Effects of undernutrition and low energy availability on reproductive functions and their underlying neuroendocrine mechanisms.

It has been well established that undernutrition and low energy availability disturb female reproductive functions in humans and many animal species. These reproductive dysfunctions are mainly caused by alterations of some hypothalamic factors, and consequent reduction of gonadotrophin-releasing hormone (GnRH) secretion. Evidence from literature suggests that increased activity of orexigenic factors and decreased activity of anorexigenic/satiety-related factors in undernourished conditions attenuate GnRH secretion in an integrated manner. Likewise, the activity of kisspeptin neurons, which is a potent stimulator of GnRH, is also reduced in undernourished conditions. In addition, it has been suggested that gonadotrophin-inhibitory hormone, which has anti-GnRH and gonadotrophic effects, may be involved in reproductive dysfunctions under several kinds of stress conditions. It should be remembered that these alterations, i.e., promotion of feeding behavior and temporary suppression of reproductive functions, are induced to prioritize the survival of individual over that of species, and that improvements in metabolic and nutritional conditions should be considered with the highest priority.

Neuroendocrine mechanisms of reproductive dysfunctions in undernourished condition.

It is well known that undernourished conditions disturb female reproductive functions in many species, including humans. These alterations are mainly caused by a reduction in gonadotrophin-releasing hormone (GnRH) secretion from the hypothalamus. Evidence from the literature suggests that some hypothalamic factors play pivotal roles in the coordination of reproductive functions and energy homeostasis in response to environmental cues and internal nutritional status. Generally, anorexigenic/satiety-related factors, such as leptin, alpha-melanocyte-stimulating hormone, and proopiomelanocortin, promote GnRH secretion, whereas orexigenic factors, such as neuropeptide Y, agouti-related protein, orexin, and ghrelin, attenuate GnRH secretion. Conversely, gonadotrophin-inhibitory hormone, which exerts anti-GnRH and gonadotrophic effects, promotes feeding behavior in many species. In addition, the activity of kisspeptin, which is a potent stimulator of GnRH, is reduced by undernourished conditions. Under normal nutritional conditions, these factors are coordinated to maintain both feeding behavior and reproductive functions. However, in undernourished conditions their activity levels are markedly altered to promote feeding behavior and temporarily suppress reproductive functions, in order to prioritize the survival of the individual over that of the species.

Hypothalamic Kisspeptin Neurons and the Control of Homeostasis.

Hypothalamic kisspeptin (Kiss1) neurons provide indispensable excitatory transmission to gonadotropin-releasing hormone (GnRH) neurons for the coordinated release of gonadotropins, estrous cyclicity, and ovulation. But maintaining reproductive functions is metabolically demanding so there must be a coordination with multiple homeostatic functions, and it is apparent that Kiss1 neurons play that role. There are 2 distinct populations of hypothalamic Kiss1 neurons, namely arcuate nucleus (Kiss1ARH) neurons and anteroventral periventricular and periventricular nucleus (Kiss1AVPV/PeN) neurons in rodents, both of which excite GnRH neurons via kisspeptin release but are differentially regulated by ovarian steroids. Estradiol (E2) increases the expression of kisspeptin in Kiss1AVPV/PeN neurons but decreases its expression in Kiss1ARH neurons. Also, Kiss1ARH neurons coexpress glutamate and Kiss1AVPV/PeN neurons coexpress gamma aminobutyric acid (GABA), both of which are upregulated by E2 in females. Also, Kiss1ARH neurons express critical metabolic hormone receptors, and these neurons are excited by insulin and leptin during the fed state. Moreover, Kiss1ARH neurons project to and excite the anorexigenic proopiomelanocortin neurons but inhibit the orexigenic neuropeptide Y/Agouti-related peptide neurons, highlighting their role in regulating feeding behavior. Kiss1ARH and Kiss1AVPV/PeN neurons also project to the preautonomic paraventricular nucleus (satiety) neurons and the dorsomedial nucleus (energy expenditure) neurons to differentially regulate their function via glutamate and GABA release, respectively. Therefore, this review will address not only how Kiss1 neurons govern GnRH release, but how they control other homeostatic functions through their peptidergic, glutamatergic and GABAergic synaptic connections, providing further evidence that Kiss1 neurons are the key neurons coordinating energy states with reproduction.

Leptin and inflammatory factors play a synergistic role in the regulation of reproduction in male mice through hypothalamic kisspeptin-mediated energy balance.

BACKGROUND: Energy balance is closely related to reproductive function, wherein hypothalamic kisspeptin mediates regulation of the energy balance. However, the central mechanism of kisspeptin in the regulation of male reproductive function under different energy balance states is unclear. Here, high-fat diet (HFD) and exercise were used to change the energy balance to explore the role of leptin and inflammation in the regulation of kisspeptin and the hypothalamic-pituitary-testis (HPT) axis. METHODS: Four-week-old male C57BL/6 J mice were randomly assigned to a normal control group (n = 16) or an HFD (n = 49) group. After 10 weeks of HFD feeding, obese mice were randomly divided into obesity control (n = 16), obesity moderate-load exercise (n = 16), or obesity high-load exercise (n = 17) groups. The obesity moderate-load exercise and obesity high-load exercise groups performed exercise (swimming) for 120 min/day and 120 min × 2 times/day (6 h interval), 5 days/week for 8 weeks, respectively. RESULTS: Compared to the mice in the normal group, in obese mice, the mRNA and protein expression of the leptin receptor, kiss, interleukin-10 (IL-10), and gonadotropin-releasing hormone (GnRH) decreased in the hypothalamus; serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels and sperm quality decreased; and serum leptin, estradiol, and tumor necrosis factor-α (TNF-α) levels and sperm apoptosis increased. Moderate- and high-load exercise effectively reduced body fat and serum leptin levels but had the opposite effects on the hypothalamus and serum IL-10 and TNF-α levels. Moderate-load exercise had anti-inflammatory effects accompanied by increased mRNA and protein expression of kiss and GnRH in the hypothalamus and increased serum FSH, LH, and testosterone levels and improved sperm quality. High-load exercise also promoted inflammation, with no significant effect on the mRNA and protein expression of kiss and GnRH in the hypothalamus, serum sex hormone level, or sperm quality. Moderate-load exercise improved leptin resistance and inflammation and reduced the inhibition of kisspeptin and the HPT axis in obese mice. The inflammatory response induced by high-load exercise may counteract the positive effect of improving leptin resistance on kisspeptin and HPT. CONCLUSION: During changes in energy balance, leptin and inflammation jointly regulate kisspeptin expression on the HPT axis.

ERα Signaling in GHRH/Kiss1 Dual-Phenotype Neurons Plays Sex-Specific Roles in Growth and Puberty.

Gonadal steroids modulate growth hormone (GH) secretion and the pubertal growth spurt via undefined central pathways. GH-releasing hormone (GHRH) neurons express estrogen receptor α (ERα) and androgen receptor (AR), suggesting changing levels of gonadal steroids during puberty directly modulate the somatotropic axis. We generated mice with deletion of ERα in GHRH cells (GHRHΔERα), which displayed reduced body length in both sexes. Timing of puberty onset was similar in both groups, but puberty completion was delayed in GHRHΔERα females. Lack of AR in GHRH cells (GHRHΔAR mice) induced no changes in body length, but puberty completion was also delayed in females. Using a mouse model with two reporter genes, we observed that, while GHRHtdTom neurons minimally colocalize with Kiss1hrGFP in prepubertal mice, ∼30% of GHRH neurons coexpressed both reporter genes in adult females, but not in males. Developmental analysis of Ghrh and Kiss1 expression suggested that a subpopulation of ERα neurons in the arcuate nucleus of female mice undergoes a shift in phenotype, from GHRH to Kiss1, during pubertal transition. Our findings demonstrate that direct actions of gonadal steroids in GHRH neurons modulate growth and puberty and indicate that GHRH/Kiss1 dual-phenotype neurons play a sex-specific role in the crosstalk between the somatotropic and gonadotropic axes during pubertal transition.SIGNIFICANCE STATEMENT Late maturing adolescents usually show delayed growth and bone age. At puberty, gonadal steroids have stimulatory effects on the activation of growth and reproductive axes, but the existence of gonadal steroid-sensitive neuronal crosstalk remains undefined. Moreover, the neural basis for the sex differences observed in the clinical arena is unknown. Lack of ERα in GHRH neurons disrupts growth in both sexes and causes pubertal delay in females. Deletion of androgen receptor in GHRH neurons only delayed female puberty. In adult females, not males, a subset of GHRH neurons shift phenotype to start producing Kiss1. Thus, direct estrogen action in GHRH/Kiss1 dual-phenotype neurons modulates growth and puberty and may orchestrate the sex differences in endocrine function observed during pubertal transition.

Metabolic regulation of kisspeptin - the link between energy balance and reproduction.

Hypothalamic kisspeptin neurons serve as the nodal regulatory centre of reproductive function. These neurons are subjected to a plethora of regulatory factors that ultimately affect the release of kisspeptin, which modulates gonadotropin-releasing hormone (GnRH) release from GnRH neurons to control the reproductive axis. The presence of sufficient energy reserves is critical to achieve successful reproduction. Consequently, metabolic factors impose a very tight control over kisspeptin synthesis and release. This Review offers a synoptic overview of the different steps in which kisspeptin neurons are subjected to metabolic regulation, from early developmental stages to adulthood. We cover an ample array of known mechanisms that underlie the metabolic regulation of KISS1 expression and kisspeptin release. Furthermore, the novel role of kisspeptin neurons as active players within the neuronal circuits that govern energy balance is discussed, offering evidence of a bidirectional role of these neurons as a nexus between metabolism and reproduction.

Kisspeptin induces LH release and ovulation in an induced ovulator†.

Kisspeptin has been implicated in the ovulatory process of several species of spontaneous ovulators but in only one induced ovulator. In contrast, NGF in semen is the principal trigger of ovulation in other species of induced ovulators-camelids. We tested the hypotheses that kisspeptin induces luteinizing hormone (LH) secretion in llamas through a hypothalamic mechanism, and kisspeptin neurons are the target of NGF in its ovulation-inducing pathway. In Experiment 1, llamas were given either NGF, kisspeptin, or saline intravenously, and LH secretion and ovulation were compared among groups. All llamas treated with NGF (5/5) or kisspeptin (5/5) had an elevation of LH blood concentrations after treatment and ovulated, whereas none of the saline group did (0/5). In Experiment 2, llamas were either pretreated with a gonadotropin-releasing hormone (GnRH) receptor antagonist or saline and treated 2 h later with kisspeptin. Llamas pretreated with saline had elevated plasma LH concentrations and ovulated (6/6) whereas llamas pretreated with cetrorelix did not (0/6). In Experiment 3, we evaluated the hypothalamic kisspeptin-GnRH neuronal network by immunohistochemistry. Kisspeptin neurons were detected in the arcuate nucleus, the preoptic area, and the anterior hypothalamus, establishing synaptic contacts with GnRH neurons. We found no colocalization between kisspeptin and NGF receptors by double immunofluorescence. Functional and morphological findings support the concept that kisspeptin is a mediator of the LH secretory pathway in llamas; however, the role of kisspeptins in the NGF ovulation-inducing pathway in camelids remains unclear since NGF receptors were not detected in kisspeptin neurons in the hypothalamus.

Clinical Translational Studies of Kisspeptin and Neurokinin B.

Kisspeptin and neurokinin B (NKB) are hypothalamic neuropeptides that are vital for reproductive health. An absence of either kisspeptin or NKB signaling results in hypogonadotrophic hypogonadism and a failure to proceed through puberty. In recent years, several studies have demonstrated potential avenues for the clinical utility of medications that act through these pathways in the assessment and treatment of reproductive disorders. Kisspeptin acts to stimulate hypothalamic gonadotrophic-releasing hormone (GnRH) secretion from the hypothalamus. Kisspeptin induces gonadotrophin secretion in both healthy men and women, and in women with reproductive disorders such as hypothalamic amenorrhea (HA). Kisspeptin-based treatments hold promise for use during in vitro fertilization (IVF) treatment; a bolus of kisspeptin-54 induces an LH surge of 12 to 14 hours of duration sufficient to induce oocyte maturation, but with markedly reduced rates of the most significant complication of IVF treatment, ovarian hyperstimulation syndrome (OHSS). Kisspeptin could also be used chronically to restore reproductive health in patients with functional hypogonadism, such as those with HA. Furthermore, kisspeptin has potential as a diagnostic test of hypothalamic function; a "kisspeptin test" could be used in children with delayed puberty to identify the subset with genetically determined deficits in hypothalamic pathways (congenital hypogonadotrophic hypogonadism [CHH]). In addition to its role in hypothalamic GnRH pulse generation, NKB plays a critical role in the occurrence of one of the most troubling symptoms of the menopause, the "hot flush." Neurokinin-3 receptor (NK3R) antagonists are highly effective as treatments for hot flushes in postmenopausal women, with several compounds now in late-phase development. Furthermore, NK3R antagonism leads to a reduction in LH secretion by reducing GnRH pulsatility in the hypothalamus and has been shown to reduce androgen levels in women with polycystic ovary syndrome (PCOS) (in whom GnRH pulsatility is often increased). In summary, although further detailed evaluation in several clinical settings is ongoing, medications based on kisspeptin and NKB pathways have prodigious potential in the assessment and treatment of reproductive disorders.

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.

Expression of progesterone receptor, estrogen receptors α and ß, and kisspeptin in the hypothalamus during perinatal development of gonad-lacking steroidogenic factor-1 knockout mice.

Gonadal hormones contribute to brain sexual differentiation. We analyzed expression of progesterone receptor (PR), estrogen receptor-α (ERα), ERß, and kisspeptin, in the preoptic area (POA) and/or the arcuate nucleus (ARC), in gonad-lacking steroidogenic factor-1 knockout (KO) mice during perinatal development. At postnatal-day (P) 0-P7, POA PR levels were higher in wild-type (WT) males compared with WT females, while those in KO males were lower than in WT males and similar to those in WT and KO females. At P14-P21, PR levels in all groups increased similarly. POA ERα levels were similar in all groups at embryonic-day (E) 15.5-P14. Those in WT but not KO males reduced during postnatal development to be significantly lower compared with females at P21. POA ERß levels were higher in WT males than in WT females, while those in KO males were lower than in WT males and similar to those in WT and KO females at P0-P21. POA kisspeptin expression was female-biased in WT mice, while levels in KO females were lower compared with WT females and similar to those in WT and KO males. ARC kisspeptin levels were equivalent among groups at E15.5-P0. At P7-P21, ARC levels in WT but not KO males became lower compared with WT females. Diethylstilbestrol exposure during P0-P6 and P7-P13 increased POA PR and ERß, and decreased POA ERα and ARC kisspeptin levels at P7 and/or P14 in both sexes of KO mice. These data further understanding of gonadal hormone action on neuronal marker expression during brain sexual development.

Glucocorticoids stimulate hypothalamic dynorphin expression accounting for stress-induced impairment of GnRH secretion during preovulatory period.

Stress-induced reproductive dysfunction is frequently associated with increased glucocorticoid (GC) levels responsible for suppressed GnRH/LH secretion and impaired ovulation. Besides the major role of the hypothalamic kisspeptin system, other key regulators may be involved in such regulatory mechanisms. Herein, we identify dynorphin as a novel transcriptional target of GC. We demonstrate that only priming with high estrogen (E2) concentrations prevailing during the late prooestrus phase enables stress-like GC concentrations to specifically stimulate Pdyn (prodynorphin) expression both in vitro (GT1-7 mouse hypothalamic cell line) and ex vivo (ovariectomized E2-supplemented mouse brains). Our results indicate that stress-induced GC levels up-regulate dynorphin expression within a specific kisspeptin neuron-containing hypothalamic region (antero-ventral periventricular nucleus), thus lowering kisspeptin secretion and preventing preovulatory GnRH/LH surge at the end of the prooestrus phase. To further characterize the molecular mechanisms of E2 and GC crosstalk, chromatin immunoprecipitation experiments and luciferase reporter gene assays driven by the proximal promoter of Pdyn show that glucocorticoid receptors bind specific response elements located within the Pdyn promoter, exclusively in presence of E2. Altogether, our work provides novel understanding on how stress affects hypothalamic-pituitary-gonadal axis and underscores the role of dynorphin in mediating GC inhibitory actions on the preovulatory GnRH/LH surge to block ovulation.

The role of kisspeptin/neurokinin B/dynorphin neurons in pathomechanism of vasomotor symptoms in postmenopausal women: from physiology to potential therapeutic applications.

Women during perimenopausal period experience a range of symptoms, which interfere with physical, sexual, and social life. About 65-75% of symptoms connected with postmenopausal period are vasomotor symptoms (VMS), such as hot flushes and night sweats. Hot flushes are subjective sensation of heat associated with cutaneous vasodilatation and drop in core temperature. It is suspected that VMS are strongly correlated with pulsatile oversecretion of gonadotropin-releasing hormone (GnRH) and subsequently luteinizing hormone (LH). Evidence has accumulated in parallel showing that lack of negative feedback of steroid hormones synthesized in ovary causes overactivation of hypertrophied kisspeptin/neurokinin B/dynorphin (KNDy) neurons, located in infundibular nucleus. Oversecretion of both kisspeptin (KISS1) and neurokinin B (NKB), as well as downregulation of dynorphin, plays dominant role in creation of GnRH pulses. This in turn causes VMS. Administration of senktide, highly potent and selective NK3R agonist, resulted in increase of serum LH concentration, induction of VMS, increase in heart rate, and skin temperature in postmenopausal women. These finding suggest that modulation of KNDy neurons may become new therapeutic approach in the treatment of VMS.

Altered aspects of anxiety-related behavior in kisspeptin receptor-deleted male mice.

The roles of kisspeptin signaling outside the hypothalamus in the brain are unknown. We examined here the impact of Kiss1r-deletion on hippocampus-related behaviors of anxiety and spatial learning in adult male mice using two mouse models. In the first, global Kiss1r-null and control mice were gonadectomized (GDX KISS1R-KO). In the second, KISS1R signalling was rescued selectively in gonadotropin-releasing hormone neurons to generate Kiss1r-null mice with normal testosterone levels (intact KISS1R-KO). Intact KISS1R-KO rescue mice were found to spend twice as much time in the open arms of the elevated plus maze (EPM) compared to controls (P < 0.01). GDX KISS1R-KO mice showed a similar but less pronounced trend. No differences were detected between intact KISS1R-KO mice and controls in the open field test (OFT), although a marked reduction in time spent in the centre quadrant was observed for all GDX mice (P < 0.001). No effects of KISS1R deletion or gonadectomy were detected in the Morris water maze. These observations demonstrate that KISS1R signalling impacts upon anxiogenic neural circuits operative in the EPM, while gonadal steroids appear important for anxiety behaviour observed in the OFT. The potential anxiogenic role of kisspeptin may need to be considered in the development of kisspeptin analogs for the clinic.

Glutamatergic Transmission to Hypothalamic Kisspeptin Neurons Is Differentially Regulated by Estradiol through Estrogen Receptor α in Adult Female Mice.

Estradiol feedback regulates gonadotropin-releasing hormone (GnRH) neurons and subsequent luteinizing hormone (LH) release. Estradiol acts via estrogen receptor α (ERα)-expressing afferents of GnRH neurons, including kisspeptin neurons in the anteroventral periventricular (AVPV) and arcuate nuclei, providing homeostatic feedback on episodic GnRH/LH release as well as positive feedback to control ovulation. Ionotropic glutamate receptors are important for estradiol feedback, but it is not known where they fit in the circuitry. Estradiol-negative feedback decreased glutamatergic transmission to AVPV and increased it to arcuate kisspeptin neurons; positive feedback had the opposite effect. Deletion of ERα in kisspeptin cells decreased glutamate transmission to AVPV neurons and markedly increased it to arcuate kisspeptin neurons, which also exhibited increased spontaneous firing rate. KERKO mice had increased LH pulse frequency, indicating loss of negative feedback. These observations indicate that ERα in kisspeptin cells is required for appropriate differential regulation of these neurons and neuroendocrine output by estradiol.SIGNIFICANCE STATEMENT The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Ovarian estradiol regulates the pattern of GnRH (negative feedback) and initiates a surge of release that triggers ovulation (positive feedback). GnRH neurons do not express the estrogen receptor needed for feedback (estrogen receptor α [ERα]); kisspeptin neurons in the arcuate and anteroventral periventricular nuclei are postulated to mediate negative and positive feedback, respectively. Here we extend the network through which feedback is mediated by demonstrating that glutamatergic transmission to these kisspeptin populations is differentially regulated during the reproductive cycle and by estradiol. Electrophysiological and in vivo hormone profile experiments on kisspeptin-specific ERα knock-out mice demonstrate that ERα in kisspeptin cells is required for appropriate differential regulation of these neurons and for neuroendocrine output.

Food restriction-induced changes in motivation differ with stages of the estrous cycle and are closely linked to RFamide-related peptide-3 but not kisspeptin in Syrian hamsters.

We tested the hypothesis that the effects of food restriction on behavioral motivation are mediated by one or both of the RFamide peptides, RFamide-related peptide-3 (RFRP-3) and kisspeptin (Kp) in female Syrian hamsters (Mesocricetus auratus). Female hamsters fed ad libitum and given a choice between food and adult male hamsters are highly motivated to visit males instead of food on all four days of the estrous cycle, but after 8days of mild food restriction (75% of ad libitum intake) they shift their preference toward food every day of the estrous cycle until the day of estrus, when they shift their preference back toward the males. In support of a role for RFRP-3 in these behavioral changes, the preference for food and the activation of RFRP-3-immunoreactive (Ir) cells in the dorsomedial hypothalamus (DMH) showed the same estrous cycle pattern in food-restricted females, but no association was observed between behavior and the activation of Kp cells in the hypothalamic arcuate nucleus or preoptic area. Next, we tested the hypothesis that food-restriction-induced activation of RFRP-3-Ir cells is modulated by high levels of ovarian steroids at the time of estrus. In support of this idea, on nonestrous days, mild food restriction increased activation of RFRP-3-Ir cells, but failed to do so on the day of estrus even though this level of food restriction did not significantly decrease circulating concentrations of estradiol or progesterone. Furthermore, in ovariectomized females, food-restriction-induced increases in activation of RFRP-3-Ir cells were blocked by systemic treatment with progesterone alone, estradiol plus progesterone, but not estradiol alone. Central infusion with RFRP-3 in ad libitum-fed females significantly decreased sexual motivation and produced significant increases in 90-minute food hoarding, in support of the hypothesis that elevated central levels of RFRP-3 are sufficient to create the shift in behavioral motivation in females fed ad libitum. Together, these results are consistent with the hypothesis that high levels of ingestive motivation are promoted during the nonfertile phase of the estrous cycle by elevated activation of RFRP-3-Ir cells, and RFRP-3-Ir cellular activation is modulated by ovarian steroids around the time of estrus, thereby diverting attention away from food and increasing sexual motivation.

Age-related alterations in hypothalamic kisspeptin, neurokinin B, and dynorphin neurons and in pulsatile LH release in female and male rats.

Pulsatile secretion of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) decreases during aging. Kisspeptin (encoded by Kiss1) neurons in the arcuate nucleus coexpress neurokinin B (Tac3) and dynorphin (Pdyn) and are critical for regulating the GnRH/LH pulse. We therefore examined kisspeptin neurons by histochemistry and pulsatile LH release in rats aged 2-3 (Young), 12-13 (Young-Middle), 19-22 (Late-Middle), and 24-26 (Old) months. Total LH concentrations, sampled for 3 hours, decreased in both sexes with aging. In females, numbers of Tac3 and Pdyn neurons were significantly reduced in all aging rats, and numbers of Kiss1 neurons were significantly reduced in Late-Middle and Old rats. In males, numbers of all 3 neuron-types were significantly decreased in all aging rats. GnRH agonist induced LH release in all animals; however, the increased LH concentration in all aging rats was less than that in Young rats. These results suggest that expression of each gene in kisspeptin neurons may be controlled individually during aging, and that reduction of their expression or change in pituitary responsiveness may cause attenuated pulsatile LH secretion.

Expression of kisspeptin/kiss1r system in developing hypothalamus of female rat and the possible effects on reproduction development and maintenance.

BACKGROUND: The kisspeptin/kiss1r system, expressed in the hypothalamic arcuate nucleus, has been proclaimed as one of the most powerful factors of the reproduction axis, according to recent researches in the reproductive field. The aim of this study was to ascertain the expression of kisspeptin, its receptor (kiss1r), and gonadotropin-releasing hormone (GnRH), and to explore the role on the development and maintenance of the reproductive function of developing female rats. METHODS: Expressions of the kisspeptin/kiss1r system were examined by immunohistochemistry and Real time Quantitative PCR (qRT-PCR). Expressions of estradiol (E2), luteinizing hormone, and follicle-stimulating hormone were analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Expression of the kisspeptin/kiss1r system increased time dependently with aging, and their peak expression was demonstrated in the adult stage. GnRH showed a similar expression pattern to that of the kisspeptin/kiss1r system. ELISA results demonstrated that the E2, luteinizing hormone, and follicle-stimulating hormone secretion increased time dependently from infancy to prepuberty to puberty. However, E2 level decreased significantly in adult rats. Morphological changes of ovaries showed that primordial follicles, primary follicles, and growing follicle inhabited the dominant status in infancy, prepuberty, and puberty stages, respectively. CONCLUSION: GnRH neurons may play an intermediate role in the activation and maintenance of the reproductive function regulated by the kisspeptin/kiss1r system, which may also indirectly regulate the serum level of luteinizing hormone, follicle-stimulating hormone, and E2.

Is the kisspeptin system involved in responses to food restriction in order to preserve reproduction in pubertal male sea bass (Dicentrarchus labrax)?

Previous works on European sea bass have determined that long-term exposure to restrictive feeding diets alters the rhythms of some reproductive/metabolic hormones, delaying maturation and increasing apoptosis during gametogenesis. However, exactly how these diets affect key genes and hormones on the brain-pituitary-gonad (BPG) axis to trigger puberty is still largely unknown. We may hypothesize that all these signals could be integrated, at least in part, by the kisspeptin system. In order to capture a glimpse of these regulatory mechanisms, kiss1 and kiss2 mRNA expression levels and those of their kiss receptors (kiss1r, kiss2r) were analyzed in different areas of the brain and in the pituitary of pubertal male sea bass during gametogenesis. Furthermore, other reproductive hormones and factors as well as the percentage of males showing full spermiation were also analyzed. Treated fish fed maintenance diets provided evidence of overexpression of the kisspeptin system in the main hypophysiotropic regions of the brain throughout the entire sexual cycle. Conversely, Gnrh1 and gonadotropin pituitary content and plasma sexual steroid levels were downregulated, except for Fsh levels, which were shown to increase during spermiation. Treated fish exhibited lower rates of spermiation as compared to control group and a delay in its accomplishment. These results demonstrate how the kisspeptin system and plasma Fsh levels are differentially affected by maintenance diets, causing a retardation, but not a full blockage of the reproductive process in the teleost fish European sea bass. This suggests that a hormonal adaptive strategy may be operating in order to preserve reproductive function in this species.

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.