AMP-activated protein kinase (AMPK) signaling in GnRH neurons links energy status and reproduction.

BACKGROUND: Reproduction is tightly coupled to body energy and metabolic status. GnRH neurons, master elements and final output pathway for the brain control of reproduction, directly or indirectly receive and integrate multiple metabolic cues to regulate reproductive function. Yet, the molecular underpinnings of such phenomenon remain largely unfolded. AMP-activated protein kinase (AMPK), the fundamental cellular sensor that becomes activated in conditions of energy deficit, has been recently shown to participate in the control of Kiss1 neurons, essential gatekeepers of the reproductive axis, by driving an inhibitory valence in situations of energy scarcity at puberty. However, the contribution of AMPK signaling specifically in GnRH neurons to the metabolic control of reproduction remains unknown. METHODS: Double immunohistochemistry (IHC) was applied to evaluate expression of active (phosphorylated) AMPK in GnRH neurons and a novel mouse line, named GAMKO, with conditional ablation of the AMPK α1 subunit in GnRH neurons, was generated. GAMKO mice of both sexes were subjected to reproductive characterization, with attention to puberty and gonadotropic responses to kisspeptin and metabolic stress. RESULTS: A vast majority (>95%) of GnRH neurons co-expressed pAMPK. Female (but not male) GAMKO mice displayed earlier puberty onset and exaggerated LH (as surrogate marker of GnRH) responses to kisspeptin-10 at the prepubertal age. In adulthood, GAMKO females retained increased LH responsiveness to kisspeptin and showed partial resilience to the inhibitory effects of conditions of negative energy balance on the gonadotropic axis. The modulatory role of AMPK in GnRH neurons required preserved ovarian function, since the differences in LH pulsatility detected between GAMKO and control mice subjected to fasting were abolished in ovariectomized animals. CONCLUSIONS: Altogether, our data document a sex-biased, physiological role of AMPK signaling in GnRH neurons, as molecular conduit of the inhibitory actions of conditions of energy deficit on the female reproductive axis.

Medium- and large-sized mammals in a steppic savanna area of the Brazilian Pampa: survey and conservation issues of a poorly known fauna.

The wildlife of the Brazilian Pampa is threatened by large-scale habitat loss, due in particular to the expansion of soybean cultivation and the conversion of grasslands areas into extensive areas of silviculture. It is essential to study how the mammal fauna copes with the highly fragmented, human-influenced, non-protected landscape. Our study presents the results of a survey of the large- and medium-sized mammals of a typical human-influenced steppic savanna area of the Pampa biome. The survey was conducted exclusively with the use of camera traps over a period of 16 months. The relative frequencies of species in the area were evaluated. We recorded 18 species, some of them locally threatened (Tamandua tetradactyla, Alouatta caraya, Leopardus colocolo, Leopardus geoffroyi, Leopardus wiedii, Puma yagouaroundi, Mazama gouazoubira and Cuniculus paca). Several species were found to thrive in the area; however, many species were considered rare, and undoubtedly new species could be recorded if we continued the sampling. Our results contribute to the knowledge of faunal diversity in the Pampa biome and associated habitats, warn about threats and provide support for conservation measures.

Medium- and large-sized mammals in a steppic savanna area of the Brazilian Pampa: survey and conservation issues of a poorly known fauna / Mamíferos de médio e grande porte em uma área de savana estépica do Pampa Brasileiro: levantamento e questões de conservação sobre uma fauna pouco conhecida

Abstract The wildlife of the Brazilian Pampa is threatened by large-scale habitat loss, due in particular to the expansion of soybean cultivation and the conversion of grasslands areas into extensive areas of silviculture. It is essential to study how the mammal fauna copes with the highly fragmented, human-influenced, non-protected landscape. Our study presents the results of a survey of the large- and medium-sized mammals of a typical human-influenced steppic savanna area of the Pampa biome. The survey was conducted exclusively with the use of camera traps over a period of 16 months. The relative frequencies of species in the area were evaluated. We recorded 18 species, some of them locally threatened (Tamandua tetradactyla, Alouatta caraya, Leopardus colocolo, Leopardus geoffroyi, Leopardus wiedii, Puma yagouaroundi, Mazama gouazoubira and Cuniculus paca). Several species were found to thrive in the area; however, many species were considered rare, and undoubtedly new species could be recorded if we continued the sampling. Our results contribute to the knowledge of faunal diversity in the Pampa biome and associated habitats, warn about threats and provide support for conservation measures.

Resumo A fauna do Pampa brasileiro está sendo ameaçada pela grande perda de habitat que vem sofrendo, em especial, devido à expansão do cultivo da soja e da conversão de áreas de pastagens em extensas áreas de silvicultura. É essencial estudar como a fauna de mamíferos lida com a paisagem altamente fragmenta e não protegida, pela influencia humana. Nosso estudo apresenta os resultados de um levantamento dos mamíferos de médio e grande porte em uma área típica do bioma Pampa com influência humana. O levantamento foi realizado exclusivamente com o uso de armadilhas fotográficas ao longo de um período de 16 meses. Foram avaliadas as frequências relativas das espécies na área. Foram registradas 18 espécies, algumas delas ameaçadas localmente (Tamandua tetradactyla, Alouatta caraya, Leopardus colocolo, Leopardus geoffroyi, Leopardus wiedii, Puma yagouaroundi, Mazama gouazoubira e Cuniculus paca). Várias espécies foram encontradas de forma abundante, no entanto, muitas espécies foram consideradas raras, sendo que novas espécies poderiam ser registradas se continuássemos a amostragem. Nossos resultados contribuem para o conhecimento da diversidade de mamíferos do bioma Pampa e habitats associados, alertam sobre ameaças e fornecem suporte para medidas de conservação.

DNA metabarcoding diet analysis for species with parapatric vs sympatric distribution: a case study on subterranean rodents.

Closely related sympatric species commonly develop different ecological strategies to avoid competition. Ctenomys minutus and C. flamarioni are subterranean rodents parapatrically distributed in the southern Brazilian coastal plain, showing a narrow sympatric zone. To gain understanding on food preferences and possible competition for food resources, we evaluated their diet composition performing DNA metabarcoding analyzes of 67 C. minutus and 100 C. flamarioni scat samples, collected along the species geographical ranges. Thirteen plant families, mainly represented by Poaceae, Araliaceae, Asteraceae and Fabaceae, were identified in the diet of C. minutus. For C. flamarioni, 10 families were recovered, with a predominance of Poaceae, Araliaceae and Asteraceae. A significant correlation between diet composition and geographical distance was detected in C. minutus, whereas the diet of C. flamarioni was quite homogeneous throughout its geographical distribution. No significant differences were observed between males and females of each species. However, differences in diet composition between species were evident according to multivariate analysis. Our results suggest some level of diet partitioning between C. flamarioni and C. minutus in the sympatric region. While the first species is more specialized on few plant items, the second showed a more varied and heterogeneous diet pattern among individuals. These differences might have been developed to avoid competition in the region of co-occurrence. Resource availability in the environment also seems to influence food choices. Our data indicate that C. minutus and C. flamarioni are generalist species, but that some preference for Poaceae, Asteraceae and Araliaceae families can be suggested for both rodents.

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.

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.

Sex steroids and the control of the Kiss1 system: developmental roles and major regulatory actions.

Kisspeptins, encoded by the Kiss1 gene, and their canonical receptor, GPR54 (also termed Kiss1R), are unanimously recognised as essential regulators of puberty onset and gonadotrophin secretion. These key reproductive functions stem from the capacity of kisspeptins to stimulate gonadotrophin-releasing hormone (GnRH) secretion in the hypothalamus, where discrete populations of Kiss1 neurones have been identified. In rodents, two major groups of hypothalamic Kiss1 neurones exist: one present in the arcuate nucleus (ARC) and the other located in the anteroventral periventricular area (AVPV/RP3V). In recent years, numerous signals have been identified as putative modulators of the hypothalamic Kiss1 system. Among them, the prominent role of sex steroids as being important regulators of Kiss1 neurones has been documented in different species and developmental stages, such as early brain sex differentiation, puberty, adulthood and senescence. These regulatory actions are (mainly) conducted via oestrogen receptor (ER)α, which is expressed in almost all Kiss1 neurones, and likely involve both classical and nonclassical pathways. The regulatory effects of sex steroids are nucleus-specific. Thus, sex steroids inhibit the expression of Kiss1/kisspeptin at the ARC, as a mechanism to conduct their negative-feedback actions on gonadotrophin secretion. By contrast, oestrogens enhance Kiss1 expression at the AVPV/RP3V in rodents, suggesting the involvement of this population in the positive-feedback actions of oestradiol to generate the preovulatory surge of gonadotrophins. In addition, sex steroids have been shown to act post-transcriptionally, modulating GnRH/gonadotrophin responsiveness to kisspeptin. Finally, sex steroids also regulate the expression of co-transmitters of Kiss1 neurones, such as neurokinin B, whose mRNA content in the ARC fluctuates in parallel to that of Kiss1 in response to changes in the circulating levels of sex steroids, therefore suggesting the contribution of this neuropeptide in the feedback control of gonadotrophin secretion. In sum, compelling experimental evidence obtained in different mammalian (and non-mammalian) species, including primates, demonstrates that sex steroids are essential regulators of hypothalamic Kiss1 neurones, which in turn operate as conduits for their effects on GnRH neurones. The physiological relevance of such regulatory phenomena is thoroughly discussed.

Regulation of NKB pathways and their roles in the control of Kiss1 neurons in the arcuate nucleus of the male mouse.

Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17ß-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.

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.

Using cardiovascular parameters and symptom severity to prescribe physical activity in women with fibromyalgia.

OBJECTIVES: This study has two main aims, firstly to define subgroups of women affected by fibromyalgia syndrome (FMS) based on symptoms and secondly to determine cardiovascular parameters in treadmill exercises in order to prescribe physical activity. METHODS: Thirty-two women (age= 53.26+/-6.61 yr) were assigned to two different groups based on their functional capacity and symptoms as measured by the Fibromyalgia Impact Questionnaire and pain. Subjects were submitted twice to a maximum treadmill incremental test until participants achieved volitional exhaustion (VO2max). Expired respiratory gases, ventilator parameters and heart rate (HR) were measured continuously through exercise, and rate perceived exertion (RPE) was assessed once a minute during the test. RESULTS: Peak VO2 values for the moderately affected group (Group 1) were significantly different from those of severely affected group (Group 2) (26.2+/-2.1 ml x kg(-1) x min(-1) (Group 1) and 22.1+/-2.5 ml x kg(-1) x min(-1) (Group 2)). Additionally taking into account VO2 at ventilatory threshold (VO2VT), significant differences between groups were found in both tests. Some notable differences in all parameters evaluated were also found. CONCLUSION: This study has demonstrated that the aerobic capacity of patients with FMS was different according to how severely affected they were by the condition; therefore, physical activity of the same intensity should not be prescribed for both groups. According to these results, health professionals could prescribe physical activity with confidence to this patient group.

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.

Follicle-stimulating hormone responses to kisspeptin in the female rat at the preovulatory period: modulation by estrogen and progesterone receptors.

Ovulation is triggered by the preovulatory surge of gonadotropins that, in rodents, is defined by the concomitant rise in circulating LH and FSH at the afternoon of proestrus (primary surge), followed by persistently elevated FSH levels at early estrus (secondary surge). In recent years, kisspeptins, products of the KiSS-1 gene that act via G protein-coupled receptor 54, have emerged as an essential hypothalamic conduit for the generation of the preovulatory LH surge by conveying positive feedback effects of estradiol onto GnRH neurons, an event that involves not only estradiol-induced transcription of the KiSS-1 gene at the anteroventral periventricular nucleus but also its ability to modulate GnRH/LH responses to kisspeptin. However, little is known about the potential modulation of FSH responsiveness to kisspeptin by sex steroids in the cyclic female. We report herein analyses on the consequences of selective blockade of estrogen receptors (ER)-alpha and -beta, as well as progesterone receptor (PR), on the ovulatory surges of FSH and their modulation by kisspeptin. Antagonism of ERalpha or PR equally blunted the primary and secondary surges of FSH and nullified FSH responses to kisspeptin at the preovulatory period. Conversely, selective blockade of ERbeta failed to induce major changes in terms of endogenous FSH surges, yet it decreased FSH responses to exogenous kisspeptin. In contrast, FSH responses to GnRH were fully conserved after ERbeta blockade and partially preserved after inhibition of ERalpha and PR signaling. Finally, secondary FSH secretion was rescued by kisspeptin in females with selective blockade of ERalpha but not PR. In sum, our results substantiate a concurrent, indispensable role of ERalpha and PR in the generation of FSH surges and the stimulation of FSH responses to kisspeptin at the ovulatory period. In addition, our data suggest that ERbeta might operate as a subtle, positive modulator of the preovulatory FSH responses to kisspeptin, a role that is opposite to its putative inhibitory action on kisspeptin-induced LH secretion and might contribute to the dissociation of gonadotropin secretion at the ovulatory phase in the cyclic female rat.