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.

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.

SIRT1 mediates obesity- and nutrient-dependent perturbation of pubertal timing by epigenetically controlling Kiss1 expression.

Puberty is regulated by epigenetic mechanisms and is highly sensitive to metabolic and nutritional cues. However, the epigenetic pathways mediating the effects of nutrition and obesity on pubertal timing are unknown. Here, we identify Sirtuin 1 (SIRT1), a fuel-sensing deacetylase, as a molecule that restrains female puberty via epigenetic repression of the puberty-activating gene, Kiss1. SIRT1 is expressed in hypothalamic Kiss1 neurons and suppresses Kiss1 expression. SIRT1 interacts with the Polycomb silencing complex to decrease Kiss1 promoter activity. As puberty approaches, SIRT1 is evicted from the Kiss1 promoter facilitating a repressive-to-permissive switch in chromatin landscape. Early-onset overnutrition accelerates these changes, enhances Kiss1 expression and advances puberty. In contrast, undernutrition raises SIRT1 levels, protracts Kiss1 repression and delays puberty. This delay is mimicked by central pharmacological activation of SIRT1 or SIRT1 overexpression, achieved via transgenesis or virogenetic targeting to the ARC. Our results identify SIRT1-mediated inhibition of Kiss1 as key epigenetic mechanism by which nutritional cues and obesity influence mammalian puberty.

Changes in keratin 8/18 expression in human granulosa cell lineage are associated to cell death/survival events: potential implications for the maintenance of the ovarian reserve.

STUDY QUESTION: Is keratin 8/18 (K8/K18) expression linked to cell death/survival events in the human granulosa cell lineage? SUMMARY ANSWER: A close association exists between changes in K8/K18 expression and cell death/survival events along the human granulosa cell lineage lifespan. WHAT IS KNOWN ALREADY: In addition to their structural and mechanical functions, K8/K18 play essential roles regulating cell death, survival and differentiation in several non-gonadal epithelial tissues. Transfection of the granulosa-like tumor KGN cells with siRNA to interfere KRT8 and KRT18 expression increases FAS-mediated apoptosis, while an inverse association between K8/K18 expression and cell death has been found in the bovine antral follicles and corpus luteum. Yet, only fragmentary and inconclusive information exists regarding K8/K18 expression in the human ovary. STUDY DESIGN, SIZE, DURATION: Expression of K8/K18 was assessed by immunohistochemistry at different stages of the granulosa cell lineage, from flattened granulosa cells in primordial follicles to fully luteinized granulosa-lutein cells in the corpus luteum (including corpus luteum of pregnancy). PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunohistochemical detection of K8/K18 was conducted in 40 archival ovarian samples from women aged 17-39 years. K8/K18 expression was analyzed at the different stages of follicle development and corpus luteum lifespan. The proportions of primordial follicles showing all K8/K18-positive, all K8/K18 negative, or a mixture of K8/K18 negative and positive granulosa cells were quantified in 18 ovaries, divided into three age groups: ≤ 25 years (N = 6), 26-30 (N = 6) and 31-36 (N = 6) years. A total number of 1793 primordial, 750 transitional and 140 primary follicles were scored. MAIN RESULTS AND THE ROLE OF CHANCE: A close association was found between changes in K8/K18 expression and cell death/cell survival events in the human granulosa cell lineage. Large secondary and early antral follicles (most of them undergoing atresia) and regressing corpora lutea displayed low/absent K8/K18 expression. Conversely, early growing and some large antral follicles, functional menstrual corpora lutea, as well as life-extended corpus luteum of pregnancy, in which cell death was scarce, showed high K8/K18 expression. Three sub-populations of primordial follicles were observed with respect to the presence of K8/K18 in their flattened granulosa cells, ranging from primordial follicles showing only positive granulosa cells [P0(+)], to others with a mixture of positive and negative cells [P0(+/-)] or follicles with only negative cells [P0(-)]. Significant age-related changes were found in the proportions of the different primordial follicle types. In relation to age, a positive correlation was found for P0(+) primordial follicles (R2= 0.7883, N = 18; P < 0.001), while negative correlations were found for P0(+/-) (R2 = 0.6853, N = 18; P < 0.001) and P0(-) (R2 = 0.6725, N = 18; P < 0.001) follicles. Furthermore, an age-related shift towards greater keratin expression was found in P0(+/-) follicles (χ2 = 19.07, P < 0.05). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a descriptive study. Hence, a cause-and-effect relationship between K8/K18 expression and cell death/survival cannot be directly established. WIDER IMPLICATIONS OF THE FINDINGS: This study describes, for the first time, the existence of sub-populations of primordial follicles on the basis of K8/K18 expression in granulosa cells, and that their proportions change with age. While a progressive increase in K8/K18 expression cannot be ruled out, our data are consistent with the hypothesis that primordial follicles expressing low levels of K8/K18 are preferentially ablated by follicle attrition, while primordial follicles showing high K8/K18 levels are those predominantly recruited into the growing pool. This suggests that K8/K18 expression could constitute a novel factor regulating primordial follicle death/survival, and raises the possibility that alterations of K8/K18 expression could be involved in the accelerated depletion of the ovarian reserve leading to premature ovarian insufficiency. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Grants BFU2011-025021 and BFU2014-57581-P (Ministerio de Economía y Competitividad, Spain; co-funded with EU funds from FEDER Program); project PIE14-00005 (Flexi-Met, Instituto de Salud Carlos III, Ministerio de Sanidad, Spain); Projects P08-CVI-03788 and P12-FQM-01943 (Junta de Andalucía, Spain); and EU research contract DEER FP7-ENV-2007-1. CIBER Fisiopatología de la Obesidad y Nutrición is an initiative of Instituto de Salud Carlos III. The authors have nothing to disclose in relation to the contents of this study.

Testicular expression of the Lin28/let-7 system: Hormonal regulation and changes during postnatal maturation and after manipulations of puberty.

The Lin28/let-7 system, which includes the RNA-binding proteins, Lin28a/Lin28b, and let-7 miRNAs, has emerged as putative regulator of puberty and male gametogenesis; yet, its expression pattern and regulation in postnatal testis remain ill defined. We report herein expression profiles of Lin28 and let-7 members, and related mir-145 and mir-132, in rat testis during postnatal maturation and in models of altered puberty and hormonal deregulation. Neonatal expression of Lin28a and Lin28b was low and rose markedly during the infantile period; yet, expression patterns diverged thereafter, with persistently elevated levels only for Lin28b, which peaked at puberty. Let-7a, let-7b, mir-132 and mir-145 showed profiles opposite to Lin28b. In fact, let-7b and mir-145 were abundant in pachytene spermatocytes, but absent in elongating spermatids, where high expression of Lin28b was previously reported. Perturbation of puberty by neonatal estrogenization reverted the Lin28/let-7 expression ratio; expression changes were also detected in other models of delayed puberty, due to early photoperiod or nutritional manipulations. In addition, hypophysectomy or growth hormone (GH) deficiency revealed regulation of this system by gonadotropins and GH. Our data document the expression profiles of the Lin28/let-7 system in rat testis along postnatal/pubertal maturation, and their perturbation in models of pubertal and hormonal manipulation.

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.

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.

Delayed puberty in spontaneously hypertensive rats involves a primary ovarian failure independent of the hypothalamic KiSS-1/GPR54/GnRH system.

Spontaneously hypertensive (SH) rats, extensively used as experimental models of essential human hypertension, display important alterations in the neuroendocrine reproductive axis, which manifest as markedly delayed puberty onset in females but whose basis remains largely unknown. We analyze herein in female SH rats: 1) possible alterations in the expression and function of KiSS-1/GPR54 and GnRH/GnRH-receptor systems, 2) the integrity of feedback mechanisms governing the hypothalamic-pituitary-ovarian axis, and 3) the control of ovarian function by gonadotropins. Our data demonstrate that, despite overtly delayed puberty, no significant decrease in hypothalamic KiSS-1, GPR54, or GnRH mRNA levels was detected in this strain. Likewise, in vivo gonadotropin responses to ovariectomy and systemic kisspeptin-10 or GnRH administration, as well as in vitro gonadotropin responses to GnRH, were fully preserved in SH rats. Moreover, circulating LH levels were grossly conserved during prepubertal maturation, whereas FSH levels were even enhanced from d 20 postpartum onwards. In striking contrast, ovarian weight and hormone (progesterone and testosterone) responses to human chorionic gonadotropin (CG) in vitro were profoundly decreased in SH rats, with impaired follicular development and delayed ovulation at puberty. Such reduced hormonal responses to human CG could not be attributed to changes in LH/CG or FSH-receptor mRNA expression but might be linked to blunted P450scc, 3beta-hydroxy steroid dehydrogenase, and aromatase mRNA levels in ovaries from SH rats. In conclusion, our results indicate that the expression and function of KiSS-1/GPR54 and GnRH/GnRH-receptor systems is normal in SH rats, whereas ovarian development, steroidogenesis, and responsiveness to gonadotropins are strongly compromised.

Superficial ovarian cortex vascularization is inversely related to the follicle reserve in normal cycling ovaries and is increased in polycystic ovary syndrome.

BACKGROUND: The superficial ovarian cortex constitutes the micro-environment where resting and early growing follicles reside. As small follicles do not possess an independent capillary network, both their survival and early growth depend on their proximity to the cortical vessels. Little is known about the possible changes in superficial ovarian cortex vascularization in normal women throughout reproductive life or in pathological conditions such as polycystic ovary syndrome (PCOS) involving abnormal early follicle growth. We studied the vascularization of the superficial and deep cortical stroma (DCS) in normal cycling ovaries from 21 to 50 years of age and in infertile women with PCOS. METHODS: We used archival ovarian samples and specific CD34 immunostaining to determine blood vessel density and to analyse correlation with age and with the ovarian follicle reserve. RESULTS: Normal cycling ovaries showed an age-related increase in the superficial cortical stroma vascularization that was inversely correlated with the density of small (primordial and primary) follicles. In contrast, blood vessel density in the DCS significantly decreased in women aged >or=40 years. Ovaries from PCOS showed a 2-fold increase in blood vessel density in both superficial cortical stroma and DCS with respect to age-matched controls. CONCLUSIONS: The increased vascularization of the superficial cortical stroma in normal ovaries in relation to age and in ovaries from PCOS could have profound effects on cortical metabolic rate, primordial follicle survival/activation and early follicle growth, and may underline changes in follicle dynamics in mid-aged women and in PCOS.

KiSS-1 in the mammalian ovary: distribution of kisspeptin in human and marmoset and alterations in KiSS-1 mRNA levels in a rat model of ovulatory dysfunction.

Kisspeptins, the products of the KiSS-1 gene acting via G protein-coupled receptor 54 (GPR54), have recently emerged as pivotal signals in the hypothalamic network triggering the preovulatory surge of gonadotropins and, hence, ovulation. Additional actions of kisspeptins at other levels of the hypothalamic-pituitary-ovarian axis have been suggested but remain to date scarcely studied. We report herein the pattern of expression of KiSS-1 and GPR54 in the human and nonhuman primate ovary and evaluate changes in ovarian KiSS-1 expression in a rat model of ovulatory dysfunction. KiSS-1 and GPR54 mRNAs were detected in human ovarian tissue and cultured granulosa-lutein cells. In good agreement, kisspeptin immunoreactivity was observed in cyclic human and marmoset ovaries, with prominent signals in the theca layer of growing follicles, corpora lutea, interstitial gland, and ovarian surface epithelium. GPR54 immunoreactivity was also found in human theca and luteal cells. Administration of indomethacin to cyclic female rats disturbed ovulation and resulted in a dramatic drop in ovarian KiSS-1, but not GPR54, cyclooxygenase-2 (COX-2), or progesterone receptor, mRNA levels at the time of ovulation; an effect mimicked by the selective COX-2 inhibitor NS398 and rescued by coadministration of PGE(2). Likewise, the stimulatory effect of human choriogonadotropin on ovarian KiSS-1 expression was partially blunted by indomethacin. In contrast, KiSS-1 mRNA levels remained unaltered in another model of ovulatory failure, i.e., the RU486-treated rat. In summary, we document for the first time the expression of KiSS-1/kisspeptin and GPR54 in the human and nonhuman primate ovary. In addition, we provide evidence for the ability of inhibitors of COX-2, known to disturb follicular rupture and ovulation, to selectively alter the expression of KiSS-1 gene in rat ovary. Altogether, our results are suggestive of a conserved role of local KiSS-1 in the direct control of ovarian functions in mammals.

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.

Novel expression and direct effects of adiponectin in the rat testis.

Adiponectin is an adipocyte hormone, with relevant roles in lipid metabolism and glucose homeostasis, recently involved in the control of different endocrine organs, such as the placenta, pituitary and, likely, the ovary. However, whether as described previously for other adipokines, such as leptin and resistin, adiponectin is expressed and/or conducts biological actions in the male gonad remains unexplored. In this study, we provide compelling evidence for the expression, putative hormonal regulation, and direct effects of adiponectin in the rat testis. Testicular expression of adiponectin was demonstrated along postnatal development, with a distinctive pattern of RNA transcripts and discernible protein levels that appeared mostly located at interstitial Leydig cells. Testicular levels of adiponectin mRNA were marginally regulated by pituitary gonadotropins but overtly modulated by metabolic signals, such as glucocorticoids, thyroxine, and peroxisome proliferator-activated receptor-gamma, whose effects were partially different from those on circulating levels of adiponectin. In addition, expression of the genes encoding adiponectin receptor (AdipoR)-1 and AdipoR2 was detected in the rat testis, with developmental changes and gonadotropin regulation for AdipoR2 mRNA, and prominent levels of AdipoR1 in seminiferous tubules. Moreover, recombinant adiponectin significantly inhibited basal and human choriogonadotropin-stimulated testosterone secretion ex vivo, whereas it failed to change relative levels of several Sertoli cell-expressed mRNAs, such as stem cell factor and anti-Müllerian hormone. In summary, our data are the first to document the expression, regulation and functional role of adiponectin in the rat testis. Taken together with its recently reported expression in the ovary and its effects on LH secretion and ovarian steroidogenesis, these results further substantiate a multifaceted role of adiponectin in the control of the reproductive axis, which might operate as endocrine integrator linking metabolism and gonadal function.

Opposite roles of estrogen receptor (ER)-alpha and ERbeta in the modulation of luteinizing hormone responses to kisspeptin in the female rat: implications for the generation of the preovulatory surge.

Ovulation is triggered by the preovulatory rise of gonadotropins, which is in turn elicited by the preceding increase in circulating estrogen. Kisspeptins, ligands of G protein-coupled receptor 54 encoded by the KiSS-1 gene, have emerged as potent stimulators of GnRH/LH secretion, and KiSS-1 neurons at the anteroventral periventricular nucleus have been shown to be involved in the generation of preovulatory LH surge, estrogen being a potent elicitor of KiSS-1 gene expression selectively at the anteroventral periventricular nucleus. Whether, in addition to transcriptional effects, estrogen influences other aspects of kisspeptin-induced GnRH/LH release in the female remains unexplored. We provide herein evidence for the specific roles of estrogen receptor (ER)-alpha and ERbeta in the modulation of LH responses to kisspeptin and the generation of the preovulatory surge. Selective blockade of ERalpha in cyclic females blunted LH responses to kisspeptin, eliminated the endogenous preovulatory rise of LH, and blocked ovulation. In contrast, antagonism of ERbeta failed to cause major changes in terms of LH surge and ovulatory rate but significantly augmented acute LH responses to kisspeptin. Notably, defective LH secretion and ovulation after ERalpha blockade were not observed after GnRH stimulation, which elicited maximal acute (<2 h) LH responses regardless of ERalpha/ERbeta signaling. In addition, net LH secretion in response to kisspeptin was decreased by ovariectomy and increased after selective activation of ERalpha but not ERbeta. Altogether, our data document the prominent positive role of ERalpha in the regulation of GnRH/LH responsiveness to kisspeptin and, thereby, ovulation. In addition, our results disclose the putative function of ERbeta as negative modifier of GnRH/LH response to kisspeptin, a phenomenon that might contribute to partially restraining LH secretion at certain physiological states.

Macrophages in human fallopian tube and ovarian epithelial inclusion cysts.

Epithelial inclusion cysts (EICs) are considered a preferential site for ovarian carcinogenesis. Local inflammation, associated to ovulatory wound repair and epithelial inflammatory conditions, facilitates EIC formation and involves activation of macrophages. The aim of this study was to analyse the presence and numbers of macrophages in the ovarian surface epithelium (OSE), in EICs, and in the fallopian tubes, as tubal metaplasia is a common finding in EICs. Immunohistochemical analysis of macrophages was performed in 25 fallopian tubes in different phases of the menstrual cycle, and in 30 ovaries showing EICs from cycling and postmenopausal women. In the fallopian tube, macrophages were abundant and underwent cyclic changes during the menstrual cycle, being particularly abundant within the epithelium at early and mid-luteal phases. Macrophages were not found in the normal OSE. However, OSE areas and EICs showing tubal metaplasia were invariably associated with infiltration by abundant macrophages. Macrophages were present among epithelial cells, infiltrating the cyst wall, as well as free in the cyst lumen. No significant differences existed between follicular and luteal phases of the cycle, or between cycling and postmenopausal women. This study has demonstrated that macrophages are associated with metaplastic EICs, and raises the possibility that these cells contribute to the particular microenvironment of EICs through secretion of cytokines and growth factors that may reach bioactive concentrations in the confined space of the EICs.

Effects of selective inhibition of cyclooxygenase and lipooxygenase pathways in follicle rupture and ovulation in the rat.

Treatment with non-steroidal anti-inflammatory drugs, either non-selective or selective cyclooxygenase-2 (COX-2) inhibitors, consistently impairs ovulation, indicating the essential role of COX-2/prostaglandins in the ovulatory process. Indomethacin, a potent inhibitor of both COX-1 and COX-2, induced several ovulatory alterations, consisting of a decrease in the number of oocytes effectively ovulated, trapping of oocytes inside the luteinized follicle, as well as abnormal follicle rupture at the basolateral sides, with release of the oocyte and follicular fluid to the interstitium. Yet, the precise role of prostaglandins in ovulation and whether some of the ovulatory defects induced by indomethacin are due to interference with additional components of the ovulatory cascade, beyond prostaglandin synthesis, are not completely understood. We have used gonadotrophin-primed immature rats to analyse whether, compared to indomethacin, selective inhibition of COX-2, with or without concomitant inhibition of COX-1, or selective inhibition of the lipooxygenase (LOX) pathway, induce similar ovulatory alterations. Immature rats (27 days of age) were injected PMSG (10 IU), and 48 h later hCG (10 IU) subcutaneously, and different anti-inflammatory drugs. Animals were killed at 21 h after hCG injection. Rats treated with the selective COX-2 inhibitor NS398 (10 mg/kg body weight, (bw)) showed alterations in follicle rupture as those treated with indomethacin (0.5 mg/rat), albeit affecting a lower number of follicles, irrespective of the concomitant inhibition of COX-1 with the selective inhibitor SC560 (10 mg/kg bw). Rats treated with the LOX inhibitor NDGA (300 mg/kg bw) did not show ovulatory alterations. These data indicate that the characteristic alterations of follicle rupture induced by indomethacin, are also induced by selective COX-2 inhibitors, strengthening the contention that prostaglandins play a crucial role in the spatial targeting of follicle rupture at the apex.

Expression of KiSS-1 in rat ovary: putative local regulator of ovulation?

Kisspeptins, the products of KiSS-1 gene, and their receptor, GPR54, have recently emerged as essential gatekeepers of reproduction, mainly through regulation of GnRH secretion at the hypothalamus. However, the profound hypogonadotropism linked to GPR54 inactivation is likely to mask additional functions of this system at other levels of the gonadal axis, in which expression of KiSS-1 and GPR54 has been preliminarily reported. We describe herein the expression of KiSS-1 gene and kisspeptin immunoreactivity (IR) in rat ovary and evaluate its developmental and hormonal regulation. KiSS-1 and GPR54 mRNAs were persistently detected in adult ovary along estrous cycle. Yet, contrary to GPR54, ovarian KiSS-1 levels fluctuated in a cyclic-dependent manner, with a robust increase in the afternoon of proestrus, i.e. preceding ovulation. In addition, kisspeptin-IR was observed in rat ovary, with strong signals in theca layers of growing follicles, corpora lutea, and interstitial gland, compartments in which modest GPR54-IR was also detected. Interestingly, the rise in ovarian KiSS-1 mRNA at proestrus was prevented by blockade of preovulatory gonadotropin surge and restored by replacement with human chorionic gonadotropin as superagonist of LH. In addition, immature ovaries showed low to negligible levels of KiSS-1 mRNA, which were significantly enhanced by gonadotropin priming. In summary, we present novel evidence for the developmental and hormonally regulated expression of the KiSS-1 gene, and the presence of kisspeptin-IR, in rat ovary. The ability of the LH surge to timely induce ovarian expression of KiSS-1 at the preovulatory period strongly suggests a previously unsuspected role of locally produced kisspeptin in the control of ovulation.

Non-steroidal anti-inflammatory drugs (NSAIDs) and ovulation: lessons from morphology.

Ovulation constitutes the central event in ovarian physiology, and ovulatory disfunction is a relevant cause of female infertility. Non-steroidal anti-inflammatory drugs (NSAIDs), widely used due to their analgesic and anti-inflammatory properties, consistently inhibit ovulation in all mammalian species investigated so far, likely due to the inhibition of cyclooxygenase 2 (COX-2), the inducible isoform of COX, that is the rate-limiting enzyme in prostaglandin (PG) synthesis. COX-2 inhibition has major effects on ovulation, fertilization and implantation, and NSAID therapy is likely implicated in human infertility and could be an important, frequently overlooked, cause of ovulatory disfunction in women. Although there is compelling evidence for a role of PGs in ovulation, the molecular targets and the precise role of these compounds in the ovulatory process are not fully understood. Morphological studies from rats treated with indomethacin (INDO), a potent inhibitor of PG synthesis, provide evidence on the actions of NSAIDs in ovulation, as well as on the possible roles of PGs in the ovulatory process. Cycling rats treated with INDO during the preovulatory period show abnormal ovulation, due to disruption of the spatial targeting of follicle rupture at the apex. Noticeably, gonadotropin-primed immature rats (widely used as a model for the study of ovulation) show age-dependent ovulatory defects similar to those of cycling rats treated with INDO. These data suggest that NSAID treatment disrupts physiological mechanisms underlying spatial targeting of follicle rupture at the apex, which are not fully established in very young rats. We summarize herein the ovulatory defects after pharmacologic COX-2 inhibition, and discuss the possible mechanisms underlying the anti-ovulatory actions of NSAIDs.

Pattern of orexin expression and direct biological actions of orexin-a in rat testis.

Orexins, hypothalamic neuropeptides initially involved in the control of food intake and sleep-wake cycle, have recently emerged as pleiotropic regulators of different biological systems, including the reproductive axis. Besides central actions, peripheral expression and functions of orexins have been reported, and prepro-orexin and orexin type-1 receptor mRNAs have been detected in the testis. However, the pattern of expression and biological actions of orexin in the male gonad remain mostly unexplored. In this study, we report analyses on testicular prepro-orexin mRNA expression and orexin-A immunoreactivity in different experimental settings, and on direct effects of orexin-A on seminiferous tubule functions. Expression of prepro-orexin mRNA was demonstrated in the rat testis at different stages of postnatal development, with negligible levels at early juvenile period and maximum values in adulthood. Likewise, orexin-A immunoreactivity was demonstrated along postnatal maturation, with strong peptide signal in Leydig cells and spermatocytes at specific stages of meiosis. Testicular expression of prepro-orexin mRNA appeared hormonally regulated; its levels decreased after hypophysectomy and increased after gonadotropin replacement and ghrelin stimulation. Finally, orexin-A suppressed the expression of key Sertoli cell genes, such as Müllerian-inhibiting substance and stem cell factor, and inhibited DNA synthesis in specific stages of the seminiferous epithelium. In conclusion, we provide evidence for the regulated expression of orexin in the rat testis and its potential involvement in the control of seminiferous tubule functions. Together with our recent results on the expression of orexin type-1 receptor in the rat testis, our data further document a novel testicular site of action of orexins in the control of male reproductive axis.

Expression of glutaredoxin (thioltransferase) in the rat ovary during the oestrous cycle and postnatal development.

Glutaredoxins (Grxs) are low-molecular-weight proteins which participate in redox events in association with glutathione (GSH) and are involved in a variety of cellular processes. It is known that oxidative stress plays important physiological roles within the ovary. In the present study, we have prepared specific antibodies against rat Grx and have used them to localize the protein in the ovaries of rats during postnatal development and during the oestrous cycle, by immunohistochemical methods. We have also performed a quantitative analysis of Grx by ELISA and Western blotting in homogenates of whole ovaries of cycling and pseudopregnant rats. We have found a prominent presence of Grx in the oocytes and in corpora lutea (CL) during developmental and oestrous cycle changes. Grx was absent from the oocytes in the first days of postnatal life when marked oocyte degeneration takes place, but its presence was very conspicuous in the cytoplasm of oocytes in healthy and attretic follicles in rats from 10 days of age onward, independently of the day of oestrous cycle. Follicular cells were negative. Grx immunostaining in the CL was strong in infiltrating macrophages and in a population of steroidogenic cells that survived the apoptotic burst in regressing CL and in CL remnants, but was faint or absent in young CL of the current cycle and in CL during pseudopregnancy. Grx content and oxidoreductase activity in whole ovaries increased significantly during the phase transition from proestrous to oestrous along the cycle. These results support a role of Grx in the maintenance of functional oocytes and in luteal cells surviving the regression process, probably as a consequence of the demonstrated deglutathionylating function of this protein in an antioxidant and antiapoptotic context.

Cyclic changes of the ovarian surface epithelium in the rat.

The ovarian surface epithelium (OSE) plays pivotal roles during ovulation and postovulatory wound repair. In this paper we describe the proliferative activity of the OSE through the estrous cycle in adult cycling rats, by immunohistochemical detection of DNA-incorporated bromodeoxyuridine (BrdU). Immunohistochemical detection of estrogen receptor alpha (ERalpha) and progesterone receptor was also performed. The cycle of the OSE consists of a proliferative phase (that lasts for two consecutive estrous cycles) and a quiescent phase of variable duration. Cyclic changes in the OSE were related to the underlying ovarian structure. OSE areas covering growing follicles entered into the proliferative phase during the transition from proestrus to estrus, with the appearance of fast-growing class 1 follicles, destined to ovulate at the end of the current estrous cycle. A labeling index (after pulse-labeling BrdU treatment) of about 7% was maintained throughout the estrous cycle in parallel to follicle growth. Cumulative BrdU-labeling (after daily BrdU treatment) indicated that about 1/3 of the total OSE cell proliferation was related to follicle growth. Following ovulation, OSE cells covering newly-formed corpora lutea showed a labeling index of about 50% that decreased through metestrus and diestrus (about 13% and 3%, respectively), returning to basal levels by proestrus. Cumulative BrdU-labeling indicated that about 2/3 of the total proliferative activity was related to ovulation repair/luteinization. The remaining OSE covering ovarian stroma or structurally regressing corpora lutea of previous cycles showed negligible BrdU labeling. The equivalent proliferative activity found in the OSE covering newly-formed corpora lutea in indomethacin-treated rats lacking rupture of the OSE at the apex, demonstrated that ovulation-triggered proliferation was not dependent on the loss of integrity of the OSE at the ovulation site. OSE cells expressed ERalpha throughout the cycle, but no differential expression was found between proliferating and quiescent OSE areas. On the contrary, OSE cells did not express PR at any time of the cycle. These data indicate the existence of a cycle of the OSE, related to the cyclic changes in the underlying ovarian structure and strongly suggest that the proliferative activity of the OSE is regulated by local microenvironmental rather than by systemic factors.