Early overnutrition sensitizes the growth hormone axis to the impact of diet-induced obesity via sex-divergent mechanisms.

In addition to its essential role in the physiological control of longitudinal growth, growth-hormone (GH) is endowed with relevant metabolic functions, including anabolic actions in muscle, lipolysis in adipose-tissue and glycemic modulation. Adult obesity is known to negatively impact GH-axis, thereby promoting a vicious circle that may contribute to the exacerbation of the metabolic complications of overweight. Yet, to what extent early-overnutrition sensitizes the somatotropic-axis to the deleterious effects of obesity remains largely unexplored. Using a rat-model of sequential exposure to obesogenic insults, namely postnatal-overfeeding during lactation and high-fat diet (HFD) after weaning, we evaluated in both sexes the individual and combined impact of these nutritional challenges upon key elements of the somatotropic-axis. While feeding HFD per se had a modest impact on the adult GH-axis, early overnutrition had durable effects on key elements of the somatotropic-system, which were sexually different, with a significant inhibition of pituitary gene expression of GH-releasing hormone-receptor (GHRH-R) and somatostatin receptor-5 (SST5) in males, but an increase in pituitary GHRH-R, SST2, SST5, GH secretagogue-receptor (GHS-R) and ghrelin expression in females. Notably, early-overnutrition sensitized the GH-axis to the deleterious impact of HFD, with a significant suppression of pituitary GH expression in both sexes and lowering of circulating GH levels in females. Yet, despite their similar metabolic perturbations, males and females displayed rather distinct alterations of key somatotropic-regulators/ mediators. Our data document a synergistic effect of postnatal-overnutrition on the detrimental impact of HFD-induced obesity on key elements of the adult GH-axis, which is conducted via mechanisms that are sexually-divergent.

Kisspeptin treatment induces gonadotropic responses and rescues ovulation in a subset of preclinical models and women with polycystic ovary syndrome.

STUDY QUESTION: Can kisspeptin treatment induce gonadotrophin responses and ovulation in preclinical models and anovulatory women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Kisspeptin administration in some anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. WHAT IS KNOWN ALREADY: PCOS is a prevalent, heterogeneous endocrine disorder, characterized by ovulatory dysfunction, hyperandrogenism and deregulated gonadotrophin secretion, in need of improved therapeutic options. Kisspeptins (encoded by Kiss1) are master regulators of the reproductive axis, acting mainly at GnRH neurons, with kisspeptins being an essential drive for gonadotrophin-driven ovarian follicular maturation and ovulation. Altered Kiss1 expression has been found in rodent models of PCOS, although the eventual pathophysiological role of kisspeptins in PCOS remains unknown. STUDY DESIGN, SIZE, DURATION: Gonadotrophin and ovarian/ovulatory responses to kisspeptin-54 (KP-54) were evaluated in three preclinical models of PCOS, generated by androgen exposures at different developmental windows, and a pilot exploratory cohort of anovulatory women with PCOS. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three models of PCOS were generated by exposure of female rats to androgens at different periods of development: PNA (prenatal androgenization; N = 20), NeNA (neonatal androgenization; N = 20) and PWA (post-weaning androgenization; N = 20). At adulthood (postnatal day 100), rats were subjected to daily treatments with a bolus of KP-54 (100 µg/kg, s.c.) or vehicle for 11 days (N = 10 per model and treatment). On Days 1, 4, 7 and 11, LH and FSH responses were assessed at different time-points within 4 h after KP-54 injection, while ovarian responses, in terms of follicular maturation and ovulation, were measured at the end of the treatment. In addition, hormonal (gonadotrophin, estrogen and inhibin B) and ovulatory responses to repeated KP-54 administration, at doses of 6.4-12.8 nmol/kg, s.c. bd for 21 days, were evaluated in a pilot cohort of anovulatory women (N = 12) diagnosed with PCOS, according to the Rotterdam criteria. MAIN RESULTS AND THE ROLE OF CHANCE: Deregulated reproductive indices were detected in all PCOS models: PNA, NeNA and PWA. Yet, anovulation was observed only in NeNA and PWA rats. However, while anovulatory NeNA rats displayed significant LH and FSH responses to KP-54 (P < 0.05), which rescued ovulation, PWA rats showed blunted LH secretion after repeated KP-54 injection and failed to ovulate. In women with PCOS, KP-54 resulted in a small rise in LH (P < 0.05), with an equivalent elevation in serum estradiol levels (P < 0.05). Two women showed growth of a dominant follicle with subsequent ovulation, one woman displayed follicle growth but not ovulation and desensitization was observed in another patient. No follicular response was detected in the other women. LIMITATIONS, REASONS FOR CAUTION: While three different preclinical PCOS models were used in order to capture the heterogeneity of clinical presentations of the syndrome, it must be noted that rat models recapitulate many but not all the features of this condition. Additionally, our pilot study was intended as proof of principle, and the number of participants is low, but the convergent findings in preclinical and clinical studies reinforce the validity of our conclusions. WIDER IMPLICATIONS OF THE FINDINGS: Our first-in-rodent and -human studies demonstrate that KP-54 administration in anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. As our rat models likely reflect the diversity of PCOS phenotypes, our results argue for the need of personalized management of anovulatory dysfunction in women with PCOS, some of whom may benefit from kisspeptin-based treatments. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research agreements between Ferring Research Institute and the Universities of Cordoba and Edinburgh. K.S. was supported by the Wellcome Trust Scottish Translational Medicine and Therapeutics Initiative (STMTI). Some of this work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. M.T.-S. is a member of CIBER Fisiopatología de la Obesidad y Nutrición, which is an initiative of Instituto de Salud Carlos III. Dr Mannaerts is an employee of Ferring International PharmaScience Center (Copenhagen, Denmark), and Drs Qi, van Duin and Kohout are employees of the Ferring Research Institute (San Diego, USA). Dr Anderson and Dr Tena-Sempere were recipients of a grant support from the Ferring Research Institute, and Dr Anderson has undertaken consultancy work and received speaker fees outside this study from Merck, IBSA, Roche Diagnostics, NeRRe Therapeutics and Sojournix Inc. Dr Skorupskaite was supported by the Wellcome Trust through the Scottish Translational Medicine and Therapeutics Initiative 102419/Z/13/A. The other authors have no competing interest.

Effects and interactions of tachykinins and dynorphin on FSH and LH secretion in developing and adult rats.

Kisspeptin/neurokinin B/dynorphin (KNDy) neurons, which coexpress kisspeptins (Kps), neurokinin B (NKB), and dynorphin (Dyn), regulate gonadotropin secretion. The KNDy model proposes that NKB (a stimulator, through NK3R) and Dyn (an inhibitor, through κ-opioid receptor) shape Kp secretion onto GnRH neurons. However, some aspects of this paradigm remain ill defined. Here we aimed to characterize the following: 1) the effects of NKB signaling on FSH secretion and 2) the role of Dyn in gonadotropin secretion after NK3R activation; 3) additionally, we explored the roles of other tachykinin receptors, NK1R and NK2R, on gonadotropin release. Thus, the effects of the NK3R agonist, senktide, on FSH release were explored across postnatal development in male and female rats; gonadotropin responses to agonists of NK1R substance P and NK2R [neurokinin A (NKA)] were also monitored. Moreover, the effects of senktide on gonadotropin secretion were assessed after antagonizing Dyn actions by nor-binaltorphimine didydrochloride. Before puberty, rats of both sexes showed increased FSH secretion to senktide (and Kp-10). Conversely, adult female rats were irresponsive to senktide in terms of FSH, despite proven LH responses, whereas the adult males did not display FSH or LH responses to senktide, even at high doses. In turn, substance P and NKA stimulated gonadotropin secretion in prepubertal rats, whereas in adults modest gonadotropin responses to NKA were detected. By pretreatment with a Dyn antagonist, adult males became responsive to senktide in terms of LH secretion and displayed elevated basal LH and FSH levels; nor-binaltorphimine didydrochloride treatment uncovered FSH responses to senktide in adult females. Furthermore, the expression of Pdyn and Opkr1 (encoding Dyn and κ-opioid receptor, respectively) in the mediobasal hypothalamus was greater in males than in females at prepubertal ages. Overall, our data contribute to refining our understanding on how the elements of the KNDy node and related factors (ie, other tachykinins) differentially participate in the control of gonadotropins at different stages of rat postnatal maturation.

Metabolic programming of puberty: sexually dimorphic responses to early nutritional challenges.

Body energy stores and metabolic cues influence the onset of puberty. However, the pubertal impact of early nutritional challenges has been only fragmentarily addressed. We evaluated here the consequences, in terms of pubertal timing and hormonal markers, of various nutritional manipulations during pre- or postnatal maturation in rats of both sexes. Males and females were submitted to gestational undernutrition (UNG) or peripubertal (SUB) subnutrition or were raised in large (LL; underfeeding) or small (SL; overfeeding) litters. In addition, groups of UNG, LL, and SL rats were fed on a high-fat diet (HFD) after weaning. Postnatal overfeeding resulted in higher body weights (BWs) during pubertal transition in both sexes, but only SL males displayed overtly advanced external signs of puberty. Postnatal underfeeding persistently decreased BW gain during puberty, yet the magnitude of pubertal delay was greater in LL males. In contrast, regardless of postnatal nutrition, HFD tended to advance the onset of puberty in females but did not alter pubertal timing in males. Likewise, SUB females displayed a marked delay in BW gain and puberty onset, whereas despite similar reduction in BW, SUB males showed normal timing of puberty. These sex divergences were also detected in various hormonal and metabolic indices so that postnatal overnutrition consistently increased LH, FSH, leptin, and insulin levels only in pubertal females, whereas HFD decreased gonadotropin levels in SL females but increased them in SL males. Notably, UNG rats did not show signs of delayed puberty but displayed a striking sex dimorphism in serum insulin/glucose levels, regardless of the diet, so that only UNG males had signs of presumable insulin resistance. Our data disclose important sex differences in the impact of various early nutritional challenges on the timing of puberty, which may help to explain the different trends of altered puberty and related comorbidities between sexes.

Changes in hypothalamic expression of the Lin28/let-7 system and related microRNAs during postnatal maturation and after experimental manipulations of puberty.

Lin28 and Lin28b are related RNA-binding proteins that inhibit the maturation of miRNAs of the let-7 family and participate in the control of cellular stemness and early embryonic development. Considerable interest has arisen recently concerning other physiological roles of the Lin28/let-7 axis, including its potential involvement in the control of puberty, as suggested by genome-wide association studies and functional genomics. We report herein the expression profiles of Lin28 and let-7 members in the rat hypothalamus during postnatal maturation and in selected models of altered puberty. The expression patterns of c-Myc (upstream positive regulator of Lin28), mir-145 (negative regulator of c-Myc), and mir-132 and mir-9 (putative miRNA repressors of Lin28, predicted by bioinformatic algorithms) were also explored. In male and female rats, Lin28, Lin28b, and c-Myc mRNAs displayed very high hypothalamic expression during the neonatal period, markedly decreased during the infantile-to-juvenile transition and reached minimal levels before/around puberty. A similar puberty-related decline was observed for Lin28b in monkey hypothalamus but not in the rat cortex, suggesting species conservation and tissue specificity. Conversely, let-7a, let-7b, mir-132, and mir-145, but not mir-9, showed opposite expression profiles. Perturbation of brain sex differentiation and puberty, by neonatal treatment with estrogen or androgen, altered the expression ratios of Lin28/let-7 at the time of puberty. Changes in the c-Myc/Lin28b/let-7 pathway were also detected in models of delayed puberty linked to early photoperiod manipulation and, to a lesser extent, postnatal underfeeding or chronic subnutrition. Altogether, our data are the first to document dramatic changes in the expression of the Lin28/let-7 axis in the rat hypothalamus during the postnatal maturation and after different manipulations that disturb puberty, thus suggesting the potential involvement of developmental changes in hypothalamic Lin28/let-7 expression in the mechanisms permitting/leading to puberty onset.

Neurokinin B and the control of the gonadotropic axis in the rat: developmental changes, sexual dimorphism, and regulation by gonadal steroids.

Neurokinin B (NKB), encoded by Tac2 in rodents, and its receptor, NK3R, have recently emerged as important regulators of reproduction; NKB has been proposed to stimulate kisspeptin output onto GnRH neurons. Accordingly, NKB has been shown to induce gonadotropin release in several species; yet, null or even inhibitory effects of NKB have been also reported. The basis for these discrepant findings, as well as other key aspects of NKB function, remains unknown. We report here that in the rat, LH responses to the NK3R agonist, senktide, display a salient sexual dimorphism, with persistent stimulation in females, regardless of the stage of postnatal development, and lack of LH responses in males from puberty onward. Such dimorphism was independent of the predominant sex steroid after puberty, because testosterone administration to adult females failed to prevent LH responses to senktide, and LH responsiveness was not restored in adult males treated with estradiol or the nonaromatizable androgen, dihydrotestosterone. Yet, removal of sex steroids by gonadectomy switched senktide effects to inhibitory, both in adult male and female rats. Sexual dimorphism was also evident in the numbers of NKB-positive neurons in the arcuate nucleus (ARC), which were higher in adult female rats. This is likely the result of differences in sex steroid milieu during early periods of brain differentiation, because neonatal exposures to high doses of estrogen decreased ARC NKB neurons at later developmental stages. Likewise, neonatal estrogenization resulted in lower serum LH levels that were normalized by senktide administration. Finally, we document that the ability of estrogen to inhibit hypothalamic Tac2 expression seems region specific, because estrogen administration decreased Tac2 levels in the ARC but increased them in the lateral hypothalamus. Altogether, our data provide a deeper insight into relevant aspects of NKB function as major regulator of the gonadotropic axis in the rat, including maturational changes, sexual dimorphism, and differential regulation by sex steroids.

Cellular distribution, regulated expression, and functional role of the anorexigenic peptide, NUCB2/nesfatin-1, in the testis.

Nesfatin-1, product of the precursor NEFA/nucleobindin2 (NUCB2), was initially identified as anorectic hypothalamic neuropeptide, acting in a leptin-independent manner. In addition to its central role in the control of energy homeostasis, evidence has mounted recently that nesfatin-1 is also produced in peripheral metabolic tissues, such as pancreas, adipose, and gut. Moreover, nesfatin-1 has been shown to participate in the control of body functions gated by whole-body energy homeostasis, including puberty onset. Yet, whether, as is the case for other metabolic neuropeptides, NUCB2/nesfatin-1 participates in the direct control of gonadal function remains unexplored. We document here for the first time the expression of NUCB2 mRNA in rat, mouse, and human testes, where NUCB2/nesfatin-1 protein was identified in interstitial mature Leydig cells. Yet in rats, NUCB2/nesfatin-1 became expressed in Sertoli cells upon Leydig cell elimination and was also detected in Leydig cell progenitors. Although NUCB2 mRNA levels did not overtly change in rat testis during pubertal maturation and after short-term fasting, NUCB2/nesfatin-1 content significantly increased along the puberty-to-adult transition and was markedly suppressed after fasting. In addition, testicular NUCB2/nesfatin-1 expression was up-regulated by pituitary LH, because hypophysectomy decreased, whereas human choriogonadotropin (super-agonist of LH receptors) replacement enhanced, NUCB2/nesfatin-1 mRNA and peptide levels. Finally, nesfatin-1 increased human choriogonadotropin-stimulated testosterone secretion by rat testicular explants ex vivo. Our data are the first to disclose the presence and functional role of NUCB2/nesfatin-1 in the testis, where its expression is regulated by developmental, metabolic, and hormonal cues as well as by Leydig cell-derived factors. Our observations expand the reproductive dimension of nesfatin-1, which may operate directly at the testicular level to link energy homeostasis, puberty onset, and gonadal function.

Acute inflammation reduces kisspeptin immunoreactivity at the arcuate nucleus and decreases responsiveness to kisspeptin independently of its anorectic effects.

Severe inflammatory challenges are frequently coupled to decreased food intake and disruption of reproductive function, the latter via deregulation of different signaling pathways that impinge onto GnRH neurons. Recently, the hypothalamic Kiss1 system, a major gatekeeper of GnRH function, was suggested as potential target for transmitting immune-mediated repression of the gonadotropic axis during acute inflammation, and yet key facets of such a phenomenon remain ill defined. Using lipopolysaccharide S (LPS)-treated male rats as model of inflammation, we document herein the pattern of hypothalamic kisspeptin immunoreactivity (IR) and hormonal responses to kisspeptin during the acute inflammatory phase. LPS injections induced a dramatic but transient drop of serum LH and testosterone levels. Suppression of gonadotropic function was associated with a significant decrease in kisspeptin-IR in the arcuate nucleus (ARC) that was not observed under conditions of metabolic stress induced by 48-h fasting. In addition, absolute responses to kisspeptin-10 (Kp-10), in terms of LH and testosterone secretion, were significantly attenuated in LPS-treated males that also displayed a decrease in food intake and body weight. Yet pair-fed males did not show similar alterations in LH and testosterone secretory responses to Kp-10, whose magnitude was preserved, if not augmented, during food restriction. In summary, our data document the impact of acute inflammation on kisspeptin content at the ARC as key center for the neuroendocrine control of reproduction. Our results also suggest that suppressed gonadotropic function following inflammatory challenges might involve a reduction in absolute responsiveness to kisspeptin that is independent of the anorectic effects of inflammation.

Characterization of the inhibitory roles of RFRP3, the mammalian ortholog of GnIH, in the control of gonadotropin secretion in the rat: in vivo and in vitro studies.

RF-amide related peptides (RFRP), as putative mammalian orthologs of the avian gonadotropin-inhibitory hormone (GnIH), have been proposed as key regulators of gonadotropin secretion in higher vertebrates. Yet considerable debate has arisen recently on their physiological relevance and potential mechanisms and sites of action. Present studies were undertaken to further characterize the effects of RFRP on LH and FSH secretion by a combination of in vivo and in vitro approaches in male and female rats. Initial screening via intracerebroventricular (icv) administration of different analogs of RFRP1 (RFRP1-12 and RFRP1-20) and RFRP3 (RFRP3-8 and RFRP3-17), as well as the related neuropeptide FF (NPFF8), to gonadectomized (GNX) female rats evidenced significant, albeit modest, inhibitory effects on LH secretion only for RFRP3-8 and RFRP3-17, which were detectable at the high dose rage (1 nmol for RFRP3-8, 5 nmol for RFRP3-17). This moderate inhibitory action was also documented after icv administration of RFRP3-8 to intact and GNX male rats. In addition, systemic (intravenous) administration of RFRP3-8 decreased the circulating levels of both gonadotropins in GNX male rats. Likewise, RFRP3-8 inhibited basal and GnRH-stimulated LH secretion by pituitaries from GNX males in vitro. This inhibitory effect was blocked by the antagonist of RFRP receptors, RF9. In summary, our results support a putative inhibitory role of RFRP3 as ortholog of GnIH in the regulation of gonadotropin secretion in mammals, which appears to involve direct pituitary actions as well as potential central (hypothalamic) effects.

Characterization of the potent gonadotropin-releasing activity of RF9, a selective antagonist of RF-amide-related peptides and neuropeptide FF receptors: physiological and pharmacological implications.

Identification of RF-amide-related peptides (RFRP), as putative mammalian orthologs of the avian gonadotropin-inhibitory hormone, has drawn considerable interest on its potential effects and mechanisms of action in the control of gonadotropin secretion in higher vertebrates. Yet, these analyses have so far relied mostly on indirect approaches, while direct assessment of their physiological roles has been hampered by the lack of suitable antagonists. RF9 was recently reported as a selective and potent antagonist of the receptors for RFRP (RFRPR) and the related neuropeptides, neuropeptide FF (NPFF) and neuropeptide AF (NPFF receptor). We show here that RF9 possesses very strong gonadotropin-releasing activities in vivo. Central administration of RF9 evoked a dose-dependent increase of LH and FSH levels in adult male and female rats. Similarly, male and female mice responded to intracerebroventricular injection of RF9 with robust LH secretory bursts. In rats, administration of RF9 further augmented the gonadotropin-releasing effects of kisspeptin, and its stimulatory effects were detected despite the prevailing suppression of gonadotropin secretion by testosterone or estradiol. In fact, blockade of estrogen receptor-alpha partially attenuated gonadotropin responses to RF9. Finally, systemic administration of RF9 modestly stimulated LH secretion in vivo, although no direct effects in terms of gonadotropin secretion were detected at the pituitary in vitro. Altogether, these data are the first to disclose the potent gonadotropin-releasing activity of RF9, a selective antagonist of RFRP (and NPFF) receptors. Our findings support a putative role of the RFRP/gonadotropin-inhibitory hormone system in the central control of gonadotropin secretion in mammals and have interesting implications concerning the potential therapeutic indications and pharmacological effects of RF9.

Critical roles of kisspeptins in female puberty and preovulatory gonadotropin surges as revealed by a novel antagonist.

Kisspeptins (Kp) have recently emerged as master regulators of the reproductive axis and among the most potent elicitors of GnRH-gonadotropin secretion. Despite their paramount importance in reproductive physiology and their potential therapeutic implications, development of Kp antagonists has remained elusive, and only recently has the first compound with the ability to block Kp actions in vitro and in vivo, namely p234, been reported. However, previous in vivo studies all used acute central injections, whereas characterization of the effects of the antagonist after continuous or systemic administration, which poses pharmacological challenges, is still pending. We report herein a comprehensive series of analyses on the impact of continuous intracerebroventricular infusion of p234 on puberty onset and the preovulatory surge of gonadotropins in the female rat. In addition, the effects of systemic (ip) administration of a tagged p234-penetratin, with a predicted higher permeability at the blood-brain barrier, on Kp-10 induced gonadotropin secretion were evaluated. Central infusion of p234 to pubertal females delayed vaginal opening and decreased uterine and ovarian weights at the expected time of puberty, without affecting body weight. Likewise, chronic intracerebroventricular administration of p234 for 4 d prevented the preovulatory surges of LH and FSH. In addition, systemic (ip) administration of p234-penetratin significantly attenuated acute LH and FSH responses to Kp-10, either after intracerebroventricular or ip injection of Kp. Our data document the validity of p234 for antagonizing Kp actions in vivo and provide direct experimental evidence for the important role of Kp signaling in the key events of female reproduction, such as puberty onset and the preovulatory surge of gonadotropins.

The mammalian target of rapamycin as novel central regulator of puberty onset via modulation of hypothalamic Kiss1 system.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that operates as sensor of cellular energy status and effector for its coupling to cell growth and proliferation. At the hypothalamic arcuate nucleus, mTOR signaling has been recently proposed as transducer for leptin effects on energy homeostasis and food intake. However, whether central mTOR also participates in metabolic regulation of fertility remains unexplored. We provide herein evidence for the involvement of mTOR in the control of puberty onset and LH secretion, likely via modulation of hypothalamic expression of Kiss1. Acute activation of mTOR by l-leucine stimulated LH secretion in pubertal female rats, whereas chronic l-leucine infusion partially rescued the state of hypogonadotropism induced by food restriction. Conversely, blockade of central mTOR signaling by rapamycin caused inhibition of the gonadotropic axis at puberty, with significantly delayed vaginal opening, decreased LH and estradiol levels, and ovarian and uterine atrophy. Inactivation of mTOR also blunted the positive effects of leptin on puberty onset in food-restricted females. Yet the GnRH/LH system retained their ability to respond to ovariectomy and kisspeptin-10 after sustained blockade of mTOR, ruling out the possibility of unspecific disruption of GnRH function by rapamycin. Finally, mTOR inactivation evoked a significant decrease of Kiss1 expression at the hypothalamus, with dramatic suppression of Kiss1 mRNA levels at the arcuate nucleus. Altogether our results unveil the role of central mTOR signaling in the control of puberty onset and gonadotropin secretion, a phenomenon that involves the regulation of Kiss1 and may contribute to the functional coupling between energy balance and gonadal activation and function.

Persistent impairment of hypothalamic KiSS-1 system after exposures to estrogenic compounds at critical periods of brain sex differentiation.

Attainment of reproductive capacity at puberty relies on a complex series of maturational events that include sexual differentiation of the brain; a hormonally driven phenomenon that takes place at early stages of development (critical period). Alterations of sex steroid milieu during such critical period disrupt pubertal maturation and gonadotropic function later in life, through mechanisms that remain partially unknown. Kisspeptins, products of the KiSS-1 gene acting via G protein-coupled receptor 54, have recently emerged as essential gatekeepers of puberty onset and reproductive function. By using rat models of neonatal administration of estrogenic compounds, we provide herein compelling evidence for the functional impairment of the hypothalamic KiSS-1 system at the time preceding puberty after early inappropriate exposures during brain sex differentiation. Neonatal injection of estradiol benzoate to male and female rats resulted in a dose-dependent decrease in hypothalamic KiSS-1 mRNA levels at the prepubertal stage, linked to lowering of serum LH concentrations. Yet, despite persistently decreased basal gonadotropin levels in estrogenized animals, intracerebral injection of kisspeptin evoked potent LH and FSH secretory responses, similar in magnitude to those of control animals. Estrogenized rats also showed defective levels of hypothalamic KiSS-1 mRNA and circulating gonadotropins in response to gonadectomy, whereas exogenous kisspeptin was capable to enhance further LH and FSH secretion in this model. Finally, protocols of neonatal exposure to high doses of an environmentally relevant estrogen, bisphenol-A, mimicked the effects of estradiol benzoate in terms of hypothalamic expression of KiSS-1 gene at the prepubertal period. Altogether, our data document the sensitivity of the hypothalamic KiSS-1 system to alterations in sex steroid milieu during critical periods of brain sex differentiation, and suggest that lowering of endogenous kisspeptin tone induced by early exposures to xeno-estrogens might be mechanistically relevant for disruption of gonadotropin secretion and puberty onset later in life.

Alterations in hypothalamic KiSS-1 system in experimental diabetes: early changes and functional consequences.

Using long-term streptozotocin (STZ)-treated male rats, we recently proposed that defective function of hypothalamic KiSS-1 system is mechanistically relevant for central hypogonadotropism of uncontrolled diabetes. However, the temporal pattern of such defects and its potential contribution to disturbed gonadotropin secretion in the diabetic female remain so far unexplored. To cover these issues, expression analyses and hormonal tests were conducted in diabetic male (1 wk after STZ; short term) and female (4 wk after STZ; long term) rats. Short-term diabetic males had lower basal testosterone levels and decreased gonadotropin responses to orchidectomy (ORX), which associated with significantly attenuated post-ORX rises of hypothalamic KiSS-1 mRNA. Yet kisspeptin administration to diabetic males was able to acutely elicit supramaximal LH and testosterone responses and normalize post-ORX gonadotropin secretion. Long-term diabetic females showed persistent anestrus and significantly decreased basal gonadotropin levels as well as blunted LH responses to ovariectomy; changes that were linked to lowering of basal and postovariectomy expression of hypothalamic KiSS-1 mRNA. Moreover, despite prevailing gonadotropin suppression, LH responses to acute kisspeptin administration were fully preserved, and even enhanced after its repeated injection, in diabetic females. In sum, our present findings further define the temporal course and mechanistic relevance of altered hypothalamic KiSS-1 system in the hypogonadotropic state of uncontrolled diabetes. Furthermore, our data provide the basis for the potential therapeutic intervention of the KiSS-1 system as adjuvant in the management of disturbed gonadotropin secretion of type 1 diabetes in the female.