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.

Combined effects of obesity and di-(2-ethylhexyl) phthalate on testosterone levels and kisspeptin/GPR54 expression in hypothalamus and testes of male mice.

BACKGROUND: This study evaluated whether obese male mice exposed to di-(2-ethylhexyl) phthalate (DEHP) showed synergistic effects on testosterone levels and the potential underlying mechanism. METHODS: Forty-eight male mice were assigned to six groups for 12-week treatments as follows: normal, DEHP100, diet-induced obesity (DIO), DIO + DEHP30, DIO + DEHP100, and DIO + DEHP300. Serum hormone levels, including testosterone (T), luteinizing hormone (LH), and leptin, were detected by ELISA. The levels of Ob-R, kisspeptin, and GPR54 protein expression in hypothalamus and testicular tissues were measured by western blot. RESULTS: There were significantly lower levels of serum T and LH, higher levels of serum leptin and Ob-R, and kisspeptin and GPR54 protein expression were reduced in hypothalamus and testicular tissues in the DIO and DEHP groups compared with controls. Moreover, serum T and leptin levels were more severe in the combined DIO and DEHP exposure group than in the single exposure groups. Serum LH levels and GPR54 expression in the testis were significantly decreased in DIO + DEHP300 mice compared with DIO mice (p < 0.05). CONCLUSION: Obesity- and DEHP-only exposure had adverse effects on testosterone levels in mice, which may be due to high leptin levels and decreased Ob-R, kisspeptin, and GPR54 expression. Obesity combined with DEHP exposure had an additive adverse effect on testosterone levels in mice. One of the potential mechanisms is higher leptin levels and decreased GPR54 expression in the testes.

Neuroendocrine disruption is associated to infertility in chronically stressed female rats.

Infertility is a growing worldwide public health problem, and stress is a main factor exerting detrimental effects on female reproduction. However, knowledge regarding the neuroendocrine changes caused by chronic stress in females is limited. Therefore, this study assessed the effects of stress on hormones that control female reproduction during the proestrus and diestrus stages of the estrous cycle, as well as its effects on fertility. Adult females were assigned to either a control or a stress group. Stress consisted of exposure, for 15 min, to cold-water immersion daily for 30 days. Estrous cyclicity, female sexual behavior, as well as hypothalamic kisspeptin, gonadotropin releasing hormone (GnRH) content, serum luteinizing hormone (LH), estradiol (E2), progesterone (P4), corticosterone (CORT) and fertility were assessed after chronic stress. The results show that chronically stressed females exhibited disrupted estrous cyclicity, decreased receptivity, low pregnancy rates and lower numbers of fetuses. The content of Kisspeptin and GnRH in the Anteroventral Periventricular/medial Preoptic Area decreased during proestrus, while Kisspeptin increased in the Arcuate nucleus in proestrus and diestrus. Serum LH decreased only during proestrus, whereas E2 and P4 concentrations decreased during proestrus and diestrus, with a concomitant increase in CORT levels in both stages. As a whole, these results indicate that chronic stress decreases Kisspeptin content in AVPV nucleus and GnRH in POA in females, and might induce disruption of the LH surge, consequently disrupting estrous cyclicity and fertility, leading to lower rates of pregnancy and number of fetuses.

Reduced Kiss­1 expression is associated with clinical aggressive feature of gastric cancer patients and promotes migration and invasion in gastric cancer cells.

Gastric cancer (GC) causes high morbidity and mortality in patients largely due to its invasion and metastasis. Kiss­1 has been shown to be a metastasis suppressor in various malignancies. However, its clinical significance and biological functions in GC have not been thoroughly investigated. The present study investigated the association between Kiss­1 expression and its methylation status and clinicopathological features in GC. Kiss­1 expression was reduced in GC and its low expression was associated with poor histological grade, lymph node metastasis and TNM III+IV stage. Kiss­1 overexpression in AGS GC cells significantly inhibited cell proliferation, migration and invasion in vitro. Kiss­1 knockdown promoted the proliferation, migration and invasion of HGC­27 cells. In summary, the data demonstrated that a low expression of Kiss­1 played a suppressive role for the proliferation, migration and invasion of GC cells. Its expression and methylation levels were associated with the clinical progression of GC. Thus, Kiss­1 is a potential diagnostic and prognostic marker as well as a new target for the treatment of GC.

Experimental characterization of a biosensor based on a tapered optical fiber for kisspeptin detection.

This paper presents the development of a biosensor based on optical fiber, using a polyclonal antibody kisspeptin receptor as a biological recognition element that is connected to puberty onset and may also help to suppress metastasis in melanoma breast cancer. The fiber surface was chemically prepared to immobilize the antibody. The structural homogeneity of the biosensor, at each stage of the self-assembly, was characterized by Fourier transform infrared spectroscopy and by measurements of the transmission at the output of the biosensor. The morphological homogeneity analysis was performed by optical microscopy and scanning electron microscopy. The biosensor developed was checked to detect kisspeptin in brain tissues by spectral transmission using a superluminescent diode. The data were analyzed using principal component analysis. The interaction of the kisspeptin with its counterpart by means of the evolution of the transmission spectrum as a function of time was observed.

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.

The dromedary camel displays annual variation in hypothalamic kisspeptin and Arg-Phe-amide-related peptide-3 according to sex, season, and breeding activity.

The dromedary camel (Camelus dromedarius) is a desert mammal whose cycles in reproductive activity ensure that the offspring's birth and weaning coincide with periods of abundant food resources and favorable climate conditions. In this study, we assessed whether kisspeptin (Kp) and arginine-phenylalanine (RF)-amide related peptide-3 (RFRP-3), two hypothalamic peptides known to regulate the mammalian hypothalamo-pituitary gonadal axis, may be involved in the seasonal control of camel's reproduction. Using specific antibodies and riboprobes, we found that Kp neurons are present in the preoptic area (POA), suprachiasmatic (SCN), and arcuate (ARC) nuclei, and that RFRP-3 neurons are present in the paraventricular (PVN), dorsomedial (DMH), and ventromedial (VMH) hypothalamic nuclei. Kp fibers are found in various hypothalamic areas, notably the POA, SCN, PVN, DMH, VMH, supraoptic nucleus, and the ventral and dorsal premammillary nucleus. RFRP-3 fibers are found in the POA, SCN, PVN, DMH, VMH, and ARC. POA and ARC Kp neurons and DMH RFRP-3 neurons display sexual dimorphism with more neurons in female than in male. Both neuronal populations display opposed seasonal variations with more Kp neurons and less RFRP-3 neurons during the breeding (December-January) than the nonbreeding (July-August) season. This study is the first describing Kp and RFRP-3 in the camel's brain with, during the winter period lower RFRP-3 expression and higher Kp expression possibly responsible for the HPG axis activation. Altogether, our data indicate the involvement of both Kp and RFRP-3 in the seasonal control of the dromedary camel's breeding activity.

Genetic dissection of the different roles of hypothalamic kisspeptin neurons in regulating female reproduction.

The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Estradiol induces negative feedback on pulsatile GnRH/luteinizing hormone (LH) release and positive feedback generating preovulatory GnRH/LH surges. Negative and positive feedbacks are postulated to be mediated by kisspeptin neurons in arcuate and anteroventral periventricular (AVPV) nuclei, respectively. Kisspeptin-specific ERα knockout mice exhibit disrupted LH pulses and surges. This knockout approach is neither location-specific nor temporally controlled. We utilized CRISPR-Cas9 to disrupt ERα in adulthood. Mice with ERα disruption in AVPV kisspeptin neurons have typical reproductive cycles but blunted LH surges, associated with decreased excitability of these neurons. Mice with ERα knocked down in arcuate kisspeptin neurons showed disrupted cyclicity, associated with increased glutamatergic transmission to these neurons. These observations suggest that activational effects of estradiol regulate surge generation and maintain cyclicity through AVPV and arcuate kisspeptin neurons, respectively, independent from its role in the development of hypothalamic kisspeptin neurons or puberty onset.

Expression of tachykinin3 and related reproductive markers in the brain of the African cichlid fish Astatotilapia burtoni.

Neurokinin B, encoded by the tachykinin3 gene, plays a crucial role in regulating reproduction in mammals via KNDy neurons and interaction with GnRH. Previous work in teleost fishes has focused on hypothalamic tac3 expression for its role in reproduction, but detailed studies on extra-hypothalamic tac3 expression are limited. Here, we identified two tac3 genes in the social African cichlid fish Astatotilapia burtoni, only one of which produces a functional protein containing the signature tachykinin motif. In situ hybridization for tac3a mRNA identified cell populations throughout the brain. Numerous tac3a cells lie in several thalamic and hypothalamic nuclei, including periventricular nucleus of posterior tuberculum, lateral tuberal nucleus (NLT), and nucleus of the lateral recess (NRL). Scattered tac3-expressing cells are also present in telencephalic parts, such as ventral (Vv) and supracomissural (Vs) part of ventral telencephalon. In contrast to other teleosts, tac3 expression was absent from the pituitary. Using double-fluorescent staining, we localized tac3a-expressing cells in relation to GnRH and kisspeptin cells. Although no GnRH-tac3a colabeled cells were observed, dense GnRH fibers surround and potentially synapse with tac3a cells in the preoptic area. Only minimal (<5%) colabeling of tac3a was observed in kiss2 cells. Despite tac3a expression in many nodes of the mesolimbic reward system, it was absent from tyrosine hydroxylase (TH)-expressing cells, but tac3a cells were located in areas with dense TH fibers. The presence of tac3a-expressing cells throughout the brain, including in socially relevant brain regions, suggest more diverse functions beyond regulation of reproductive physiology that may be conserved across vertebrates.

Evaluation of the correlation of MACC1, CD44, Twist1, and KiSS-1 in the metastasis and prognosis for colon carcinoma.

BACKGROUND: Metastasis-associated in colon cancer 1 (MACC1) has been reported to promote tumor cell invasion and metastasis. Cancer stem cells and epithelial-mesenchymal transition (EMT) have also been reported to promote tumor cell proliferation, invasion, and metastasis. KiSS-1, a known suppressor of metastasis, has been reported to be down-regulated in various tumors. However, the associations of MACC1, CD44, Twist1, and KiSS-1 in colonic adenocarcinoma (CAC) invasion and metastasis remain unclear. The purpose of this study is to investigate the roles of MACC1, CD44, Twist1, and KiSS-1 in CAC invasion and metastasis and their associations with each other and with the clinicopathological characteristics of CAC patients. METHODS: Immunohistochemistry and multivariate analysis were carried out to explore the expression of MACC1, CD44, Twist1, and KiSS-1 in 212 whole-CAC-tissue specimens and the corresponding normal colon mucosa tissues. Demographic, clinicopathological, and follow-up data were also collected. RESULTS: The results of this study showed MACC1, CD44, and Twist1 expression to be up-regulated, and KiSS-1 expression was down-regulated in CAC tissues. Positive expression of MACC1, CD44, and Twist1 was found to be positively correlated with invasion, tumor grades, and lymph- node-metastasis (LNM) stages and tumor-node-metastasis (TNM) stages for patients with CAC. Positive expression of KiSS-1 was inversely associated with invasion, tumor size, LNM stage, and TNM stage. The KiSS-1-positive expression group had significantly more favorable OS than did the KiSS-1-negative group. Univariate analysis indicated that overexpression of MACC1, CD44, and Twists1 was negatively associated with longer overall survival (OS) time, and there was a positive relationship between KiSS-1-positive expression and OS time for patients with CAC. Multivariate Cox analysis demonstrated that overexpression of MACC1, CD44, Twist1, and low expression of KiSS-1 and LNM and TNM stages were independent predictors of prognosis in patients with CAC. CONCLUSIONS: The results in this study indicated that levels of expression of MACC1, CD44, Twist1, and KiSS-1 are related to the duration of OS in patients with CAC. MACC1, CD44, Twist1, and KiSS-1 may be suitable for use as biomarkers and therapeutic targets in CAC.

The expression of kisspeptin and its receptor in the domestic cat ovary and uterus in different stages of the ovarian cycle.

Kisspeptin is well known for its indispensable role in the regulation of reproduction, mainly through controlling the release of GnRH at the hypothalamic level. Recent studies have shown that kisspeptin and the kisspeptin receptor are expressed in the ovary and uterus, indicating an additional local function in reproduction at the extra-hypothalamic level. In this study, we aimed: (1) to investigate the localization pattern of kisspeptin and its receptor in the domestic cat ovary and uterus throughout the ovarian cycle using immunohistochemistry; and (2) to compare the relative expression of ovarian Kiss1 mRNA levels at different ovarian stages with qPCR analysis. Ovaries and uteri were collected and classified into three ovarian stages (inactive, follicular and luteal stages (n = 7 in each stage)) according to the ovarian morphology, vaginal cytology and serum progesterone. Kisspeptin immunoreactivity (Kp-IR) and kisspeptin receptor immunoreactivity (KpR-IR) were present in the ovaries and uteri at all ovarian stages, with no notable differences in the localization patterns between the ovarian stages. In the ovary, Kp-IR and KpR-IR were present in various ovarian compartments, including the follicles at all classes and the corpus luteum (CL). In the follicles, Kp-IR and KpR-IR were present in the oocytes, granulosa cells and theca cells. Kp-IR was also detected in the follicular fluid of antral follicles. In CL, a strong intensity of Kp-IR was present in the periphery CL of development/maintenance, with a relatively fainter intensity in the central CL. By contrast, KpR-IR was present in both peripheral and central CL at the same intensity. In the uterus, Kp-IR and KpR-IR were present in the uterine glands, myometrium and perimetrium. The relative ovarian Kiss1 mRNA level was higher in the follicular stage than in the luteal stage (P < 0.05). We concluded that kisspeptin and its receptor are present in the cat ovary and uterus, suggesting possible local functions of kisspeptin at the extra-hypothalamic level, such as folliculogenesis, oocyte survival and uterine adenogenesis.

Presence and function of kisspeptin/KISS1R system in swine ovarian follicles.

Kisspeptin and its receptor KISS1R are involved in the neuroendocrine regulation of mammalian reproduction and their role on follicular development and function can be hypothesized. The present work was designed to confirm the immunopresence of kisspeptin and its receptor in the ovary of swine and to study the effects of kisspeptin 10 and its antagonist, kisspeptin 234, on main functional parameters of granulosa cells (i.e. cell proliferation, steroid production, and redox status) as well as their modulatory action on angiogenesis. The immunopresence of kisspeptin and KISS1R were detected in granulosa cells. Kisspeptin 10 stimulated progesterone in vitro production, thus indirectly suggesting that it can have a role in the luteinization process of granulosa cells. Kisspeptin 10 displayed potentiating effects on non-enzymatic scavenging activity, thus supporting its involvement in the control of the antioxidant defense system of ovarian follicles. In addition, results from the angiogenesis bioassay suggest that kisspeptin may have a role in the physiological development of new ovarian vessels. Additional studies are needed to confirm the functional significance of the kisspeptin/KISS1R system within the swine ovary.

Expression of GnRH receptor in the canine corpus luteum, and luteal function following deslorelin acetate-induced puberty delay.

The goals of this study were as follows: (Experiment 1) to examine the basic capability of canine corpora lutea (CL) to respond to GnRH by assessing expression of gonadotropin-releasing hormone receptor (GnRH-R) in luteal samples collected throughout the luteal lifespan from non-pregnant dogs, and (Experiment 2) to investigate the effects of pre-pubertal application of the GnRH agonist deslorelin acetate on luteal function following the first oestrus. Mature CL were collected during the mid-luteal phase (days 30-45) from treated and control bitches. Transcript levels of several factors were determined: estrogen receptors (ESR1/ERα, ESR2/ERß), progesterone (P4)-receptor (PGR), prolactin receptor (PRLR), PGE2-synthase (PTGES) and PGE2 receptors (PTGER2/EP2, PTGER4/EP4), vascular endothelial growth factor (VEGFA) and VEGF receptors (VEGFR1 and VEGFR2), cyclooxygenase 2 (COX2/PTGS2), steroidogenic acute regulatory protein (STAR) and 3ß-hydroxysteroid dehydrogenase (3ßHSD). Additionally, levels of Kisspeptin 1 (Kiss1) and its receptor (KISS1-R) were evaluated. Although generally low, GnRH-R expression was time dependent and was elevated during early dioestrus, with a significant decrease towards luteal regression. In deslorelin-treated and control dogs, its expression was either low or frequently below the detection limit. EP2 and VEGFR1 were higher in the treated group, which could be caused by a feedback mechanism after long-term suppression of reproductive activity. Despite large individual variations, 3ßHSD was higher in the deslorelin-treated group. This, along with unchanged STAR expression, was apparently not mirrored in increased luteal functionality, because similar P4 levels were detected in both groups. Finally, the deslorelin-mediated long-term delay of puberty does not have negative carry-over effects on subsequent ovarian functionality in bitches.

Hypothalamic transcriptional expression of the kisspeptin system and sex steroid receptors differs among polycystic ovary syndrome rat models with different endocrine phenotypes.

OBJECTIVES:: Polycystic ovary syndrome is a heterogeneous endocrine disorder that affects reproductive-age women. The mechanisms underlying the endocrine heterogeneity and neuroendocrinology of polycystic ovary syndrome are still unclear. In this study, we investigated the expression of the kisspeptin system and gonadotropin-releasing hormone pulse regulators in the hypothalamus as well as factors related to luteinizing hormone secretion in the pituitary of polycystic ovary syndrome rat models induced by testosterone or estradiol. METHODS:: A single injection of testosterone propionate (1.25 mg) (n=10) or estradiol benzoate (0.5 mg) (n=10) was administered to female rats at 2 days of age to induce experimental polycystic ovary syndrome. Controls were injected with a vehicle (n=10). Animals were euthanized at 90-94 days of age, and the hypothalamus and pituitary gland were used for gene expression analysis. RESULTS:: Rats exposed to testosterone exhibited increased transcriptional expression of the androgen receptor and estrogen receptor-ß and reduced expression of kisspeptin in the hypothalamus. However, rats exposed to estradiol did not show any significant changes in hormone levels relative to controls but exhibited hypothalamic downregulation of kisspeptin, tachykinin 3 and estrogen receptor-α genes and upregulation of the gene that encodes the kisspeptin receptor. CONCLUSIONS:: Testosterone- and estradiol-exposed rats with different endocrine phenotypes showed differential transcriptional expression of members of the kisspeptin system and sex steroid receptors in the hypothalamus. These differences might account for the different endocrine phenotypes found in testosterone- and estradiol-induced polycystic ovary syndrome rats.

Hypothalamic transcriptional expression of the kisspeptin system and sex steroid receptors differs among polycystic ovary syndrome rat models with different endocrine phenotypes

OBJECTIVES: Polycystic ovary syndrome is a heterogeneous endocrine disorder that affects reproductive-age women. The mechanisms underlying the endocrine heterogeneity and neuroendocrinology of polycystic ovary syndrome are still unclear. In this study, we investigated the expression of the kisspeptin system and gonadotropin-releasing hormone pulse regulators in the hypothalamus as well as factors related to luteinizing hormone secretion in the pituitary of polycystic ovary syndrome rat models induced by testosterone or estradiol. METHODS: A single injection of testosterone propionate (1.25 mg) (n=10) or estradiol benzoate (0.5 mg) (n=10) was administered to female rats at 2 days of age to induce experimental polycystic ovary syndrome. Controls were injected with a vehicle (n=10). Animals were euthanized at 90-94 days of age, and the hypothalamus and pituitary gland were used for gene expression analysis. RESULTS: Rats exposed to testosterone exhibited increased transcriptional expression of the androgen receptor and estrogen receptor-β and reduced expression of kisspeptin in the hypothalamus. However, rats exposed to estradiol did not show any significant changes in hormone levels relative to controls but exhibited hypothalamic downregulation of kisspeptin, tachykinin 3 and estrogen receptor-α genes and upregulation of the gene that encodes the kisspeptin receptor. CONCLUSIONS: Testosterone- and estradiol-exposed rats with different endocrine phenotypes showed differential transcriptional expression of members of the kisspeptin system and sex steroid receptors in the hypothalamus. These differences might account for the different endocrine phenotypes found in testosterone- and estradiol-induced polycystic ovary syndrome rats.

Visualisation of Kiss1 Neurone Distribution Using a Kiss1-CRE Transgenic Mouse.

Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE-GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss-CRE heterozygous mice with CRE-activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80-90% of tdTomato positive neurones in the ARC were co-labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato-labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre-optic areas and to the lateral hypothalamus.

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.

Kisspeptin is involved in ovarian follicular development during aging in rats.

We have previously reported that kisspeptin (KP) may be under the control of the sympathetic innervation of the ovary. Considering that the sympathetic activity of the ovary increases with aging, it is possible that ovarian KP also increases during this period and participates in follicular development. To evaluate this possibility, we determined ovarian KP expression and its action on follicular development during reproductive aging in rats. We measured ovarian KP mRNA and protein levels in 6-, 8-, 10- and 12-month-old rats. To evaluate follicular developmental changes, intraovarian administration of KP or its antagonist, peptide 234 (P234), was performed using a mini-osmotic pump, and to evaluate FSH receptor (FSHR) changes in the senescent ovary, we stimulated cultured ovaries with KP, P234 and isoproterenol (ISO). Our results shows that KP expression in the ovary was increased in 10- and 12-month-old rats compared with 6-month-old rats, and this increase in KP was strongly correlated with the increase in ovarian norepinephrine observed with aging. The administration of KP produced an increase in corpora lutea and type III follicles in 6- and 10-month-old rats, which was reversed by P234 administration at 10 months. In addition, KP decreased the number and size of antral follicles in 6- and 10-month-old rats, while P234 administration produced an increase in these structures at the same ages. In ovarian cultures KP prevented the induction of FSHR by ISO. These results suggest that intraovarian KP negatively participates in the acquisition of FSHR, indicating a local role in the regulation of follicular development and ovulation during reproductive aging.

Unpredictable chronic stress-induced reproductive suppression associated with the decrease of kisspeptin immunoreactivity in male mice.

Environmental stress affects various parts of mammals typically through the circulation of stress hormones. It has been identified as one of the possible reasons for male reproductive difficulties, but the complex mechanisms responsible for stress-induced reproductive suppression are poorly understood. Here, we examined the relationship between chronic environmental stress and hypothalamic kisspeptin, a recently discovered upstream regulator of the reproductive endocrine feedback system. We studied male mice under an unpredictable chronic stress procedure to replicate the situation of animals under chronic stress. Histological and immunohistochemical analyses were performed focusing on kisspeptin neurons in the arcuate hypothalamic nucleus (ARC) and DNA fragmented cells in seminiferous tubules. Although the ARC was not morphologically altered in either the stressed or non-stressed group, granular kisspeptin immunoreactivities decreased slightly in the stress group. In the testes of the stress group, several signs of testicular degeneration were observed, including increased numbers of ssDNA-positive cells per seminiferous tubule, thinning, vacuoled seminiferous epithelia and multinucleated giant cells. The decreases in kisspeptin in the stress group might be due to other hypothalamic peptides, such as corticotropin-releasing hormone and leptin, whose receptors are known to coexpress in the ARC. In addition, environmental stress directly and indirectly affects testicular function through stress hormones and gonadotropins. In summary, our findings enhance the understanding of stress-induced reproductive suppression possibly mediated by kisspeptin in the ARC.

Kisspeptin, c-Fos and CRFR type 2 co-expression in the hypothalamus after insulin-induced hypoglycaemia.

Normal reproductive function is dependent upon availability of glucose and insulin-induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28 h after progesterone withdrawal (PW), animals received saline (n = 6) or insulin (4 IU/kg; n = 5) and were subsequently killed at 31 h after PW (i.e., 3 h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c-Fos (a marker of neuronal activation) or CRFR type 2. Within 3 h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p < 0.05 for both). In the arcuate nucleus (ARC), insulin-treated ewes had an increased expression of c-Fos. Furthermore, the percentage of kisspeptin cells co-expressing c-Fos increased in the ARC (from 11 to 51%; p < 0.05), but there was no change in the medial pre-optic area (mPOA; 14 vs 19%). CRFR type 2 expression in the lower part of the ARC and the median eminence was not altered by insulin treatment. Thus, disruption of the LH surge after IIH in the follicular phase is not associated with decreased kisspeptin cell activation or an increase in CRFR type 2 in the ARC but may involve other cell types located in the ARC nucleus which are activated in response to IIH.