daily, and a stress+ethanol group that was injected with ethanol daily and was exposed to restraint stress, simultaneously. Furthermore, testis tissue was evaluated histomorphometrically and immunohistochemically for apoptosis using a TUNEL assay after 12 days. Epididymis sperm analysis was done. Blood cortisol and testosterone were measured and expression of hypothalamic kisspeptin (Kiss1), RFRP-3, and MC4R mRNA were evaluated. Ethanol exposure during restraint stress did not alter body weight. Ethanol exposure decreased the cellular diameter and area, and stress increased the cellular diameter and area, in comparison with the control group. In the stress group, in comparison with the other groups, the number of seminiferous tubules decreased and the numerical density of seminiferous tubules increased. In addition, ethanol exposure and/or stress reduced semen analysis parameters (sperm viability and motility), but did not change serum testosterone concentrations. Apoptosis increased in spermatogonia with ethanol exposure, but spermatocytes were not affected. Our data present the novel finding that ethanol and stress reduced hypothalamic Kiss1 mRNA expression, while ethanol exposure decreased hypothalamic RFRP-3 and MC4R mRNA expression. Ethanol decreased cortisol hormone level during the restraint stress condition and attenuated hypothalamic reproductive-related gene expressions. Therefore, ethanol exposure may induce reduction of sperm viability, increased sperm mortality, and increased apoptosis, with long-term effects, and may induce permanent male subfertility.

Distinct effects of growth hormone deficiency and disruption of hypothalamic kisspeptin system on reproduction of male mice

Growth hormone (GH) deficiency is a common cause of late sexual maturation and fertility issues. To determine whether GH-induced effects on reproduction are associated with alterations in hypothalamic kisspeptin system, we studied the male reproduction in two distinct GH deficiency mouse models. In the first model, mice present GH deficiency secondary to arcuate nucleus of the hypothalamus (ARH) lesions induced by posnatal monosodium glutamate (MSG) injections. MSG-induced ARH lesions led to significant reductions in hypothalamic Ghrh mRNA expression and consequently growth. Hypothalamic Kiss1 mRNA expression and Kiss1-expressing cells in the ARH were disrupted in the MSG-treated mice. In contrast, kisspeptin immunoreactivity remained preserved in the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) of MSG-treated mice. Importantly, ARH lesions caused late sexual maturation and infertility in male mice. In our second mouse model, we studied animals profound GH deficiency due to a loss-of-function mutation in the Ghrhr gene (Ghrhrlit/lit mice). Interestingly, although Ghrhrlit/lit mice exhibited late puberty onset, hypothalamic Kiss1 mRNA expression and hypothalamic kisspeptin fiber density were normal in Ghrhrlit/lit mice. Despite presenting dwarfism, the majority of Ghrhrlit/lit male mice were fertile. These findings suggest that spontaneous GH deficiency during development does not compromise the kisspeptin system. Furthermore, ARH Kiss1-expressing neurons are required for fertility, while AVPV/PeN kisspeptin expression is sufficient to allow maturation of the hypothalamic-pituitary-gonadal axis in male mice.

Kisspeptin Neurons Excitability Is Not Modulate by Fasting

Abstract Kisspeptin is the most important neuromodulator of gonadotrophin releasing hormone neurons. Hypothalamic Kiss1-expressing neurons can be found in the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) and in the arcuate nucleus of the hypothalamus (ARH). Food intake and energy balance are modulated by leptin, which acts primarily in the ARH. Leptin acts inhibiting orexigen neurotransmitters and stimulating anorectic neurotransmitters. Few ARH kisspeptin cells (10-15%) coexpress the leptin receptor and have their resting membrane potential (RMP) depolarized by leptin. Since the reproductive axis is sensitive to metabolic disorders, in the present study we used immunohistochemical and electrophysiological approach to understand the effects of fasting on kisspeptin neurons activity. To determine whether AVPV/PeN and ARH kisspeptin neurons activity are modulated by fasting, Kiss1/hrGFP female mice were fed regular chow ad libitum or fasted for 24 hours. Mice were euthanized after 24h. Fasting induced a significant increase of Fos protein on the ARH nucleus, as expected (rostral level of ARH, control: 19.3 ± 3.5 vs fasted: 81.3 ± 3.5 cells; caudal level, control: 15.7 ± 1.3 vs fasted: 103.3 ± 6.2 cells, P &amp;lt;0.0001, n=3/6 mice per group). Despite the significant increase of Fos-immunoreactive in the ARH nucleus, kisspeptin neurons did not co-express this neuronal marker. Next, we determined the RMP of kisspeptin cells obtained from control or fasted mice. Compared to the control group (AVPV/PeN, -56.5 ± 2.0 mV, n=13 cells from 4 animals; ARH, - 49.8 ± 2.4 mV, n= 9 cells from 6 animals), fasting was not sufficient to induce changes in RMP (AVPV/PeN, -56.1 ± 4.6 mV, n=6 cells from 3 animals, P=0.95; ARH, - 50.9 ± 2.6 mV, n=9 cells from 3 animals, P=0.11). The frequency (freq) and amplitude (amp) of the excitatory postsynaptic currents acting on kisspeptin neurons was further investigated. No significant difference was observed by comparing data obtained from control (AVPV/PeN: freq 0.76 ± 0.1 Hz, n=19; amp, 28.6 ± 1.8 pA, n=16; ARH: freq 0.36 ± 0.1 Hz, n=15; amp, 28.5 ± 2 pA, n=15) and fasted mice (AVPV/PeN: freq 0.7 ± 0.1 Hz, n=18; amp, 28.5 ± 1.1 pA; n=18; ARH: freq 0.5 ± 0.2 Hz, n=24; amp, 27.9 ± 1.3 pA; n=24; P &amp;gt; 0.5, 7/9 animals per group). Considering that 24hr fasting is not enough to inhibit estrous cyclicity, even though it is sufficient to induce a significant reduction of hypothalamic Kiss1 mRNA expression (unpublished data), our results suggest that prolonged periods of food restriction may be required to disturb excitatory inputs into kisspeptin cells.

Effects of Recombinant Human Growth Hormone on the Onset of Puberty, Leydig Cell Differentiation, Spermatogenesis and Hypothalamic KISS1 Expression in Immature Male Rats.

PurposeRecombinant human growth hormone (rhGH) has been used to treat short stature and rhGH-related syndromes. However, there are concerns that rhGH-treatment may cause precocious puberty. We investigated the effects of rhGH-treatment on the puberty onset, sexual maturation, androgen production, and hypothalamic gene expression in prepubertal male rats.Materials and MethodsSprague-Dawley male rats were injected subcutaneously daily with 1 or 2 IU/kg/d rhGH or 0.1 mL saline from postnatal day (PND) 21 to 30. At PND 31 bodyweight, reproductive organs weight, preputial separation, testis histology, circulating testosterone, and expression of testicular steroidogenic pathway genes and hypothalamic Kiss1 were examined.ResultsBy day 4 of injection bodyweights of rhGH groups were significantly higher than those of controls. rhGH 2 IU group showed earlier preputial separation compared to the control group. At PND 31, the weights of testes, epididymides, seminal vesicles, prostates, and preputial glands of the 2 IU-rhGH group were significantly higher than control group. Serum testosterone levels of the 2 IU-rhGH group were significantly higher than control group. Testicular steroidogenic pathway gene Hsd17b3 and Nr5a1 mRNA and cell counts and areas of Leydig cells in rhGH groups were significantly higher than control group, suggesting functional differentiation of Leydig cells. Hypothalamic Kiss1 mRNA levels of the 1 IU-rhGH group were significantly lower than control group, suggesting negative feedback of Kiss1 by elevated testosterone.ConclusionsPrepubertal rhGH-treatment in male rats may induce early onset of puberty, sexual maturation, elevation of testosterone, and spermatogenesis, and accompanies downregulation of hypothalamic KISS1.

Kisspeptin-52 partially rescues the activity of the hypothalamus-pituitary-gonadal axis in underweight male rats dosed with an anti-obesity compound

Energy metabolism and reproduction are closely linked and reciprocally regulated. The detrimental effect of underweight on reproduction complicates the safety evaluation of anti-obesity drugs, making it challenging to distinguish pathological changes mediated through the intended drug-induced weight loss from direct drug effects on reproductive organs. Four-weeks dosing of normal weight Sprague Dawley rats with a glucagon-like peptide 1 (GLP-1)/glucagon receptor co-agonist induced a robust weight loss, accompanied by histological findings in prostate, seminal vesicles, mammary glands, uterus/cervix and vagina. Characterization of the hypothalamus-pituitary-gonadal (HPG) axis in male rats revealed reduced hypothalamic Kiss1 mRNA levels and decreased serum luteinizing hormone (LH) and testosterone concentrations following co-agonist dosing. These alterations resemble hypogonadotropic hypogonadism typically seen in adverse energy deprived conditions, like chronic food restriction. Concomitant daily administration of kisspeptin-52 from day 21 to the end of the four-week co-agonist dosing period evoked LH and testosterone responses without normalizing histological findings. This incomplete rescue by kisspeptin-52 may be due to the rather short kisspeptin-52 treatment period combined with a desensitization observed on testosterone responses. Concomitant leptin treatment from day 21 did not reverse co-agonist induced changes in HPG axis activity. Furthermore, a single co-agonist injection in male rats slightly elevated LH levels but left testosterone unperturbed, thereby excluding a direct acute inhibitory effect on the HPG axis. Our data suggest that the reproductive phenotype after repeated co-agonist administration was driven by the intended weight loss, however, we cannot exclude a direct organ related effect in chronically treated rats.

SAT-290 Food Restriction Effects on the Hypothalamus-Pituitary-Gonadal Axis

It is well known that nutritional status affects the reproduction, since an adequate amount of energy is necessary for puberty onset and fertility. However, the neural mechanisms by which energy homeostasis affects reproduction is not completely elucidated. To determine if acute or chronic food restriction (FR) are able to modulate the estrous cycle, adult female mice were used in the experiments. The estrous cycle was evaluated by daily observation of vaginal smear. To determine the effects of an acute FR protocol on estrous cycle, females were individualized and kept on ad libitum diet (control, n=17) or fasted for 24 hours (n= 21). A subgroup of animals was euthanized shortly after the 24-hours test to collect hypothalamus and determinate Kiss1 mRNA levels, while another group of mice were regrouped and fed ad libitum. To determine the effects of a chronic FR protocol on estrous cycle, control mice were individualized and maintained with 100% of daily food content (average of 5 g per day, n = 6), or submitted to 60% of FR (n= 12). Animals were fed ad libitum after test. As expected, mice fasted for 24-hours exhibited a significant weight loss (control: 21.7 g ± 0.5 vs 21.6 ± 0.5 g; fasted: 22.7g ± 0.5 vs 18.7g ± 0.4, P=0.0001). This effect was followed by a significant reduction of hypothalamic Kiss1 mRNA expression (control: 1.0 ± 0.2; fasted: 0.3 ± 0.05, P=0.04, n=4/4 per group). Surprisingly, even under lower Kiss1 mRNA levels, 24-hours fasting induced no changes on estrous cycle. On the other hand, chronic FR induced a gradual weight loss (body weight at the 5th day of FR, control: 21.5g ± 0.2; FR: 17.3g ± 0.7, P=0.0002). The chronic FR was follow by the disruption of estrous cyclicity. While control mice exhibited a regular pattern of cyclicity during the period of evaluation, only leukocytes were identified in the vaginal smear of mice submitted to 60% of FR, even though they had a normal cycling pattern before the test. Therefore, by comparing 30 days of estrous cycle evaluation, including the period before chronic FR, while control mice exhibited cornified cells in the vaginal smear 58.5 ± 4.9% of days, female mice submitted to FR exhibited cornified cells in 38.3 ± 3.8% of days (P= 0.0068). Approximately 3-4 days after the end of the chronic FR females returned to exhibit estrous cyclicity, however the length of the estrous cycle was prolonged compared to control group. Our data suggest that chronic nutritional status variations are required to disrupt the hypothalamus-pituitary-gonadal axis and therefore the estrous cyclicity.

Mating-induced increase in &lt;i&gt;Kiss1&lt;/i&gt; 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.

Experimental obesity and diabetes reduce male fertility: Potential involvement of hypothalamic Kiss-1, pituitary nitric oxide, serum vaspin and visfatin

Reproductive dysfunction is a common consequence of both obesity and diabetes. This study investigated the impact of obesity and diabetes, alone or combined, on physiological reproductive parameters in male rats. Twenty-four male Wistar Albino rats were divided into four groups: Control; obese non-diabetic; diabetic; and obese diabetic. Obesity was provoked by consumption of a high-fat diet (HFD) consisting of 40% energy from fat for 90 days. Diabetes was induced by an intraperitoneal injection of streptozotocin at a dose of 40 mg/kg/day for three consecutive days. Semen, histopathological, and morphometric analyses were carried out. Serum testosterone, luteinizing hormone (LH), and vaspin and visfatin were measured using ELISA kits. Hypothalamic Kiss-1 mRNA was detected using qPCR and pituitary nitric oxide (NO) was determined using Griess reagent. Our results showed a decrease in semen quality parameters, testosterone, and LH levels with degenerative changes in the testes in experimental groups when compared to control group. This had a positive correlation with hypothalamic Kiss-1 and a negative correlation with pituitary NO and serum vaspin and visfatin. In addition, adverse effects were more pronounced in animals with obesity and diabetes combined compared to rats who were either diabetic or obese. In conclusion, obesity and diabetes, alone or combined, had a negative impact on male rat fertility. Moreover, obesity and diabetes combined had more harmful effects on male fertility when compared with obesity alone. Hypothalamic Kiss-1, pituitary NO, and serum vaspin and visfatin may play a role in the pathophysiology of male infertility-associated with obesity and diabetes.

OR-006 Effect of Moderate-intensity Exercise on the Expression of Hypothalamic KiSS-1 and GPR54 mRNA in Diet Induced Obesity Rats

Objective To observe the effect of high fat diet on the hypothalamic expression of KiSS-1and the G-protein coupled receptor (GPR) 54 mRNA and explore the modulatory role of moderate-intensity exercise in the diet induced obesity male rats.&#x0D; Methods After 8 weeks high fat feeding, 20 obesity 11-weeks SD rats were randomly assigned to high-fat diet sedentary (FS, n=8) and high-fat diet exercise (FE, n=8) groups, 20 normal diet 11-weeks SD rats also were randomly assigned to sedentary (SS, n=8) and exercise (SE, n=8) groups. During the following 8 weeks, obesity rats were continued expose to high-fat-diet. SE and FE groups did the 60%-70%V(•)O2max treadmill training (5 days/week, 1 hour/day). The V(•)O2 max of exercise groups were remeasured every two weeks. The hypothalamic expression of KiSS-1 and GPR54 mRNA were tested in each group.&#x0D; Results &#x0D; After the first 8-weeks high fat feeding, the obesity rats were heavier than normal diet group (491.74±26.19g vs. 410.05±45.77g, p&lt;0.01). After 8-weeks training, the FE group was lighter than FS group (590.23±35.74g vs. 681±52.56, p&lt;0.01). The FS group had higher hypothalamic expression of KiSS-1 mRNA (1.51±0.66 vs 0.75±0.27, p&lt;0.05) and GPR54 mRNA (2.45±0.38 vs 0.61±0.15, p&lt;0.01) than SS group. The FE group had lower hypothalamic expression of KiSS-1 mRNA (0.69±0.13, p&gt;0.05) and GPR54 mRNA (0.58±0.10, p&lt;0.01) than FS group.&#x0D; Conclusions &#x0D; There is stimulating effect of high-fat diet induced obesity on hypothalamic expression of KiSS-1and GPR54 mRNA. 8-weeks 60%-70%V (•) O2max treadmill training could cure this effect.&#x0D;

Participation of hypothalamic CB1 receptors in reproductive axis disruption during immune challenge.

Immune challenge inhibits reproductive function and endocannabinoids (eCB) modulate sexual hormones. However, no studies have been performed to assess whether the eCB system mediates the inhibition of hormones that control reproduction as a result of immune system activation during systemic infections. For that reason, we evaluated the participation of the hypothalamic cannabinoid receptor CB1 on the hypothalamic-pituitary-gonadal (HPG) axis activity in rats submitted to immune challenge. Male adult rats were treated i.c.v. administration with a CB1 antagonist/inverse agonist (AM251) (500ng/5 μL), followed by an i.p. injection of lipopolysaccharide (LPS) (5mg/kg) 15minutes later. Plasmatic, hypothalamic and adenohypophyseal pro-inflammatory cytokines, hormones and neuropeptides were assessed 90 or 180minutes post-LPS. The plasma concentration of tumour necrosis factor α and adenohypophyseal mRNA expression of Tnfα and Il1β increased 90 and 180minutes post i.p. administration of LPS. However, cytokine mRNA expression in the hypothalamus increased only 180minutes post-LPS, suggesting an inflammatory delay in this organ. CB1 receptor blockade with AM251 increased LPS inflammatory effects, particularly in the hypothalamus. LPS also inhibited the HPG axis by decreasing gonadotrophin-releasing hormone hypothalamic content and plasma levels of luteinising hormone and testosterone. These disruptor effects were accompanied by decreased hypothalamic Kiss1 mRNA expression and prostaglandin E2 content, as well as by increased gonadotrophin-inhibitory hormone (Rfrp3) mRNA expression. All these disruptive effects were prevented by the presence of AM251. In summary, our results suggest that, in male rats, eCB mediate immune challenge-inhibitory effects on reproductive axis at least partially via hypothalamic CB1 activation. In addition, this receptor also participates in homeostasis recovery by modulating the inflammatory process taking place after LPS administration.

Intracerebroventricular injection of RFRP-3 delays puberty onset and stimulates growth hormone secretion in female rats

BackgroundPuberty onset is a complex, organized biological process with multilevel regulation, and its physiopathological mechanisms are yet to be elucidated. RFRP-3, the mammalian ortholog to gonadotropin-inhibitory hormone, is implicated in inhibiting the synthesis and release of gonadotropin in mammals. However, it is unclear whether RFRP-3 participates in regulating pubertal development.MethodsThis study investigated the functional significance and regulatory mechanism of hypothalamic RFRP-3 neuropeptide in the onset of puberty in young female rats. On postnatal day 22, we implanted cannulas into the lateral ventricles of female rat pups. From postnatal day 28 to postnatal day 36, the intracerebroventricular injection of RFRP-3, or vehicle, was conducted twice a day. To investigate whether puberty onset was affected, we examined the body weight, age of vaginal opening, serum hormone levels, uterus and ovary development, and hypothalamic Kiss-1 mRNA expression.ResultsIntracerebroventricular injection of RFRP-3 significantly decreased the serum concentrations of luteinizing hormone and estradiol, delayed uterine maturation, and postponed the time of vaginal opening. This study suggests that RFRP-3 can delay the onset of puberty in young female rats; the expression of Kiss-1 mRNA is potently inhibited in the RFRP-3 group. Moreover, our data show that RFRP-3 elevates serum growth hormone levels.ConclusionsThese data suggest that intracerebroventricular injection of RFRP-3 significantly delays the onset of puberty in female rats. Additionally, RFRP-3 may be associated with prepubertal rise in the secretion of growth hormone.

Blockage of neonatal leptin signaling induces changes in the hypothalamus associated with delayed pubertal onset and modifications in neuropeptide expression during adulthood in male rats

The neonatal leptin surge, occurring from postnatal day (PND) 5 to 13 and peaking at PND9 in rodents, is important for the development of neuroendocrine circuits involved in metabolic control and reproductive function. We previously demonstrated that treatment with a leptin antagonist from PND 5 to 9, coincident with peak leptin levels in the neonatal surge, modified trophic factors and markers of cell turnover and neuronal maturation in the hypothalamus of peri-pubertal rats. The kisspeptin system and metabolic neuropeptide and hormone levels were also modified. Here our aim was to investigate if the timing of pubertal onset is altered by neonatal leptin antagonism and if the previously observed peripubertal modifications in hormones and neuropeptides persist into adulthood and affect male sexual behavior. To this end, male Wistar rats were treated with a pegylated super leptin antagonist (5mg/kg, s.c.) from PND 5 to 9 and killed at PND102–103. The appearance of external signs of pubertal onset was delayed. Hypothalamic kiss1 mRNA levels were decreased in adult animals, but sexual behavior was not significantly modified. Although there was no effect on body weight or food intake, circulating leptin, insulin and triglyceride levels were increased, while hypothalamic leptin receptor, POMC and AgRP mRNA levels were decreased. In conclusion, alteration of the neonatal leptin surge can modify the timing of pubertal onset and have long-term effects on hypothalamic expression of reproductive and metabolic neuropeptides.