Sex-specific hypothalamic expression of kisspeptin, gonadotropin releasing hormone, and kisspeptin receptor in progressive demyelination model.

Demyelinating diseases, such as multiple sclerosis, decrease the quality of life of patients and can affect reproduction. Assisted reproductive therapies are available, which although effective, aggravate motor symptoms. For this reason, it is important to determine how the control of the hypothalamus-pituitary-gonadal axis is affected in order to develop better strategies for these patients. One way to determine this is using animal models such as the taiep rat, which shows progressive demyelination of the central nervous system, and was used in the present study to characterize the expression of gonadotrophin-releasing hormone (GnRH), Kisspeptin, and kisspeptin receptor (Kiss1R) and luteinizing hormone (LH) secretion. The expression of kisspeptin, GnRH, and Kiss1R was determined at the hypothalamic level by immunofluorescence and serum LH levels were determined by ELISA. The expression of kisspeptin at the hypothalamic level showed sexual dimorphism, where there was an increase in males and a decrease in females during oestrus. There was no change in the expression of GnRH or kisspeptin receptor, regardless of sex. However, a decrease in serum LH concentration was observed in both sexes. The taiep rat showed changes in the expression of kisspeptin at the hypothalamic level. These changes are different from those reported in the literature with the use of animals with experimental allergic encephalomyelitis, this is because both animal models represent different degrees of progression of multiple sclerosis. Our results suggest that the effects on the hypothalamus-pituitary-gonadal axis depend on the differences between the demyelinating processes, their progression, and even individual factors, and it is thus important that fertility treatments are individualized to maximize therapeutic effects.

Mating-induced increase in Kiss1 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.

The Impact of Morphine on Reproductive Activity in Male Rats Is Regulated by Rf-Amid-Related Peptide-3 and Substance P Adjusting Hypothalamic Kisspeptin Expression.

Obviously, opiates (e.g., morphine) are associated with the suppression and dysfunction of reproductive axis. It has been reported that substance P (SP) and RF-amid-related peptide-3 (RFRP-3) can exhibit anti-opioid effects in some regions of the nervous system. Moreover, SP and RFRP-3 are deemed as neuropeptides which exert modulatory and regulatory impacts on the function of the reproductive axis. The precise interactions of morphine with SP or RFRP-3 on the parameters of the reproductive activity, however, are not fully known. The present study was aimed to determine the impacts of the interaction of morphine either with SP or RFRP-3 on the hormonal and behavioral parameters of reproductive activity in male rats. In addition, it was aimed at determining whether the effects of these interactions rely on kisspeptin/G protein coupled receptor 54 (GPR54) pathway as the main upstream pulse generator and the mediator of the function of many inputs of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) system or not. Altogether, the resulted data from the sexual behavior tests, radioimmunoassay of LH/testosterone, and real-time quantitative PCR for the assessment of the expression of hypothalamic Kiss1, Gpr54, and Gnrh1 genes following concomitant administration of morphine with SP or RFRP-3 revealed that the suppressing effects of morphine on the parameters of reproductive axis activity can be affected by the administration of either RFRP-3 or SP. It is advocated that SP and RFRP-3, by the modulation of the expression of hypothalamic Kiss1, can possibly antagonize the effects of morphine on GnRH/LH system and sexual behavior.

Dynamic Regulation of Hypothalamic DMXL2, KISS1, and RFRP Expression During Postnatal Development in Non-Human Primates.

The neurobiological mechanism of puberty onset in primates is currently only partly understood. A recent study reported an important role of Dmx-like 2 (DMXL2), a gene encoding rabconnectin-3α vesicular protein, in human subjects with mental retardation and neuroendocrine impairment of reproduction. To further characterize the potential role of DMXL2 in the regulation of reproduction, we analyzed the expression of DMXL2 in hypothalami of newborn, infantile, juvenile, pubertal, and postpubertal female and male common marmoset monkeys. Additionally, as the relative hypothalamic levels of gonadotropin-inhibitory hormone (GnIH) transcript during postnatal development are unknown in primates, we also quantified messenger RNA (mRNA) levels of RFRP, a gene encoding GnIH. Moreover, the transcript levels of kisspeptin, a well-known regulator of the hypothalamic neurohormonal axis controlling reproduction, were also checked. Transcript and protein levels of DMXL2 and Kiss1 transcript levels increase from the newborn to the infantile and from the juvenile (prepubertal) to the pubertal and the postpubertal period. We also noted a clear upsurge in RFRP transcript levels in the prepubertal period. In conclusion, the hypothalamic expressions of Kiss1 and DMXL2 mRNA increase during infantile, pubertal, and adult stages compared to newborn and juvenile stages in common marmoset monkeys. In contrast, the expression of RFRP mRNA upsurges in juvenile monkeys. Further mechanistic studies are needed to characterize the potential inhibitory role of the GnIH-GPR147 signaling in the prepubertal period and the role of DMXL2 in the molecular cascade regulating the neuroendocrine reproductive axis in primates.

Differential Effects of Alcohol on Excitatory and Inhibitory Puberty-Related Peptides in the Basal Hypothalamus of the Female Rat.

BACKGROUND: An increase in development of excitatory inputs along with a decline in inhibitory inputs ultimately govern the timely increased secretion of hypothalamic luteinizing hormone-releasing hormone (LHRH) at the time of puberty. As chronic alcohol (ALC) exposure acts at the hypothalamic level to suppress LHRH secretion and delay puberty, we assessed its ability to differentially affect the expression of key puberty-related proteins. METHODS: ALC was administered to female rats from days 27 to 33, at which time animals were killed and tissues collected for protein expression. In the medial basal hypothalamus (MBH), we assessed kisspeptin (Kp) 10, an excitatory peptide critical for prepubertal LHRH secretion, and Lin28b, a peptide with an inhibitory influence on puberty. As a direct mechanism of action of Lin28b was not known, we determined whether its central administration could induce dynorphin (DYN), a peptide that is inhibitory on LHRH secretion. Also, ALC's effect on DYN protein expression was assessed, as well as its effect on DYN release in vitro. RESULTS: ALC markedly suppressed (p < 0.01) the expression of the excitatory Kp protein, while at the same time increased (p < 0.001) the expression of inhibitory Lin28b protein. Subsequently, we showed for the first time that the central administration of Lin28b stimulated (p < 0.01) the synthesis of DYN. Finally, ALC also induced (p < 0.01) the protein expression and stimulated (p < 0.01) the in vitro release of DYN from the MBH. CONCLUSIONS: These results indicate that ALC can simultaneously and differentially alter both excitatory and inhibitory influences governing pubertal development, show for the first time a mechanism of action by which Lin28b exerts its prepubertal inhibitory tone, and further demonstrate the negative influences of ALC on the pubertal process.

The Critical Hormone-Sensitive Window for the Development of Delayed Effects Extends to 10 Days after Birth in Female Rats Postnatally Exposed to 17alpha-Ethynylestradiol.

Neonatal exposure to estrogens is known to cause delayed effects, a late-occurring adverse effect on adult female reproductive functions, such as early onset of age-matched abnormal estrous cycling. However, the critical period in which neonates are sensitive to delayed effects inducible by exogenous estrogen exposure has not been clearly identified. To clarify this window, we examined the intensity and timing of delayed effects using rats exposed to ethynylestradiol (EE) at various postnatal ages. After subcutaneous administration of a single dose of EE (20 µg/kg, which induces delayed effects) on Postnatal Day (PND) 0, 5, 10, or 14 in Wistar rats, hypothalamic and hormonal alterations in young adults and long-term estrous cycling status were investigated as indicators of delayed effects. In young adults, peak luteinizing hormone concentrations at the time of the luteinizing hormone surge showed a decreasing trend, and KiSS1 mRNA expression of the anterior hypothalamus and number of KiSS1-positive cells in the anteroventral periventricular nucleus were significantly decreased in the PND 0, 5, and 10 groups. The reduction in KiSS1 mRNA and KiSS1-postive cells was inversely correlated with age at time of exposure. These groups also exhibited early onset of abnormal estrous cycling, starting from 17 wk of age in the PND0 group and 19 wk of age in the PND5 and 10 groups. These indicators were not apparent in the PND14 group. Our results suggest that PND0-PND10 is the critical window of susceptibility for delayed effects, and PND14 is presumed to be the provisional endpoint of the window.

A Novel Letrozole Model Recapitulates Both the Reproductive and Metabolic Phenotypes of Polycystic Ovary Syndrome in Female Mice.

Polycystic ovary syndrome (PCOS) pathophysiology is poorly understood, due partly to lack of PCOS animal models fully recapitulating this complex disorder. Recently, a PCOS rat model using letrozole (LET), a nonsteroidal aromatase inhibitor, mimicked multiple PCOS phenotypes, including metabolic features absent in other models. Given the advantages of using genetic and transgenic mouse models, we investigated whether LET produces a similar PCOS phenotype in mice. Pubertal female C57BL/6N mice were treated for 5 wk with LET, which resulted in increased serum testosterone and normal diestrus levels of estradiol, similar to the hyperandrogenemia and follicular phase estrogen levels of PCOS women. As in PCOS, ovaries from LET mice were larger, polycystic, and lacked corpora lutea versus controls. Most LET females were acyclic, and all were infertile. LET females displayed elevated serum LH levels and higher Lhb mRNA in the pituitary. In contrast, serum FSH and Fshb were significantly reduced in LET females, demonstrating differential effects on gonadotropins, as in PCOS. Within the ovary, LET females had higher Cyp17, Cyp19, and Fsh receptor mRNA expression. In the hypothalamus, LET females had higher kisspeptin receptor mRNA expression but lower progesterone receptor mRNA levels. LET females also gained more weight than controls, had increased abdominal adiposity and adipocyte size, elevated adipose inflammatory mRNA levels, and impaired glucose tolerance, mirroring the metabolic phenotype in PCOS women. This is the first report of a LET paradigm in mice that recapitulates both reproductive and metabolic PCOS phenotypes and will be useful to genetically probe the PCOS condition.

Nutritional Programming of Accelerated Puberty in Heifers: Involvement of Pro-Opiomelanocortin Neurones in the Arcuate Nucleus.

The timing of puberty and subsequent fertility in female mammals are dependent on the integration of metabolic signals by the hypothalamus. Pro-opiomelanocortin (POMC) neurones in the arcuate nucleus (ARC) comprise a critical metabolic-sensing pathway controlling the reproductive neuroendocrine axis. α-Melanocyte-stimulating hormone (αMSH), a product of the POMC gene, has excitatory effects on gonadotrophin-releasing hormone (GnRH) neurones and fibres containing αMSH project to GnRH and kisspeptin neurones. Because kisspeptin is a potent stimulator of GnRH release, αMSH may also stimulate GnRH secretion indirectly via kisspeptin neurones. In the present work, we report studies conducted in young female cattle (heifers) aiming to determine whether increased nutrient intake during the juvenile period (4-8 months of age), a strategy previously shown to advance puberty, alters POMC and KISS1 mRNA expression, as well as αMSH close contacts on GnRH and kisspeptin neurones. In Experiment 1, POMC mRNA expression, detected by in situ hybridisation, was greater (P < 0.05) in the ARC in heifers that gained 1 kg/day of body weight (high-gain, HG; n = 6) compared to heifers that gained 0.5 kg/day (low-gain, LG; n = 5). The number of KISS1-expressing cells in the middle ARC was reduced (P < 0.05) in HG compared to LG heifers. In Experiment 2, double-immunofluorescence showed limited αMSH-positive close contacts on GnRH neurones, and the magnitude of these inputs was not influenced by nutritional status. Conversely, a large number of kisspeptin-immunoreactive cells in the ARC were observed in close proximity to αMSH-containing varicosities. Furthermore, HG heifers (n = 5) exhibited a greater (P < 0.05) percentage of kisspeptin neurones in direct apposition to αMSH fibres and an increased (P < 0.05) number of αMSH close contacts per kisspeptin cell compared to LG heifers (n = 6). These results indicate that the POMC-kisspeptin pathway may be important in mediating the nutritional acceleration of puberty in heifers.

Kisspeptin cell-specific PI3K signaling regulates hypothalamic kisspeptin expression and participates in the regulation of female fertility.

Hypothalamic kisspeptin neurons integrate and translate cues from the internal and external environments that regulate gonadotropin-releasing hormone (GnRH) secretion and maintain fertility in mammals. However, the intracellular signaling pathways utilized to translate such information into changes in kisspeptin expression, release, and ultimately activation of the kisspeptin-receptive GnRH network have not yet been identified. PI3K is an important signaling node common to many peripheral factors known to regulate kisspeptin expression and GnRH release. We investigated whether PI3K signaling regulates hypothalamic kisspeptin expression, pubertal development, and adult fertility in mice. We generated mice with a kisspeptin cell-specific deletion of the PI3K catalytic subunits p110α and p110ß (kiss-p110α/ß-KO). Using in situ hybridization, we examined Kiss1 mRNA expression in gonad-intact, gonadectomized (Gdx), and Gdx + steroid-replaced mice. Kiss1 cell number in the anteroventral periventricular hypothalamus (AVPV) was significantly reduced in intact females but not in males. In contrast, compared with WT and regardless of steroid hormone status, Kiss1 cell number was lower in the arcuate (ARC) of kiss-p110α/ß-KO males, but it was unaffected in females. Both intact Kiss-p110α/ß-KO males and females had reduced ARC kisspeptin-immunoreactive (IR) fibers compared with WT animals. Adult kiss-p110α/ß-KO males had significantly lower circulating luteinizing hormone (LH) levels, whereas pubertal development and fertility were unaffected in males. Kiss-p110α/ß-KO females exhibited a reduction in fertility despite normal pubertal development, LH levels, and estrous cyclicity. Our data show that PI3K signaling is important for the regulation of hypothalamic kisspeptin expression and contributes to normal fertility in females.

Hypothalamic Kiss1 and RFRP gene expressions are changed by a high dose of lipopolysaccharide in female rats.

Reproductive function is suppressed by several types of stress. Hypothalamic kisspeptin, which is a product of the Kiss1 gene, and GnIH/RFRP have pivotal roles in the regulation of GnRH and gonadotropins through their receptors Kiss1r and GPR147 in many species. However, alterations of these factors under stress conditions have not been fully evaluated. This study investigated the mechanisms of immune stress-induced reproductive dysfunction, especially focusing on the changes of Kiss1 and RFRP gene expression. Serum LH levels and hypothalamic Kiss1 and GnRH mRNA levels were decreased, while hypothalamic RFRP and GPR147 mRNA levels were increased by administration of a high dose of LPS (5mg/kg) in both ovariectomized and gonadal intact female rats. In this condition, Kiss1 and/or RFRP mRNA levels were positively and negatively correlated with GnRH expression, respectively. In contrast, hypothalamic Kiss1, RFRP, and GPR147 mRNA levels were not changed by administration of a moderate dose of LPS (500µg/kg) in ovariectomized rats. Rats with high-dose LPS injection showed more prolonged fever responses and severe anorexia compared with rats with moderate-dose LPS injection, indicating that more energy was used for the immune response in the former. These results suggest that the underlying mechanisms of dysfunction of gonadotropin secretion are changed according to the severity of immune stress, and that changes of some reserved factors, such as kisspeptin and RFRP, begin to participate in the suppression of GnRH and gonadotropin in severe conditions. As reproduction needs a large amount of energy, dysfunction of gonadotropin secretion under immune stress may be a biophylatic mechanism by which more energy is saved for the immune response.

Hypothalamic KISS1 expression, gonadotrophin-releasing hormone and neurotransmitter innervation vary with stress and sensitivity in macaques.

The present study examined the effect of short-term psychosocial and metabolic stress in a monkey model of stress-induced amenorrhaea on the hypothalamic-pituitary-gonadal axis. KISS1 expression was determined by in situ hybridisation in the infundibular arcuate nucleus. Downstream of KISS1, gonadotrophin-releasing hormone (GnRH) axons in lateral areas rostral to the infundibular recess, serum luteinising hormone (LH) and serum oestradiol were measured by immunohistochemistry and radioimmunoassay. Upstream of KISS1, norepinephrine axons in the rostral arcuate nucleus and serotonin axons in the anterior hypothalamus and periaqueductal grey were measured by immunohistochemistry. Female cynomolgus macaques (Macaca fascicularis) characterised as highly stress resilient (HSR) or stress sensitive (SS) were examined. After characterisation of stress sensitivity, monkeys were either not stressed, or mildly stressed for 5 days before euthanasia in the early follicular phase. Stress consisted of 5 days of 20% food reduction in a novel room with unfamiliar conspecifics. There was a significant increase in KISS1 expression in HSR and SS animals in the presence versus absence of stress (P = 0.005). GnRH axon density increased with stress in HSR and SS animals (P = 0.015), whereas LH showed a gradual but nonsignificant increase with stress. Oestradiol trended higher in HSR animals and there was no effect of stress (P = 0.83). Norepinephrine axon density (marked with dopamine ß-hydroxylase) increased with stress in both HSR and SS groups (P ≤ 0.002), whereas serotonin axon density was higher in HSR compared to SS animals and there was no effect of stress (P = 0.03). The ratio of dopamine ß-hydroxylase/oestradiol correlated with KISS1 (P = 0.052) and GnRH correlated with serum LH (P = 0.039). In conclusion, oestradiol inhibited KISS1 in the absence of stress, although stress increased norepinephrine, which may over-ride oestradiol inhibition of KISS1 expression. We speculate that neural pathways transduce stress to KISS1 neurones, which changes their sensitivity to oestradiol.

Effects of estradiol on kisspeptin neurons during puberty.

The activation of the gonadotropin-releasing hormone (GnRH) neurons from a state of relative quiescence is critical for initiating puberty in mammals. Kisspeptin and its G-protein coupled receptor Gpr54 are essential for puberty, with disruption to either resulting in failed puberty in humans and mice. Robust data from several species indicate that Kiss1 mRNA and/or kisspeptin peptide expression within the hypothalamus increases during pubertal development. Kisspeptin fiber innervation of GnRH neurons and kisspeptin release within the hypothalamus also increase during pubertal development, indicating that there is increased kisspeptinergic drive to GnRH neurons during pubertal development. It is becoming increasingly apparent that gonadal steroids play important roles in the regulation of kisspeptin expression during pubertal development, and in particular, estradiol signaling through estrogen receptor alpha appears to be necessary for these changes to occur. This review focuses on the role that estradiol plays in the regulation of kisspeptin expression during pubertal development.

Lesions of the ventral premammillary nucleus disrupt the dynamic changes in Kiss1 and GnRH expression characteristic of the proestrus-estrus transition.

We have recently demonstrated that the ventral premammillary nucleus (PMV) plays a key role in the metabolic control of the female reproductive axis. However, whether PMV neurons modulate the reproductive neural circuitry and/or the expression of sexual behaviors has not been determined. Here, we showed that the expression of estrogen and progesterone receptors in the PMV is modulated by changing levels of sex steroids across the estrous cycle. We also showed that sexual behavior, not the high physiologic levels of sex steroids, induces Fos in PMV neurons. Bilateral lesions of the PMV caused no significant changes in proceptive behavior but a high percentage of PMV-lesioned rats failed to exhibit lordosis behavior when exposed to a sexually experienced male rat (50% vs. 18% in the control group). Notably, lesions of the PMV disrupted the physiologic fluctuations of Kiss1 and GnRH mRNA expression characteristic of the proestrus-to-estrus transition. This neurochemical imbalance may ultimately alter female reproductive behavior. Our findings suggest that the PMV is a component of the neural circuitry that modulates the physiologic fluctuations of key neuroendocrine players (i.e., Kiss1 and GnRH) in the control of the female reproductive physiology.

Effect of food restriction on reproductive-related genes and reproductive hormones in adult female rats.

OBJECTIVES: A number of factors involved in the control of energy balance and metabolism act as modulators of gonadal axis. Ghrelin, a peptide secreted from the stomach and hypothalamus, has emerged as an orexigenic food intake controlling signal acting upon hypothalamus. Recently, the potential reproductive role of ghrelin has received great attention. This study was designed to investigate the influence of food restriction and consequent metabolic hormone (ghrelin) on the level and gene expression of female reproductive hormones in adult rats. MATERIALS AND METHODS: To study the effect of chronic food restriction on ghrelin level in adult female rats and its relation to female reproductive hormones, 32 adult female Sprague Dawley rats divided into 4 groups: Group I (control group) comprised 8 rats fed ad libitum for 30 days, Group II, III and IV (food-restricted groups for 10, 20 and 30 days respectively) each consisted of 8 rats fed 50% of ad libitum intake determined by the amount of food consumed by the control group. RESULTS: Mean body weight of food restricted rats was observed to decrease during the period of the experiment. Food restriction produced significant increase of serum ghrelin with significant decrease of both gastric and hypothalamic ghrelin accompanied with significant increase in its gene expression in stomach and hypothalamus. Estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels showed significant decrease correlated with down-regulation of gonadotropins, cyclin-dependent kinase (cdc2), cyclin B and kisspeptin (Kiss1) genes in food restricted rats compared with control group. CONCLUSIONS: Ghrelin could be one of the hormones responsible for the suppression of female reproductive axis in case of negative energy balance. Thus, ghrelin may operate as an autocrine/paracrine regulator of ovarian function. Overall, ghrelin may represent an additional link between body weight homeostasis and reproductive function.

Hyperprolactinemia-induced ovarian acyclicity is reversed by kisspeptin administration.

Hyperprolactinemia is the most common cause of hypogonadotropic anovulation and is one of the leading causes of infertility in women aged 25-34. Hyperprolactinemia has been proposed to block ovulation through inhibition of GnRH release. Kisspeptin neurons, which express prolactin receptors, were recently identified as major regulators of GnRH neurons. To mimic the human pathology of anovulation, we continuously infused female mice with prolactin. Our studies demonstrated that hyperprolactinemia in mice induced anovulation, reduced GnRH and gonadotropin secretion, and diminished kisspeptin expression. Kisspeptin administration restored gonadotropin secretion and ovarian cyclicity, suggesting that kisspeptin neurons play a major role in hyperprolactinemic anovulation. Our studies indicate that administration of kisspeptin may serve as an alternative therapeutic approach to restore the fertility of hyperprolactinemic women who are resistant or intolerant to dopamine agonists.

Hypothalamic kiss1 mRNA and kisspeptin immunoreactivity are reduced in a rat model of polycystic ovary syndrome (PCOS).

An intact hypothalamic kiss1/kisspeptin/kiss1r complex is a prerequisite for reproductive competence, and kisspeptin treatment could be a practical therapeutic approach to some problems of infertility. One such disorder is polycystic ovarian syndrome (PCOS), a common cause of infertility affecting more than 100 million women. A rodent model of PCOS is the prepubertal female rat treated for a prolonged period with dihydrotestosterone (DHT), which induces many of the metabolic characteristics of the syndrome. We hypothesized that hypothalamic kiss1 mRNA levels, and kisspeptin immunoreactivity (ir), would be abnormal in these rats. Prepubertal female rats were exposed to DHT for 60 days. Rats were killed in two groups: at 26 and 60 days of DHT exposure. Kiss1 mRNA was quantified in hypothalamus, pituitary, ovary and visceral adipose tissue. Separate groups of rats provided brain tissue for immunohistochemical analysis of kisspeptin-ir. At 26 days of DHT exposure, hypothalamic kiss1 mRNA was severely depleted. In contrast DHT had no effect on pituitary kiss1 expression but it significantly increased levels of kiss1 mRNA in fat (+9-fold; p<0.01) and in ovary (+3-fold; p<0.05). At 60days, kiss1 expression had reverted to normal in hypothalamus and ovary but remained elevated in fat (+4-fold; p<0.05). Immunohistochemical analysis revealed that after 26 days of exposure to DHT, kisspeptin-ir was almost completely absent in the arcuate nucleus and a large depletion in kisspeptin +ve fibers was also seen in the paraventricular nucleus, supraoptic nucleus and in the anteroventral periventricular area. At 60 days, despite restored normal levels of kiss1 mRNA, hypothalamic kisspeptin-ir remained depleted in the treated rats. In summary Kiss1 gene expression is differentially affected in various tissues by chronic exposure to dihydrotestosterone in a rat model of polycystic ovary syndrome. In hypothalamus, specifically, kiss1 mRNA, and levels of kisspeptin immunoreactivity, are significantly reduced. Since these rats exhibit many of the characteristics of polycystic ovary syndrome, we suggest that atypical kiss1 expression may contribute to the multiple tissue abnormalities observed in women with this disorder. However, and of some importance, our data do not appear to be consistent with the elevated levels of LH seen in women with PCOS; i.e. reduced levels of hypothalamic kiss1 mRNA and kisspeptin immunoreactivity observed in DHT-treated rats are unlikely to produce elevated LH secretion.

Preweaning over- and underfeeding alters onset of puberty in the rat without affecting kisspeptin.

The perinatal nutritional environment can permanently influence body weight, potentially leading to changes in puberty onset and reproductive function. We hypothesized that perinatal under- or overfeeding would alter puberty onset and influence concentrations of a neuropeptide crucial for successful puberty, kisspeptin. We manipulated Wistar rat litter sizes to derive small (SL), control (CL), and large (LL) litters containing 4, 12, and 20 rat pups respectively. This manipulation results in an overweight phenotype in SL rats and a lean phenotype in LL that persists throughout life. To investigate whether successful puberty onset is affected by neonatal under- or overfeeding, we examined indices of growth and development, including the onset of puberty, as well as the central expression of Kiss1 mRNA in these pups. Male LL rats reached puberty later than those from CL. These males also had reduced plasma testosterone and elevated 17beta-estradiol concentrations at puberty. The age at puberty onset was not affected in SL males despite accelerated growth. In females, puberty onset was not significantly delayed by having a lean phenotype, and steroid hormones were not affected. The age at onset was, however, younger in the SL females. Kiss1 mRNA in the hypothalamus was not affected by neonatal nutrition either at puberty or 7 days later. Our findings show early life underfeeding in males and overfeeding in females significantly affects puberty onset, altering steroid hormone concentrations in males, but this is not related to changes in hypothalamic kisspeptin.

Kisspeptin neurons co-express met-enkephalin and galanin in the rostral periventricular region of the female mouse hypothalamus.

It is now well established that the kisspeptin neurons of the hypothalamus play a key role in regulating the activity of gonadotropin-releasing hormone (GnRH) neurons. The population of kisspeptin neurons residing in the rostral periventricular region of the third ventricle (RP3V), encompassing the anteroventral periventricular (AVPV) and periventricular preoptic nuclei (PVpo), are implicated in the generation of the preovulatory GnRH surge mechanism and puberty onset in female rodents. The present study examined whether these kisspeptin neurons may express other neuropeptides in the adult female mouse. Initially, the distribution of galanin, neurotensin, met-enkephalin (mENK), and cholecystokinin (CCK)-immunoreactive cells was determined within the RP3V of colchicine-treated mice. Subsequent experiments, using a new kisspeptin-10 antibody raised in sheep, examined the relationship of these neuropeptides to kisspeptin neurons. No evidence was found for expression of neurotensin or CCK by RP3V kisspeptin neurons, but subpopulations of kisspeptin neurons were observed to express galanin and mENK. Dual-labeled RP3V kisspeptin/galanin cells represented 7% of all kisspeptin and 21% of all galanin neurons whereas dual-labeled kisspeptin/mENK cells represented 28-38% of kisspeptin neurons and 58-68% of the mENK population, depending on location within the AVPV or PVpo. Kisspeptin neurons in the arcuate nucleus were also found to express galanin but not mENK. These observations indicate that, like the kisspeptin population of the arcuate nucleus, kisspeptin neurons in the RP3V also co-express a range of neuropeptides. This pattern of co-expression should greatly increase the dynamic range with which kisspeptin neurons can modulate the activity of their afferent neurons.