Targeted inhibition of kisspeptin neurons reverses hyperandrogenemia and abnormal hyperactive LH secretion in a preclinical mouse model of polycystic ovary syndrome.

STUDY QUESTION: Do hyperactive kisspeptin neurons contribute to abnormally high LH secretion and downstream hyperandrogenemia in polycystic ovary syndrome (PCOS)-like conditions and can inhibition of kisspeptin neurons rescue such endocrine impairments? SUMMARY ANSWER: Targeted inhibition of endogenous kisspeptin neuron activity in a mouse model of PCOS reduced the abnormally hyperactive LH pulse secretion and hyperandrogenemia to healthy control levels. WHAT IS KNOWN ALREADY: PCOS is a reproductive disorder characterized by hyperandrogenemia, anovulation, and/or polycystic ovaries, along with a hallmark feature of abnormal LH hyper-pulsatility, but the mechanisms underlying the endocrine impairments remain unclear. A chronic letrozole (LET; aromatase inhibitor) mouse model recapitulates PCOS phenotypes, including polycystic ovaries, anovulation, high testosterone, and hyperactive LH pulses. LET PCOS-like females also have increased hypothalamic kisspeptin neuronal activation which may drive their hyperactive LH secretion and hyperandrogenemia, but this has not been tested. STUDY DESIGN, SIZE, DURATION: Transgenic KissCRE+/hM4Di female mice or littermates Cre- controls were treated with placebo, or chronic LET (50 µg/day) to induce a PCOS-like phenotype, followed by acute (once) or chronic (2 weeks) clozapine-N-oxide (CNO) exposure to chemogenetically inhibit kisspeptin cells (n = 6 to 10 mice/group). PARTICIPANTS/MATERIALS, SETTING, METHODS: Key endocrine measures, including in vivo LH pulse secretion patterns and circulating testosterone levels, were assessed before and after selective kisspeptin neuron inhibition and compared between PCOS groups and healthy controls. Alterations in body weights were measured and pituitary and ovarian gene expression was determined by qRT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Acute targeted inhibition of kisspeptin neurons in PCOS mice successfully lowered the abnormally hyperactive LH pulse secretion (P < 0.05). Likewise, chronic selective suppression of kisspeptin neuron activity reversed the previously high LH and testosterone levels (P < 0.05) down to healthy control levels and rescued reproductive gene expression (P < 0. 05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ovarian morphology was not assessed in this study. Additionally, mouse models can offer mechanistic insights into neuroendocrine processes in PCOS-like conditions but may not perfectly mirror PCOS in women. WIDER IMPLICATIONS OF THE FINDINGS: These data support the hypothesis that overactive kisspeptin neurons can drive neuroendocrine PCOS-like impairments, and this may occur in PCOS women. Our findings complement recent clinical investigations using NKB receptor antagonists to lower LH in PCOS women and suggest that pharmacological dose-dependent modulation of kisspeptin neuron activity may be a valuable future therapeutic target to clinically treat hyperandrogenism and lower elevated LH in PCOS women. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by NIH grants R01 HD111650, R01 HD090161, R01 HD100580, P50 HD012303, R01 AG078185, and NIH R24 HD102061, and a pilot project award from the British Society for Neuroendocrinology. There are no competing interests.

Mistimed restricted feeding disrupts circadian rhythms of male mating behavior and female preovulatory LH surges in mice.

In rodents, eating at atypical circadian times, such as during the biological rest phase when feeding is normally minimal, reduces fertility. Prior findings suggest this fertility impairment is due, at least in part, to reduced mating success. However, the physiological and behavioral mechanisms underlying this reproductive suppression are not known. In the present study, we tested the hypothesis that mistimed feeding-induced infertility is due to a disruption in the normal circadian timing of mating behavior and/or the generation of pre-ovulatory luteinizing hormone (LH) surges (estrogen positive feedback). In the first experiment, male+female mouse pairs, acclimated to be food restricted to either the light (mistimed feeding) or dark (control feeding) phase, were scored for mounting frequency and ejaculations over 96 h. Male mounting behavior and ejaculations were distributed much more widely across the day in light-fed mice than in dark-fed controls and fewer light-fed males ejaculated. In the second experiment, the timing of the LH surge, a well characterized circadian event driven by estradiol (E2) and the SCN, was analyzed from serial blood samples taken from ovariectomized and E2-primed female mice that were light-, dark-, or ad-lib-fed. LH concentrations peaked 2 h after lights-off in both dark-fed and ad-lib control females, as expected, but not in light-fed females. Instead, the normally clustered LH surges were distributed widely with high inter-mouse variability in the light-fed group. These data indicate that mistimed feeding disrupts the temporal control of the neural processes underlying both ovulation and mating behavior, contributing to infertility.

Conditional knockout of kisspeptin signaling in brown adipose tissue increases metabolic rate and body temperature and lowers body weight.

The peptide kisspeptin and its receptor, Kiss1r, act centrally to stimulate reproduction. Evidence indicates that kisspeptin signaling is also important for body weight (BW) and metabolism. We recently reported that Kiss1r KO mice develop obesity, along with reduced metabolism and energy expenditure, independent of estradiol levels. Outside the brain, Kiss1r is expressed in several metabolic tissues, including brown adipose tissue (BAT), but it is unknown which specific tissue is responsible for the metabolic phenotype in Kiss1r KOs. We first determined that global Kiss1r KO mice have significant alterations in body temperature and BAT thermogenic gene expression, perhaps contributing to their obesity. Next, to test whether kisspeptin signaling specifically in BAT influences BW, metabolism, or body temperature, we used Cre/lox technology to generate conditional Kiss1r knockout exclusively in BAT (BAT-Kiss1r KO). Unlike global Kiss1r KOs, BAT-Kiss1r KOs (lacking Kiss1r in just BAT) were not hypogonadal, as expected. Surprisingly, however, BAT-Kiss1r KOs of both sexes displayed significantly lower BW and adiposity than controls. This novel BAT-Kiss1r KO phenotype was of greater magnitude in females and was associated with improved glucose tolerance, increased metabolism, energy expenditure, and locomotor activity, along with increased body temperature and BAT gene expression, specifically Cox8b. Our findings suggest that the previously observed obesity and decreased metabolism in global Kiss1r KOs reflect impaired kisspeptin signaling in non-BAT tissues. However, the novel finding of increased metabolism and body temperature and lower BW in BAT-Kiss1r KOs reveal a previously unidentified role for endogenous kisspeptin signaling in BAT in modulating metabolic and thermogenic physiology.

Primary cilia enhance kisspeptin receptor signaling on gonadotropin-releasing hormone neurons.

Most central neurons in the mammalian brain possess an appendage called a primary cilium that projects from the soma into the extracellular space. The importance of these organelles is highlighted by the fact that primary cilia dysfunction is associated with numerous neuropathologies, including hyperphagia-induced obesity, hypogonadism, and learning and memory deficits. Neuronal cilia are enriched for signaling molecules, including certain G protein-coupled receptors (GPCRs), suggesting that neuronal cilia sense and respond to neuromodulators in the extracellular space. However, the impact of cilia on signaling to central neurons has never been demonstrated. Here, we show that the kisspeptin receptor (Kiss1r), a GPCR that is activated by kisspeptin to regulate the onset of puberty and adult reproductive function, is enriched in cilia projecting from mouse gonadotropin-releasing hormone (GnRH) neurons. Interestingly, GnRH neurons in adult animals are multiciliated and the percentage of GnRH neurons possessing multiple Kiss1r-positive cilia increases during postnatal development in a progression that correlates with sexual maturation. Remarkably, disruption of cilia selectively on GnRH neurons leads to a significant reduction in kisspeptin-mediated GnRH neuronal activity. To our knowledge, this result is the first demonstration of cilia disruption affecting central neuronal activity and highlights the importance of cilia for proper GPCR signaling.

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.

Organizational and activational effects of sex steroids on kisspeptin neuron development.

Kisspeptin, encoded by the Kiss1 gene, is a neuropeptide required for puberty and adult reproductive function. Understanding the regulation and development of the kisspeptin system provides valuable knowledge about the physiology of puberty and adult fertility, and may provide insights into human pubertal or reproductive disorders. Recent studies, particularly in rodent models, have assessed how kisspeptin neurons develop and how hormonal and non-hormonal factors regulate this developmental process. Exposure to sex steroids (testosterone and estradiol) during critical periods of development can induce organizational (permanent) effects on kisspeptin neuron development, with respect to both sexually dimorphic and non-sexually dimorphic aspects of kisspeptin biology. In addition, sex steroids can also impart activational (temporary) effects on kisspeptin neurons and Kiss1 gene expression at various times during neonatal and peripubertal development, as they do in adulthood. Here, we discuss the current knowledge--and in some cases, lack thereof--of the influence of hormones and other factors on kisspeptin neuronal development.

Examination of the influence of leptin and acute metabolic challenge on RFRP-3 neurons of mice in development and adulthood.

BACKGROUND: The neuropeptide RFamide-related peptide-3 (RFRP-3; mammalian ortholog to gonadotropin-inhibiting hormone) can inhibit luteinizing hormone (LH) release and increases feeding, but the regulation and development of RFRP-3 neurons remains poorly characterized, especially in mice. METHODS AND RESULTS: We first confirmed that peripheral injections of murine RFRP-3 peptide could markedly suppress LH secretion in adult mice, as in other species. Second, given RFRP-3's reported orexigenic properties, we performed double-label in situ hybridization for metabolic genes in Rfrp neurons of mice. While Rfrp neurons did not readily coexpress neuropeptide Y, thyrotropin-releasing hormone, or MC4R, a small subset of Rfrp neurons did express the leptin receptor in both sexes. Surprisingly, we identified no changes in Rfrp expression or neuronal activation in adult mice after acute fasting. However, we determined that Rfrp mRNA levels in the dorsal-medial nucleus were significantly reduced in adult obese (Ob) mice of both sexes. Given the lower Rfrp levels observed in adult Ob mice, we asked whether leptin might also regulate RFRP-3 neuron development. Rfrp gene expression changed markedly over juvenile development, correlating with the timing of the juvenile 'leptin surge' known to govern hypothalamic feeding circuit development. However, the dramatic developmental changes in juvenile Rfrp expression did not appear to be leptin driven, as the pattern and timing of Rfrp neuron development were unaltered in Ob juveniles. CONCLUSION: Leptin status modulates RFRP-3 expression in adulthood, but is not required for normal development of the RFRP-3 system. Leptin's regulation of adult RFRP-3 neurons likely occurs primarily via indirect signaling, and may be secondary to obesity, as only a small subset of RFRP-3 neurons express the long form of the leptin receptor (LepRb).

Androgen Inhibition of Reproductive Neuroendocrine Function in Females and Transgender Males.

Ovarian function is controlled by pituitary secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH), which in turn are governed by gonadotropin releasing hormone (GnRH) secreted from the brain. A fundamental principle of reproductive axis regulation is negative feedback signaling by gonadal sex steroids back to the brain to fine-tune GnRH and gonadotropin secretion. Endogenous negative feedback effects can be mimicked by exogenous steroid treatments, including androgens, in both sexes. Indeed, a growing number of clinical and animal studies indicate that high levels of exogenous androgens, in the typically male physiological range, can inhibit LH secretion in females, as occurs in males. However, the mechanisms by which male-level androgens inhibit GnRH and LH secretion still remain poorly understood, and this knowledge gap is particularly pronounced in transgender men (individuals designated female at birth but identifying as male). Indeed, many transgender men take long-term gender-affirming hormone therapy that mimics male-level testosterone levels. The impact of such gender-affirming testosterone on the reproductive axis, both at the ovarian and neuroendocrine level, is a long-understudied area that still requires further investigation. Importantly, the few concepts of androgen actions in females mostly come from studies of polycystic ovary syndrome, which does not recapitulate a similar androgen milieu or a pathophysiology of inhibited LH secretion as occurs in testosterone-treated transgender men. This review summarizes clinical evidence indicating that exogenous androgens can impair neuroendocrine reproductive function in both female individuals and transgender men and highlights emerging experimental data supporting this in recently developed transgender rodent models.

Analysis of multiple positive feedback paradigms demonstrates a complete absence of LH surges and GnRH activation in mice lacking kisspeptin signaling.

Kisspeptin stimulates gonadotropin-releasing hormone (GnRH) neurons via the kisspeptin receptor, Kiss1r. In rodents, estrogen-responsive kisspeptin neurons in the rostral hypothalamus have been postulated to mediate estrogen-induced positive feedback induction of the preovulatory luteinizing hormone (LH) surge. However, conflicting evidence exists regarding the ability of mice lacking Kiss1r to display LH surges in response to exogenous hormones. Whether the discrepancy reflects different mouse strains used and/or utilization of different surge-induction paradigms is unknown. Here, we tested multiple hormonal paradigms in one Kiss1r knockout (KO) model to see which paradigms, if any, could generate circadian-timed LH surges. Kiss1r KO and wild-type (WT) females were ovariectomized, given sex steroids in various modes, and assessed several days later for LH levels in the morning or evening (when surges occur). Serum LH levels were very low in all morning animals, regardless of genotype or hormonal paradigm. In each paradigm, virtually all WT females displayed clear LH surges in the evening, whereas none of the KO females demonstrated LH surges. The lack of LH surges in KO mice reflects a lack of GnRH secretion rather than diminished pituitary responsiveness from a lifetime lack of GnRH exposure because KO mice responded to GnRH priming with robust LH secretion. Moreover, high cfos-GnRH coexpression was detected in WT females in the evening, whereas low cfos-GnRH coexpression was present in KO females at all time points. Our findings conclusively demonstrate that WT females consistently display LH surges under multiple hormonal paradigms, whereas Kiss1r KO mice do not, indicating that kisspeptin-Kiss1r signaling is mandatory for GnRH/LH surge induction.

Lack of functional GABAB receptors alters Kiss1 , Gnrh1 and Gad1 mRNA expression in the medial basal hypothalamus at postnatal day 4.

BACKGROUND/AIMS: Adult mice lacking functional GABAB receptors (GABAB1KO) show altered Gnrh1 and Gad1 expressions in the preoptic area-anterior hypothalamus (POA-AH) and females display disruption of cyclicity and fertility. Here we addressed whether sexual differentiation of the brain and the proper wiring of the GnRH and kisspeptin systems were already disturbed in postnatal day 4 (PND4) GABAB1KO mice. METHODS: PND4 wild-type (WT) and GABAB1KO mice of both sexes were sacrificed; tissues were collected to determine mRNA expression (qPCR), amino acids (HPLC), and hormones (RIA and/or IHC). RESULTS: GnRH neuron number (IHC) did not differ among groups in olfactory bulbs or OVLT-POA. Gnrh1 mRNA (qPCR) in POA-AH was similar among groups. Gnrh1 mRNA in medial basal hypothalamus (MBH) was similar in WTs but was increased in GABAB1KO females compared to GABAB1KO males. Hypothalamic GnRH (RIA) was sexually different in WTs (males > females), but this sex difference was lost in GABAB1KOs; the same pattern was observed when analyzing only the MBH, but not in the POA-AH. Arcuate nucleus Kiss1 mRNA (micropunch-qPCR) was higher in WT females than in WT males and GABAB1KO females. Gad1 mRNA in MBH was increased in GABAB1KO females compared to GABAB1KO males. Serum LH and gonadal estradiol content were also increased in GABAB1KOs. CONCLUSION: We demonstrate that GABABRs participate in the sexual differentiation of the ARC/MBH, because sex differences in several reproductive genes, such as Gad1, Kiss1 and Gnrh1, are critically disturbed in GABAB1KO mice at PND4, probably altering the organization and development of neural circuits governing the reproductive axis.

The development of kisspeptin circuits in the Mammalian brain.

The neuropeptide kisspeptin, encoded by the Kiss1 gene, is required for mammalian puberty and fertility. Examining the development of the kisspeptin system contributes to our understanding of pubertal progression and adult reproduction and sheds light on possible mechanisms underlying the development of reproductive disorders, such as precocious puberty or hypogonadotropic hypogonadism. Recent work, primarily in rodent models, has begun to study the development of kisspeptin neurons and their regulation by sex steroids and other factors at early life stages. In the brain, kisspeptin is predominantly expressed in two areas of the hypothalamus, the anteroventral periventricular nucleus and neighboring periventricular nucleus (pre-optic area in some species) and the arcuate nucleus. Kisspeptin neurons in these two hypothalamic regions are differentially regulated by testosterone and estradiol, both in development and in adulthood, and also display differences in their degree of sexual dimorphism. In this chapter, we discuss what is currently known and not known about the ontogeny, maturation, and sexual differentiation of kisspeptin neurons, as well as their regulation by sex steroids and other factors during development.

The molecular phenotype of kisspeptin neurons in the medial amygdala of female mice.

Reproduction is regulated through the hypothalamic-pituitary-gonadal (HPG) axis, largely via the action of kisspeptin neurons in the hypothalamus. Importantly, Kiss1 neurons have been identified in other brain regions, including the medial amygdala (MeA). Though the MeA is implicated in regulating aspects of both reproductive physiology and behavior, as well as non-reproductive processes, the functional roles of MeA Kiss1 neurons are largely unknown. Additionally, besides their stimulation by estrogen, little is known about how MeA Kiss1 neurons are regulated. Using a RiboTag mouse model in conjunction with RNA-seq, we examined the molecular profile of MeA Kiss1 neurons to identify transcripts that are co-expressed in MeA Kiss1 neurons of female mice and whether these transcripts are modulated by estradiol (E2) treatment. RNA-seq identified >13,800 gene transcripts co-expressed in female MeA Kiss1 neurons, including genes for neuropeptides and receptors implicated in reproduction, metabolism, and other neuroendocrine functions. Of the >13,800 genes co-expressed in MeA Kiss1 neurons, only 45 genes demonstrated significantly different expression levels due to E2 treatment. Gene transcripts such as Kiss1, Gal, and Oxtr increased in response to E2 treatment, while fewer transcripts, such as Esr1 and Cyp26b1, were downregulated by E2. Dual RNAscope and immunohistochemistry was performed to validate co-expression of MeA Kiss1 with Cck and Cartpt. These results are the first to establish a profile of genes actively expressed by MeA Kiss1 neurons, including a subset of genes regulated by E2, which provides a useful foundation for future investigations into the regulation and function of MeA Kiss1 neurons.

Neuroendocrine mechanisms underlying estrogen positive feedback and the LH surge.

A fundamental principle in reproductive neuroendocrinology is sex steroid feedback: steroid hormones secreted by the gonads circulate back to the brain to regulate the neural circuits governing the reproductive neuroendocrine axis. These regulatory feedback loops ultimately act to modulate gonadotropin-releasing hormone (GnRH) secretion, thereby affecting gonadotropin secretion from the anterior pituitary. In females, rising estradiol (E2) during the middle of the menstrual (or estrous) cycle paradoxically "switch" from being inhibitory on GnRH secretion ("negative feedback") to stimulating GnRH release ("positive feedback"), resulting in a surge in GnRH secretion and a downstream LH surge that triggers ovulation. While upstream neural afferents of GnRH neurons, including kisspeptin neurons in the rostral hypothalamus, are proposed as critical loci of E2 feedback action, the underlying mechanisms governing the shift between E2 negative and positive feedback are still poorly understood. Indeed, the precise cell targets, neural signaling factors and receptors, hormonal pathways, and molecular mechanisms by which ovarian-derived E2 indirectly stimulates GnRH surge secretion remain incompletely known. In many species, there is also a circadian component to the LH surge, restricting its occurrence to specific times of day, but how the circadian clock interacts with endocrine signals to ultimately time LH surge generation also remains a major gap in knowledge. Here, we focus on classic and recent data from rodent models and discuss the consensus knowledge of the neural players, including kisspeptin, the suprachiasmatic nucleus, and glia, as well as endocrine players, including estradiol and progesterone, in the complex regulation and generation of E2-induced LH surges in females.

Developmental sex differences in the peri-pubertal pattern of hypothalamic reproductive gene expression, including Kiss1 and Tac2, may contribute to sex differences in puberty onset.

The mechanisms regulating puberty still remain elusive, as do the underlying causes for sex differences in puberty onset (girls before boys) and pubertal disorders. Neuroendocrine puberty onset is signified by increased pulsatile GnRH secretion, yet how and when various upstream reproductive neural circuits change developmentally to govern this process is poorly understood. We previously reported day-by-day peri-pubertal increases (Kiss1, Tac2) or decreases (Rfrp) in hypothalamic gene expression of female mice, with several brain mRNA changes preceding external pubertal markers. However, similar pubertal measures in males were not previously reported. Here, to identify possible neural sex differences underlying sex differences in puberty onset, we analyzed peri-pubertal males and directly compared them with female littermates. Kiss1 expression in male mice increased over the peri-pubertal period in both the AVPV and ARC nuclei but with lower levels than in females at several ages. Likewise, Tac2 expression in the male ARC increased between juvenile and older peri-pubertal stages but with levels lower than females at most ages. By contrast, both DMN Rfrp expressionand Rfrp neuronal activation strongly decreased in males between juvenile and peri-pubertal stages, but with similar levels as females. Neither ARC KNDy neuronal activation nor Kiss1r expression in GnRH neurons differed between males and females or changed with age. These findings delineate several peri-pubertal changes in neural populations in developing males, with notable sex differences in kisspeptin and NKB neuron developmental patterns. Whether these peri-pubertal hypothalamic sex differences underlie sex differences in puberty onset deserves future investigation.

Metabolic actions of kisspeptin signaling: Effects on body weight, energy expenditure, and feeding.

Kisspeptin (encoded by the Kiss1 gene) and its receptor, KISS1R (encoded by the Kiss1r gene), have well-established roles in stimulating reproduction via central actions on reproductive neural circuits, but recent evidence suggests that kisspeptin signaling also influences metabolism and energy balance. Indeed, both Kiss1 and Kiss1r are expressed in many metabolically-relevant peripheral tissues, including both white and brown adipose tissue, the liver, and the pancreas, suggesting possible actions on these tissues or involvement in their physiology. In addition, there may be central actions of kisspeptin signaling, or factors co-released from kisspeptin neurons, that modulate metabolic, feeding, or thermoregulatory processes. Accumulating data from animal models suggests that kisspeptin signaling regulates a wide variety of metabolic parameters, including body weight and energy expenditure, adiposity and adipose tissue function, food intake, glucose metabolism, respiratory rates, locomotor activity, and thermoregulation. Herein, the current evidence for the involvement of kisspeptin signaling in each of these physiological parameters is reviewed, gaps in knowledge identified, and future avenues of important research highlighted. Collectively, the discussed findings highlight emerging non-reproductive actions of kisspeptin signaling in metabolism and energy balance, in addition to previously documented roles in reproductive control, but also emphasize the need for more research to resolve current controversies and uncover underlying molecular and physiological mechanisms.

Conditional Deletion of KOR (Oprk1) in Kisspeptin Cells Does Not Alter LH Pulses, Puberty, or Fertility in Mice.

Classic pharmacological studies suggested that endogenous dynorphin-KOR signaling is important for reproductive neuroendocrine regulation. With the seminal discovery of an interconnected network of hypothalamic arcuate neurons co-expressing kisspeptin, neurokinin B, and dynorphin (KNDy neurons), the KNDy hypothesis was developed to explain how gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) pulses are generated. Key to this hypothesis is dynorphin released from KNDy neurons acting in a paracrine manner on other KNDy neurons via kappa opioid receptor (KOR) signaling to terminate neural "pulse" events. While in vitro evidence supports this aspect of the KNDy hypothesis, a direct in vivo test of the necessity of KOR signaling in kisspeptin neurons for proper LH secretion has been lacking. We therefore conditionally knocked out KOR selectively from kisspeptin neurons of male and female mice and tested numerous reproductive measures, including in vivo LH pulse secretion. Surprisingly, despite validating successful knockout of KOR in kisspeptin neurons, we found no significant effect of kisspeptin cell-specific deletion of KOR on any measure of puberty, LH pulse parameters, LH surges, follicle-stimulating hormone (FSH) levels, estrous cycles, or fertility. These outcomes suggest that the KNDy hypothesis, while sufficient normally, may not be the only neural mechanism for sculpting GnRH and LH pulses, supported by recent findings in humans and mice. Thus, besides normally acting via KOR in KNDy neurons, endogenous dynorphin and other opioids may, under some conditions, regulate LH and FSH secretion via KOR in non-kisspeptin cells or perhaps via non-KOR pathways. The current models for GnRH and LH pulse generation should be expanded to consider such alternate mechanisms.

Kisspeptin impacts on circadian and ultradian rhythms of core body temperature: Evidence in kisspeptin receptor knockout and kisspeptin knockdown mice.

Kisspeptin is vital for the regulation of both fertility and metabolism. Kisspeptin receptor (Kiss1r) knockout (KO) mice exhibit increased adiposity and reduced energy expenditure in adulthood. Kiss1r mRNA is expressed in brown adipose tissue (BAT) and Kiss1r KO mice exhibit reduced Ucp1 mRNA in BAT and impaired thermogenesis. We hypothesised that mice with diminished kisspeptin signalling would exhibit reduced core body temperature (Tc) and altered dynamics of circadian and ultradian rhythms of Tc. Tc was recorded every 15-min over 14-days in gonadectomised wild-type (WT), Kiss1r KO, and also Kiss1-Cre (95% reduction in Kiss1 transcription) mice. Female Kiss1r KOs had higher adiposity and lower Ucp1 mRNA in BAT than WTs. No change was detected in Kiss1-Cre mice. Mean Tc during the dark phase was lower in female Kiss1r KOs versus WTs, but not Kiss1-Cre mice. Female Kiss1r KOs had a lower mesor and amplitude of the circadian rhythm of Tc than did WTs. In WT mice, there were more episodic ultradian events (EUEs) of Tc during the dark phase than the light phase, but this measure was similar between dark and light phases in Kiss1r KO and Kiss1-Cre mice. The amplitude of EUEs was higher in the dark phase in female Kiss1r KO and male Kiss1-Cre mice. Given the lack of clear metabolic phenotype in Kiss1-Cre mice, 5% of Kiss1 transcription may be sufficient for proper metabolic control, as was shown for fertility. Moreover, the observed alterations in Tc suggest that kisspeptin has a role in circadian and ultradian rhythm-driven pathways.

Estrogen Regulation of the Molecular Phenotype and Active Translatome of AVPV Kisspeptin Neurons.

In females, ovarian estradiol (E2) exerts both negative and positive feedback regulation on the neural circuits governing reproductive hormone secretion, but the cellular and molecular mechanisms underlying this remain poorly understood. In rodents, estrogen receptor α-expressing kisspeptin neurons in the hypothalamic anteroventral periventricular region (AVPV) are prime candidates to mediate E2 positive feedback induction of preovulatory gonadotropin-releasing hormone and luteinizing hormone (LH) surges. E2 stimulates AVPV Kiss1 expression, but the full extent of estrogen effects in these neurons is unknown; whether E2 stimulates or inhibits other genes in AVPV Kiss1 cells has not been determined. Indeed, understanding of the function(s) of AVPV kisspeptin cells is limited, in part, by minimal knowledge of their overall molecular phenotype, as only a few genes are currently known to be co-expressed in AVPV Kiss1 cells. To provide a more detailed profiling of co-expressed genes in AVPV Kiss1 cells, including receptors and other signaling factors, and test how these genes respond to E2, we selectively isolated actively translated mRNAs from AVPV Kiss1 cells of female mice and performed RNA sequencing (RNA-seq). This identified >13 000 mRNAs co-expressed in AVPV Kiss1 cells, including multiple receptor and ligand transcripts positively or negatively regulated by E2. We also performed RNAscope to validate co-expression of several transcripts identified by RNA-seq, including Pdyn (prodynorphin), Penk (proenkephalin), Vgf (VGF), and Cartpt (CART), in female AVPV Kiss1 cells. Given the important role of AVPV kisspeptin cells in positive feedback, E2 effects on identified genes may relate to the LH surge mechanism and/or other physiological processes involving these cells.

Progesterone Receptors in AVPV Kisspeptin Neurons Are Sufficient for Positive Feedback Induction of the LH Surge.

Kisspeptin, encoded by Kiss1, stimulates gonadotropin-releasing hormone neurons to govern reproduction. In female rodents, estrogen-sensitive kisspeptin neurons in the rostral anteroventral periventricular (AVPV) hypothalamus are thought to mediate estradiol (E2)-induced positive feedback induction of the preovulatory luteinizing hormone (LH) surge. AVPV kisspeptin neurons coexpress estrogen and progesterone receptors (PGRs) and are activated during the LH surge. While E2 effects on kisspeptin neurons have been well studied, progesterone's regulation of kisspeptin neurons is less understood. Using transgenic mice lacking PGR exclusively in kisspeptin cells (termed KissPRKOs), we previously demonstrated that progesterone action specifically in kisspeptin cells is essential for ovulation and normal fertility. Unlike control females, KissPRKO females did not generate proper LH surges, indicating that PGR signaling in kisspeptin cells is required for positive feedback. However, because PGR was knocked out from all kisspeptin neurons in the brain, that study was unable to determine the specific kisspeptin population mediating PGR action on the LH surge. Here, we used targeted Cre-mediated adeno-associated virus (AAV) technology to reintroduce PGR selectively into AVPV kisspeptin neurons of adult KissPRKO females, and tested whether this rescues occurrence of the LH surge. We found that targeted upregulation of PGR in kisspeptin neurons exclusively in the AVPV is sufficient to restore proper E2-induced LH surges in KissPRKO females, suggesting that this specific kisspeptin population is a key target of the necessary progesterone action for the surge. These findings further highlight the critical importance of progesterone signaling, along with E2 signaling, in the positive feedback induction of LH surges and ovulation.

Discovery of potent kisspeptin antagonists delineate physiological mechanisms of gonadotropin regulation.

Neurons that produce gonadotropin-releasing hormone (GnRH) are the final common pathway by which the brain regulates reproduction. GnRH neurons are regulated by an afferent network of kisspeptin-producing neurons. Kisspeptin binds to its cognate receptor on GnRH neurons and stimulates their activity, which in turn provides an obligatory signal for GnRH secretion, thus gating down-stream events supporting reproduction. We have developed kisspeptin antagonists to facilitate the direct determination of the role of kisspeptin neurons in the neuroendocrine regulation of reproduction. In vitro and in vivo studies of analogues of kisspeptin-10 with amino substitutions have identified several potent and specific antagonists. A selected antagonist was shown to inhibit the firing of GnRH neurons in the brain of the mouse and to reduce pulsatile GnRH secretion in female pubertal monkeys; the later supporting a key role of kisspeptin in puberty onset. This analog also inhibited the kisspeptin-induced release of luteinizing hormone (LH) in rats and mice and blocked the postcastration rise in LH in sheep, rats, and mice, suggesting that kisspeptin neurons mediate the negative feedback effect of sex steroids on gonadotropin secretion in mammals. The development of kisspeptin antagonists provides a valuable tool for investigating the physiological and pathophysiological roles of kisspeptin in the regulation of reproduction and could offer a unique therapeutic agent for treating hormone-dependent disorders of reproduction, including precocious puberty, endometriosis, and metastatic prostate cancer.