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Polycystic ovary syndrome (PCOS) is a complex disease affecting up to 15% of women of reproductive age. Women with PCOS suffer from reproductive dysfunctions with excessive androgen secretion and irregular ovulation, leading to reduced fertility and pregnancy complications. The syndrome is associated with a wide range of comorbidities including type 2 diabetes, obesity, and psychiatric disorders. Despite the high prevalence of PCOS, its etiology remains unclear. To understand the pathophysiology of PCOS, how it is inherited, and how to predict PCOS, and prevent and treat women with the syndrome, animal models provide an important approach to answering these fundamental questions. This minireview summarizes recent investigative efforts on PCOS-like rodent models aiming to define underlying mechanisms of the disease and provide guidance in model selection. The focus is on new genetic rodent models, on a naturally occurring rodent model, and provides an update on prenatal and peripubertal exposure models.

The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable regulator of adrenal and gonadal formation, playing roles in sex determination, hypothalamic development, and pituitary function. This study aimed to identify the roles of SF-1 in postnatal female reproductive function. Using a progesterone receptor–driven Cre recombinase, we developed a novel murine model, characterized by conditional depletion of SF-1 [PR-Cre;Nr5a1f/f; conditional knockout (cKO)] in the hypothalamic-pituitary-gonadal axis. Mature female cKO were infertile due to the absence of ovulation. Reduced gonadotropin concentrations in the pituitary gland that were nevertheless sufficient to maintain regular estrous cycles were observed in mature cKO females. The cKO ovaries showed abnormal lipid accumulation in the stroma, associated with an irregular expression of cholesterol homeostatic genes such as Star, Scp2, and Acat1. The depletion of SF-1 in granulosa cells prevented appropriate cumulus oöphorus expansion, characterized by reduced expression of Areg, Ereg, and Ptgs2. Exogenous delivery of gonadotropins to cKO females to induce ovulation did not restore fertility and was associated with impaired formation and function of corpora lutea accompanied by reduced expression of the steroidogenic genes Cyp11a1 and Cyp19a1 and attenuated progesterone production. Surgical transplantation of cKO ovaries to ovariectomized control animals (Nr5a1f/f) resulted in 2 separate phenotypes, either sterility or apparently normal fertility. The deletion of SF-1 in the pituitary and in granulosa cells near the moment of ovulation demonstrated that this nuclear receptor functions across the pituitary-gonadal axis and plays essential roles in gonadotropin synthesis, cumulus expansion, and luteinization.

Female mice homozygous for an engineered Gnrhr E90K mutation have reduced gonadotropin-releasing hormone signaling, leading to infertility. Their ovaries have numerous antral follicles but no corpora lutea, indicating a block to ovulation. These mutants have high levels of circulating estradiol and low progesterone, indicating a state of persistent estrus. This mouse model provided a unique opportunity to examine the lack of cyclic levels of ovarian hormones on uterine gland biology. Although uterine gland development appeared similar to controls during prepubertal development, it was compromised during adolescence in the mutants. By age 20 weeks, uterine gland development was comparable to controls, but pathologies, including cribriform glandular structures, were observed. Induction of ovulations by periodic human chorionic gonadotropin treatment did not rescue postpubertal uterine gland development. Interestingly, progesterone receptor knockout mice, which lack progesterone signaling, also have defects in postpubertal uterine gland development. However, progesterone treatment did not rescue postpubertal uterine gland development. These studies indicate that chronically elevated levels of estradiol with low progesterone and therefore an absence of cyclic ovarian hormone secretion disrupts postpubertal uterine gland development and homeostasis.

In the liver, the sterol response element binding protein (SREBP) and the SREBP cleavage-activated protein (SCAP) complex upregulate cholesterol biosynthesis by gene induction of de novo cholesterol synthetic enzymes (Hmgcr, Cyp51, and Dhcr7). Insulin induced gene 1 (INSIG1) negatively regulates cholesterol biosynthesis by the inhibition of de novo cholesterol biosynthetic gene expression. In the ovary, cholesterol is de novo synthesized; however, the roles of SREBP and its regulators (SCAP and INSIG1) are not well understood. In this study, when immature mice were treated with gonadotropins (eCG followed by hCG), eCG induced and hCG maintained the expression of SREBP-1a, -2, and SCAP granulosa cells, whereas INSIG1 expression was dramatically downregulated after hCG injection. Downregulation of INSIG1 led to generate the SREBPs active form and translocate the SREBPs active form to nuclei. Inhibition of generation of the SREBPs active form by fatostatin or Scap siRNA in both in vivo and in vitro significantly decreased the expressions of de novo cholesterol biosynthetic enzymes, cholesterol accumulation, and progesterone (P4) production compared with the control group. Fatostatin treatment inhibited the ovulation and increased the formation of abnormal corpus luteum which trapped the matured oocyte in the corpus luteum; however, the phenomenon was abolished by P4 administration. The results showed that decreasing INSIG1 level after hCG stimulation activated SREBP-induced de novo cholesterol biosynthesis in granulosa cells of preovulatory follicles, which is essential for P4 production and the rupture of matured oocyte during ovulation process.

The ovulatory luteinizing hormone (LH) surge induces rapid changes of gene expression and cellular functions in granulosa cells (GCs) undergoing luteinization. However, it remains unclear how the changes in genome-wide gene expression are regulated. H3K4me3 histone modifications are involved in the rapid alteration of gene expression. In this study, we investigated genome-wide changes of transcriptome and H3K4me3 status in mouse GCs undergoing luteinization. GCs were obtained from mice treated with equine chorionic gonadotropin (hCG) before, 4 hours, and 12 hours after human chorionic gonadotropin injection. RNA-sequencing identified a number of upregulated and downregulated genes, which could be classified into 8 patterns according to the time-course changes of gene expression. Many genes were transiently upregulated or downregulated at 4 hours after hCG stimulation. Gene Ontology terms associated with these genes included steroidogenesis, ovulation, cumulus-oocyte complex (COC) expansion, angiogenesis, immune system, reactive oxygen species (ROS) metabolism, inflammatory response, metabolism, and autophagy. The cellular functions of DNA repair and cell growth were newly identified as being activated during ovulation. Chromatin immunoprecipitation–sequencing revealed a genome-wide and rapid change in H3K4me3 during ovulation. Integration of transcriptome and H3K4me3 data identified many H3K4me3-associated genes that are involved in steroidogenesis, ovulation, COC expansion, angiogenesis, inflammatory response, immune system, ROS metabolism, lipid and glucose metabolism, autophagy, and regulation of cell size. The present results suggest that genome-wide changes in H3K4me3 after the LH surge are associated with rapid changes in gene expression in GCs, which enables GCs to acquire a lot of cellular functions within a short time that are required for ovulation and luteinization.

FOS, a subunit of the activator protein-1 (AP-1) transcription factor, has been implicated in various cellular changes. In the human ovary, the expression of FOS and its heterodimeric binding partners JUN, JUNB, and JUND increases in periovulatory follicles. However, the specific role of the FOS/AP-1 remains elusive. The present study determined the regulatory mechanisms driving the expression of FOS and its partners and functions of FOS using primary human granulosa/lutein cells (hGLCs). Human chorionic gonadotropin (hCG) induced a biphasic increase in the expression of FOS, peaking at 1 to 3 hours and 12 hours. The levels of JUN proteins were also increased by hCG, with varying expression patterns. Coimmunoprecipitation analyses revealed that FOS is present as heterodimers with all JUN proteins. hCG immediately activated protein kinase A and p42/44MAPK signaling pathways, and inhibitors for these pathways abolished hCG-induced increases in the levels of FOS, JUN, and JUNB. To identify the genes regulated by FOS, high-throughput RNA sequencing was performed using hGLC treated with hCG ± T-5224 (FOS inhibitor). Sequencing data analysis revealed that FOS inhibition affects the expression of numerous genes, including a cluster of genes involved in the periovulatory process such as matrix remodeling, prostaglandin synthesis, glycolysis, and cholesterol biosynthesis. Quantitative PCR analysis verified hCG-induced, T-5224-regulated expression of a selection of genes involved in these processes. Consistently, hCG-induced increases in metabolic activities and cholesterol levels were suppressed by T-5224. This study unveiled potential downstream target genes of and a role for the FOS/AP-1 complex in metabolic changes and cholesterol biosynthesis in granulosa/lutein cells of human periovulatory follicles.

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

Sirtuin 1 (Sirt1) is an NAD-dependent class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, evidence suggests that SIRT1 in neurons plays a role in the central regulation of energy balance and reproduction, but no studies have addressed the contribution of astrocytes. We show here that overexpression of SIRT1 in astrocytes causes markedly increased food intake, body weight gain, and glucose intolerance, but expression of a deacetylase-deficient SIRT1 mutant decreases food intake and body weight and improves glucose tolerance, particularly in female mice. Paradoxically, the effect of these SIRT1 mutants on insulin tolerance was reversed, with overexpression showing greater insulin sensitivity. The mice overexpressing SIRT1 were more active, generated more heat, and had elevated oxygen consumption, possibly in compensation for the increased food intake. The female overexpressing mice were also more sensitive to diet-induced obesity. Reproductively, the mice expressing the deacetylase-deficient SIRT1 mutant had impaired estrous cycles, decreased LH surges, and fewer corpora lutea, indicating decreased ovulation. The GnRH neurons were responsive to kisspeptin stimulation, but hypothalamic expression of Kiss1 was reduced in the mutant mice. Our results showed that SIRT1 signaling in astrocytes can contribute to metabolic and reproductive regulation independent of SIRT1 effects in neurons. Fil: Choi, Irene. VA San Diego Healthcare System; Estados Unidos Fil: Rickert, Emily. University of California at San Diego; Estados Unidos Fil: Fernandez, Marina Olga. University of California at San Diego; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Webster, Nicholas J.G.. VA San Diego Healthcare System; Estados Unidos. University of California at San Diego; Estados Unidos

Chronic undernutrition is a type of metabolic stress that impairs reproduction in multiple species. Although energy balance and female reproductive capacity is recognized as tightly coupled, the neuroendocrine loci and molecular mechanisms that mediate ovarian cycle dysfunction during chronic undernutrition in adult females remain poorly understood. Here, we present a series of studies in which we tested the hypothesis that inhibition of kisspeptin (Kiss1) neurons, which are critical for controlling luteinizing hormone (LH) pulses and the preovulatory LH surge in females, underlies the impairment of the ovarian cycle by undernutrition. We first investigated the effect of chronic undernutrition (70% of unrestricted feed intake) on estrous cyclicity in intact female c57bl6 mice. Undernutrition caused a rapid cessation of ovarian cyclicity during the 2-week treatment, suppressing ovarian steroidogenesis and inhibiting ovulation. Using 2 well-defined estradiol-replacement paradigms, we directly tested the hypothesis that undernutrition inhibits Kiss1 neurons in the arcuate nucleus (ARCKiss1), which are required for LH pulses and in the anteroventral periventricular nucleus (AVPVKiss1), which are necessary for LH surge secretion. Undernutrition prevented LH pulses and impaired ARCKiss1 neuronal activation, using c-Fos as a marker, in ovariectomized females subcutaneously implanted with a pellet containing a diestrus-like level of estradiol. In addition, undernutrition completely blocked the estradiol-induced LH surge and diminished Kiss1 messenger RNA abundance, without decreasing estradiol receptor α (Erα), in micropunches of the AVPV. Collectively, these studies demonstrate that undernutrition disrupts ovarian cyclicity in females via impairment both of ARCKiss1 control of LH pulses and AVPVKiss1 induction of the LH surge.

Mouse models with altered gonadotropin functions have provided invaluable insight into the functions of these hormones/receptors. Here we describe the repurposing of the infertile and hypogonadal luteinizing hormone receptor (LHR) knockout mouse model (LuRKO), to address outstanding questions in reproductive physiology. Using crossbreeding strategies and physiological and histological analyses, we first addressed the physiological relevance of forced LHR homomerization in female mice using BAC expression of 2 ligand-binding and signaling deficient mutant LHR, respectively, that have previously shown to undergo functional complementation and rescue the hypogonadal phenotype of male LuRKO mice. In female LuRKO mice, coexpression of signaling and binding deficient LHR mutants failed to rescue the hypogonadal and anovulatory phenotype. This was apparently due to the low-level expression of the 2 mutant LHR and potential lack of luteinizing hormone (LH)/LHR-dependent pleiotropic signaling that has previously been shown at high receptor densities to be essential for ovulation. Next, we utilized a mouse model overexpressing human chorionic gonadotropin (hCG) with increased circulating “LH/hCG”-like bioactivity to ~40 fold higher than WT females, to determine if high circulating hCG in the LuRKO background could reveal putative LHR-independent actions. No effects were found, thus, suggesting that LH/hCG mediate their gonadal and non-gonadal effects solely via LHR. Finally, targeted expression of a constitutively active follicle stimulating hormone receptor (FSHR) progressed antral follicles to preovulatory follicles and displayed phenotypic markers of enhanced estrogenic activity but failed to induce ovulation in LuRKO mice. This study highlights the critical importance and precise control of functional LHR and FSHR for mediating ovarian functions and of the potential repurposing of existing genetically modified mouse models in answering outstanding questions in reproductive physiology.

Coordination of ovulation and behavior is critical to reproductive success in many species. During the female estrous cycle, the preovulatory gonadotropin surge occurs when ovarian follicles reach maturity and, in rodents, it begins just before the daily onset of activity, ensuring that ovulation coincides with sex behavior. Timing of the surge relies on projections from the suprachiasmatic nucleus (SCN), the locus of the central circadian clock, to hypothalamic circuits that regulate gonadotropin secretion. The cellular mechanisms through which the SCN controls these circuits and gates the preovulatory surge to the appropriate estrous cycle stage, however, are poorly understood. We investigated in mice the functional impact of SCN arginine-vasopressin (AVP) neuron projections to kisspeptin (Kiss1) neurons in the rostral periventricular area of the third ventricle (RP3VKiss1), responsible for generating the preovulatory surge. Conditional anterograde tracing revealed that SCNAVP neurons innervate approximately half of the RP3VKiss1 neurons. Optogenetic activation of SCNAVP projections in brain slices caused an AVP-mediated stimulation of RP3VKiss1 action potential firing in proestrus, the cycle stage when the surge is generated. This effect was less prominent in diestrus, the preceding cycle stage, and absent in estrus, following ovulation. Remarkably, in estrus, activation of SCNAVP projections resulted in GABA-mediated inhibition of RP3VKiss1 neuron firing, an effect rarely encountered in other cycle stages. Together, these data reveal functional plasticity in SCNAVP neuron output that drives opposing effects on RP3VKiss1 neuron activity across the ovulatory cycle. This might contribute to gating activation of the preovulatory surge to the appropriate estrous cycle stage.

Polybrominated diphenyl ethers (PBDEs), a major class of flame retardants incorporated into numerous consumer products, leach out into dust resulting in widespread exposure. There is evidence from in vitro and in vivo animal studies that PBDEs affect ovarian granulosa cell function and follicular development, yet human studies of their association with female infertility are inconclusive. Here, we tested the hypothesis that exposure to the PBDEs in follicular fluid is associated with dysregulation of gene expression in the mural and cumulus granulosa cells collected from women undergoing in vitro fertilization by intracytoplasmic sperm injection. The median concentration of the ∑ 10PBDEs detected in the follicular fluid samples (n = 37) was 15.04 pg/g wet weight. RNA microarray analyses revealed that many genes were differentially expressed in mural and cumulus granulosa cells. Highest vs lowest quartile exposure to the Σ 10PBDEs or to 2 predominant PBDE congeners, BDE-47 or BDE-153, was associated with significant effects on gene expression in both cell types. Mural granulosa cells were generally more sensitive to PBDE exposure compared to cumulus cells. Overall, gene expression changes associated with BDE-47 exposure were similar to those for ∑ 10PBDEs but distinct from those associated with BDE-153 exposure. Interestingly, exposure to BDE-47 and ∑ 10PBDEs activated the expression of genes in pathways that are important in innate immunity and inflammation. To the best of our knowledge, this is the first demonstration that exposure to these environmental chemicals is associated with the dysregulation of pathways that play an essential role in ovulation.

Progesterone receptor (PGR) is indispensable for pregnancy in mammals. Uterine PGR responds to the heightened levels of ovarian progesterone (P4) after ovulation and regulates uterine gene transcription for successful embryo implantation. Although epithelial and stromal P4-PGR signaling may interact with each other to form appropriate endometrial milieu for uterine receptivity and the subsequent embryo attachment, it remains unclear what the specific roles of epithelial P4-PGR signaling in the adult uterus are. Here we generated mice with epithelial deletion of Pgr in the adult uterus (Pgrfl/flLtfCre/+ mice) by crossing Pgr-floxed and Ltf-Cre mice. Pgrfl/flLtfCre/+ mice are infertile due to the impairment of embryo attachment. Pgrfl/flLtfCre/+ uteri did not exhibit epithelial growth arrest, suggesting compromised uterine receptivity. Both epithelial and stromal expressions of P4-responsive genes decreased in Pgrfl/flLtfCre/+ mice during the peri-implantation period, indicating that epithelial Pgr deletion affects not only epithelial but stromal P4 responsiveness. In addition, uterine LIF, an inducer of embryo attachment, was decreased in Pgrfl/flLtfCre/+ mice. The RNA-seq analysis using luminal epithelial specimens dissected out by laser capture microdissection revealed that the signaling pathways related to extracellular matrix, cell adhesion, and cell proliferation are altered in Pgr fl/flLtf Cre/+ mice. These findings suggest that epithelial PGR controls both epithelial and stromal P4 responsiveness and epithelial cell differentiation, which provides normal uterine receptivity and subsequent embryo attachment.

Inhibin was first characterized in mammals as a gonadal dimeric protein that inhibited pituitary follicle-stimulating hormone (FSH) secretion. As in mammals, the inhibin-specific α subunit (INHA/Inha/inha) has also been characterized in teleosts; however, its functions and physiological importance in fish reproduction remain unknown. Using CRISPR/Cas9 method, we generated an inha-deficient zebrafish line and analyzed its reproductive performance. As expected, pituitary expression of fshb increased significantly in both the young and the adult inha mutant. The expression of lhb also increased in the mutant, but only in sexually mature adults. Interestingly, the expression of activin βA (inhbaa) increased significantly in both the ovary and the testis of inha mutant, and the expression of ovarian aromatase (cyp19a1a) also increased dramatically in the mutant ovary. The juvenile female mutant showed clear signs of early follicle activation or precocious puberty onset. However, the adult female mutant was infertile with follicles arrested at the full-grown stage without final oocyte maturation and ovulation. Although follicle growth was normal overall in the mutant, the size and distribution of yolk granules in oocytes were distinct and some follicles showed granulosa cell hypertrophy. In contrast to females, inha-null males showed normal spermatogenesis and fertility. As reported in mammals, we also found sporadic tumor formation in inha mutants. Taken together, our study not only confirmed some conserved roles of inhibin across vertebrates, such as inhibition of FSH biosynthesis and tumor formation, but also revealed novel aspects of inhibin functions such as disruption of folliculogenesis and female infertility but no obvious involvement in spermatogenesis in fish.

In female mammals, cycles in reproductive function depend both on the biological clock synchronized to the light/dark cycle and on a balance between the negative and positive feedbacks of estradiol, whose concentration varies during oocyte maturation. In women, studies report that chronodisruptive environments such as shiftwork may impair fertility and gestational success. The objective of this study was to explore the effects of shifted light/dark cycles on both the robustness of the estrous cycles and the timing of the preovulatory luteinizing hormone (LH) surge in female mice. When mice were exposed to a single 10-hour phase advance or 10-hour phase delay, the occurrence and timing of the LH surge and estrous cyclicity were recovered at the third estrous cycle. By contrast, when mice were exposed to chronic shifts (successive rotations of 10-hoursour phase advances for 3 days followed by 10-hour phase delays for 4 days), they exhibited a severely impaired reproductive activity. Most mice had no preovulatory LH surge at the beginning of the chronic shifts. Furthermore, the gestational success of mice exposed to chronic shifts was reduced, because the number of pups was 2 times lower in shifted than in control mice. In conclusion, this study reports that exposure of female mice to a single phase shift has minor reproductive effects, whereas exposure to chronically disrupted light/dark cycles markedly impairs the occurrence of the preovulatory LH surge, leading to reduced fertility.

Follicle development is the most crucial step toward female fertility and is controlled mainly by follicle-stimulating hormone (FSH). In ovarian granulosa cells (GCs), FSH activates protein kinase A by increasing 3′,5′-cyclic adenosine 5′-monophosphate (cAMP). Since cAMP signaling is impinged in part by salt-inducible kinases (SIKs), we examined the role of SIKs on the regulation of FSH actions. Here, we report that SIKs are essential for normal ovarian function and female fertility. All SIK isoforms are expressed in human and rodent GCs at different levels (SIK3>SIK2>SIK1). Pharmacological inhibition of SIK activity potentiated the stimulatory effect of FSH on markers of GC differentiation in mouse, rat, and human GCs and estradiol production in rat GCs. In humans, SIK inhibition strongly enhanced FSH actions in GCs of patients with normal or abnormal ovarian function. The knockdown of SIK2, but not SIK1 or SIK3, synergized with FSH on the induction of markers of GC differentiation. SIK inhibition boosted gonadotropin-induced GC differentiation in vivo, while the genomic knockout of SIK2 led to a significant increase in the number of ovulated oocytes. Conversely, SIK3 knockout females were infertile, FSH insensitive, and had abnormal folliculogenesis. These findings reveal novel roles for SIKs in the regulation of GC differentiation and female fertility, and contribute to our understanding of the mechanisms regulated by FSH. Furthermore, these data suggest that specific pharmacological modulation of SIK2 activity could be of benefit to treat ovulatory defects in humans and to increase the propagation of endangered species and farm mammals.

Elevated and sustained estradiol concentrations cause a gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) surge that is necessary for ovulation. In sheep, several different neural systems have been implicated in this stimulatory action of estradiol and this study focused on somatostatin (SST) neurons in the ventral lateral region of the ventral medial nucleus (vlVMN) which express c-Fos during the surge. First, we determined if increased activity of SST neurons could be related to elevated GnRH secretion by assessing SST synapses onto GnRH neurons and neurons coexpressing kisspeptin, neurokinin B, dynorphin (KNDy). We found that the percentage of preoptic area GnRH neurons that receive SST input increased during the surge compared with other phases of the cycle. However, since SST is generally inhibitory, and pharmacological manipulation of SST signaling did not alter the LH surge in sheep, we hypothesized that nitric oxide (NO) was also produced by these neurons to account for their activation during the surge. In support of this hypothesis we found that (1) the majority of SST cells in the vlVMN (>80%) contained neuronal nitric oxide synthase (nNOS); (2) the expression of c-Fos in dual-labeled SST-nNOS cells, but not in single-labeled cells, increased during the surge compared with other phases of the cycle; and (3) intracerebroventricular (ICV) infusion of the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester, completely blocked the estrogen-induced LH surge. These data support the hypothesis that the population of SST-nNOS cells in the vlVMN are a source of NO that is critical for the LH surge, and we propose that they are an important site of estradiol positive feedback in sheep.

Ovulation is the appropriately timed release of a mature, developmentally competent oocyte from the ovary into the oviduct, where fertilization occurs. Importantly, ovulation is tightly linked with oocyte maturation, demonstrating the interdependency of these two parallel processes, both essential for female fertility. Initiated by pituitary gonadotropins, the ovulatory process is mediated by intrafollicular paracrine factors from the theca, mural, and cumulus granulosa cells, as well as the oocyte itself. The result is the induction of cumulus expansion, proteolysis, angiogenesis, inflammation, and smooth muscle contraction, which are each required for follicular rupture. These complex intercellular communication networks and the essential ovulatory genes have been well defined in mouse models and are highly conserved in primates, including humans. Importantly, recent discoveries in regulation of ovulation highlight new areas of investigation.

The luteinizing hormone (LH) surge is essential for ovulation, but the intrafollicular factors induced by LH that mediate ovulatory processes (e.g., angiogenesis) are poorly understood, especially in women. The role of secretogranin II (SCG2) and its cleaved bioactive peptide, secretoneurin (SN), were investigated as potential mediators of ovulation by testing the hypothesis that SCG2/SN is induced in granulosa cells by human chorionic gonadotropin (hCG), via a downstream LH receptor signaling mechanism, and stimulates ovarian angiogenesis. Humans, nonhuman primates, and rodents were treated with hCG in vivo resulting in a significant increase in the messenger RNA and protein levels of SCG2 in granulosa cells collected early during the periovulatory period and just prior to ovulation (humans: 12 to 34 hours; monkeys: 12 to 36 hours; rodents: 4 to 12 hours post-hCG). This induction by hCG was recapitulated in an in vitro culture system utilizing granulosa-lutein cells from in vitro fertilization patients. Using this system, inhibition of downstream LH receptor signaling pathways revealed that the initial induction of SCG2 is regulated, in part, by epidermal growth factor receptor signaling. Further, human ovarian microvascular endothelial cells were treated with SN (1 to 100 ng/mL) and subjected to angiogenesis assays. SN significantly increased endothelial cell migration and new sprout formation, suggesting induction of ovarian angiogenesis. These results establish that SCG2 is increased in granulosa cells across species during the periovulatory period and that SN may mediate ovulatory angiogenesis in the human ovary. These findings provide insight into the regulation of human ovulation and fertility.

Core binding factor β (CBFβ) is a non–DNA-binding partner of all RUNX proteins and critical for transcription activity of CBF transcription factors (RUNXs/CBFβ). In the ovary, the expression of Runx1 and Runx2 is highly induced by the luteinizing hormone (LH) surge in ovulatory follicles, whereas Cbfb is constitutively expressed. To investigate the physiological significance of CBFs in the ovary, the current study generated two different conditional mutant mouse models in which granulosa cell expression of Cbfb and Runx2 was reduced by Cre recombinase driven by an Esr2 promoter. Cbfb(gc−/−) and Cbfb(gc−/−) × Runx2(gc+/−) mice exhibited severe subfertility and infertility, respectively. In the ovaries of both mutant mice, follicles develop normally, but the majority of preovulatory follicles failed to ovulate either in response to human chorionic gonadotropin administration in pregnant mare serum gonadotropin–primed immature animals or after the LH surge at 5 months of age. Morphological and physiological changes in the corpus luteum of these mutant mice revealed the reduced size, progesterone production, and vascularization, as well as excessive lipid accumulation. In granulosa cells of periovulatory follicles and corpora lutea of these mice, the expression of Edn2, Ptgs1, Lhcgr, Sfrp4, Wnt4, Ccrl2, Lipg, Saa3, and Ptgfr was also drastically reduced. In conclusion, the current study provided in vivo evidence that CBFβ plays an essential role in female fertility by acting as a critical cofactor of CBF transcription factor complexes, which regulate the expression of specific key ovulatory and luteal genes, thus coordinating the ovulatory process and luteal development/function in mice.

Placental growth factor (PGF) is member of the vascular endothelial growth factor (VEGF) family of angiogenesis regulators. VEGFA is an established regulator of ovulation and formation of the corpus luteum. To determine whether PGF also mediates aspects of ovulation and luteinization, macaques received gonadotropins to stimulate multiple follicular development. Ovarian biopsies and whole ovaries were collected before (0 hours) and up to 36 hours after human chorionic gonadotropin (hCG) administration to span the ovulatory interval. PGF and VEGFA were expressed by both granulosa cells and theca cells. In follicular fluid, PGF and VEGFA levels were lowest before hCG. PGF levels remained low until 36 hours after hCG administration, when PGF increased sevenfold to reach peak levels. Follicular fluid VEGFA increased threefold to reach peak levels at 12 hours after hCG, then dropped to intermediate levels. To explore the roles of PGF and VEGFA in ovulation, luteinization, and follicular angiogenesis in vivo, antibodies were injected into the follicular fluid of naturally developed monkey follicles; ovariectomy was performed 48 hours after hCG, with ovulation expected about 40 hours after hCG. Intrafollicular injection of control immunoglobulin G resulted in no retained oocytes, follicle rupture, and structural luteinization, including granulosa cell hypertrophy and capillary formation in the granulosa cell layer. PGF antibody injection resulted in oocyte retention, abnormal rupture, and incomplete luteinization, with limited and disorganized angiogenesis. Injection of a VEGFA antibody resulted in oocyte retention and very limited follicle rupture or structural luteinization. These studies demonstrate that PGF, in addition to VEGFA, is required for ovulation, luteinization, and follicular angiogenesis in primates.

In women, the window of implantation is limited to a brief 2- to 3-day period characterized by optimal levels of circulating ovarian hormones and a receptive endometrium. Although the window of implantation is assumed to occur 8 to 10 days after ovulation in women, molecular markers of endometrial receptivity are necessary to determine optimal timing prior to embryo transfer. Previous studies showed that members of the bone morphogenetic protein (BMP) family are expressed in the uterus necessary for female fertility; however, the role of BMP7 during implantation and in late gestation is not known. To determine the contribution of BMP7 to female fertility, we generated Bmp7flox/flox-Pgr-cre+/– [BMP7 conditional knockout (cKO)] mice. We found that absence of BMP7 in the female reproductive tract resulted in subfertility due to uterine defects. At the time of implantation, BMP7 cKO females displayed a nonreceptive endometrium with elevated estrogen-dependent signaling. These implantation-related defects also affected decidualization and resulted in decreased expression of decidual cell markers such as Wnt4, Cox2, Ereg, and Bmp2. We also observed placental abnormalities in pregnant Bmp7 cKO mice, including excessive parietal trophoblast giant cells and absence of a mature placenta at 10.5 days post coitum. To establish possible redundant roles of BMP5 and BMP7 during pregnancy, we generated double BMP5 knockout/BMP7 cKO [BMP5/7 double knockout (DKO)] mice; however, we found that the combined deletion had no additive disruptive effect on fertility. Our studies indicate that BMP7 is an important factor during the process of implantation that contributes to healthy embryonic development.

Follicle-stimulating hormone (FSH)-induced growth of ovarian follicles is independent of follicular vascularization. Recent evidence has indicated that follicular vascularization is critical to ovarian follicle development and survival. FSH, a gonadotropin that induces follicular growth and development, also acts as the major survival factor for antral follicles. FSH has been reported to stimulate angiogenesis in the theca layers mediated in part by the vascular endothelial growth factor A (VEGFA) and the transcription factor hypoxia inducible factor 1α (HIF-1α). However, it remains largely undetermined whether FSH-dependent growth and survival of antral follicles relies on FSH-induced vascularization. Here, we first demonstrated that induction of angiogenesis through the FSH–HIF–1α-VEGFA axis is not required for FSH-stimulated follicular growth in mouse ovary. FSH increased the total number of blood vessels in mouse ovarian follicles, which was correlated with elevated expression of VEGFA and HIF-1α in granulosa cells. In contrast, blocking of follicular angiogenesis using inhibitors against the HIF-1α-VEGFA pathway repressed vasculature formation in follicles despite FSH administration. Interestingly, by measuring follicular size and ovarian weight, we found that the suppression of angiogenesis via HIF-1α–VEGFA pathway did not influence FSH-mediated follicular growth. However, inhibition of FSH-induced follicular vascularization by PX-478, a small-molecule inhibitor that suppresses HIF-1α activity, blocked ovulation and triggered atresia in large follicles. On the other hand, PX-478 injection reduced oocyte quality via impairing the meiotic apparatus, showing a prominently defective spindle assembly and actin dynamics. Collectively, our findings unveiled a vascularization-independent effect of FSH on follicular growth, whereas follicular survival, ovulation, and oocyte development relies on FSH-mediated angiogenesis in the follicles.

Exposure to bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical (EDC), is known to produce variable effects on female puberty and ovulation. This variability of effects is possibly due to differences in dose and period of exposure. Little is known about the effects of adult exposure to environmentally relevant doses of this EDC and the differences in effect after neonatal exposure. This study sought to compare the effects of neonatal vs adult exposure to a very low dose or a high dose of BPA for 2 weeks on ovulation and folliculogenesis and to explore the hypothalamic mechanisms involved in such disruption by BPA. One-day-old and 90-day-old female rats received daily subcutaneous injections of corn oil (vehicle) or BPA (25 ng/kg/d or 5 mg/kg/d) for 15 days. Neonatal exposure to both BPA doses significantly disrupted the estrous cycle and induced a decrease in primordial follicles. Effects on estrous cyclicity and folliculogenesis persisted into adulthood, consistent with a disruption of organizational mechanisms. During adult exposure, both doses caused a reversible decrease in antral follicles and corpora lutea. A reversible disruption of the estrous cycle associated with a delay and a decrease in the amplitude of the LH surge was also observed. Alterations of the hypothalamic expression of the clock gene Per1 and the reproductive peptide phoenixin indicated a disruption of the hypothalamic control of the preovulatory LH surge by BPA., info:eu-repo/semantics/published

WNT signaling regulates a variety of ovarian processes, including follicle development, granulosa cell (GC) proliferation and differentiation, steroidogenesis, and ovulation. The secreted frizzled-related proteins (SFRPs) comprise a family of WNT signaling antagonists. Sfrp4 expression was previously reported to be induced in ovarian GCs and cumulus cells in vivo following human chorionic gonadotropin treatment, suggesting that it may play key roles in cumulus expansion, ovulation/luteinization, and corpus luteum (CL) function. In this study, we aimed to define the physiological roles of Sfrp4 in the ovary by gene targeting. Sfrp4-null female mice were found to produce larger litters than did their wild-type littermates. Although previous studies had suggested roles of Sfrp4 in luteal cell survival, no differences in CL formation, morphology, steroidogenesis, involution, or luteal cell apoptosis were found in Sfrp4-null mice. Likewise, cumulus expansion occurred normally in Sfrp4-null mice, with minimal changes in cumulus cell gene expression. Hyperfertility in the Sfrp4-null model was ultimately attributed to decreased antral follicle atresia, leading to an enhanced ovulatory rate. Increased expression of FSH- and LH-responsive genes was found in GCs from Sfrp4-null mice, and GCs isolated from Sfrp4-null mice were found to be hyperresponsive to FSH and LH in vitro. Although Sfrp2 was found to be overexpressed in the GCs of Sfrp4-null mice (suggesting a compensatory mechanism), Sfrp2-null mice had normal fertility and ovulatory rates, and Sfrp2/4 double knockout mice did not differ from Sfrp4-null mice. Taken together, our results suggest that SFRP4 acts to attenuate GC responsiveness to gonadotropins, thereby decreasing follicle survival, ovulatory rate, and fertility.

Increased proteolytic activity is a key event that aids in breakdown of the follicular wall to permit oocyte release. How the protease activity is regulated is still unknown. We hypothesize that tissue factor pathway inhibitor 2 (TFPI2), a Kunitz-type serine protease inhibitor, plays a role in regulating periovulatory proteolytic activity as in other tissues. TFPI2 is secreted into the extracellular matrix (ECM) where it is postulated to regulate physiological ECM remodeling. The expression profile of TFPI2 during the periovulatory period was assessed utilizing a well-characterized human menstrual cycle model and a gonadotropin-primed rat model. Administration of an ovulatory dose of human chorionic gonadotropin (hCG) increased TFPI2 expression dramatically in human and rat granulosa and theca cells. This increase in Tfpi2 expression in rat granulosa cells required hCG-mediated epidermal growth factor, protein kinase A, mitogen-activated protein kinase (MAPK) 1/2, p38 MAPK and protease activated receptor 1-dependent cell signaling. A small interferingRNA-mediated knockdown of TFPI2 in rat granulosa cells resulted in increased plasmin activity in the granulosa cell conditioned media. Knockdown of TFPI2 also reduced expression of multiple genes including interleukin 6 (Il6) and amphiregulin (Areg). Overexpression of TFPI2 using an adenoviral vector partially restored the expression of Il6 and Areg in TFPI2 siRNA treated rat granulosa cells. These data support the hypothesis that TFPI2 is important for moderating plasmin activity and regulating granulosa cell gene expression during the periovulatory period. We, therefore, propose that through these actions, TFPI2 aids in the tissue remodeling taking place during follicular rupture and corpus luteum formation.

The hydroxysteroid (17beta) dehydrogenase (HSD17B)12 gene belongs to the hydroxysteroid (17β) dehydrogenase superfamily, and it has been implicated in the conversion of estrone to estradiol as well as in the synthesis of arachidonic acid (AA). AA is a precursor of prostaglandins, which are involved in the regulation of female reproduction, prompting us to study the role of HSD17B12 enzyme in the ovarian function. We found a broad expression of HSD17B12 enzyme in both human and mouse ovaries. The enzyme was localized in the theca interna, corpus luteum, granulosa cells, oocytes, and surface epithelium. Interestingly, haploinsufficiency of the HSD17B12 gene in female mice resulted in subfertility, indicating an important role for HSD17B12 enzyme in the ovarian function. In line with significantly increased length of the diestrous phase, the HSD17B+/- females gave birth less frequently than wild-type females, and the litter size of HSD17B12+/- females was significantly reduced. Interestingly, we observed meiotic spindle formation in immature follicles, suggesting defective meiotic arrest in HSD17B12+/- ovaries. The finding was further supported by transcriptome analysis showing differential expression of several genes related to the meiosis. In addition, polyovular follicles and oocytes trapped inside the corpus luteum were observed, indicating a failure in the oogenesis and ovulation, respectively. Intraovarian concentrations of steroid hormones were normal in HSD17B12+/- females, whereas the levels of AA and its metabolites (6-keto prostaglandin F1alpha, prostaglandin D2, prostaglandin E2, prostaglandin F2α, and thromboxane B2) were decreased. In conclusion, our study demonstrates that HSD17B12 enzyme plays an important role in female fertility through its role in AA metabolism.

The anteroventral periventricular nucleus (AVPV) orchestrates the neuroendocrine-positive feedback response that triggers ovulation in female rodents. The AVPV is larger and more cell-dense in females than in males, and during puberty, only females develop the capacity to show a positive feedback response. We previously reported a potential new mechanism to explain this female-specific gain of function during puberty, namely a female-biased sex difference in the pubertal addition of new cells to the rat AVPV. Here we first asked whether this sex difference is due to greater cell proliferation and/or survival in females. Female and male rats received the cell birthdate marker 5-bromo-2′-deoxyuridine (BrdU; 200 mg/kg, ip) on postnatal day (P) 30; brains were collected at short and long intervals after BrdU administration to assess cell proliferation and survival, respectively. Overall, females had more BrdU-immunoreactive cells in the AVPV than did males, with no sex differences in the rate of cell attrition over time. Thus, the sex difference in pubertal addition of AVPV cells appears to be due to greater cell proliferation in females. Next, to determine the phenotype of pubertally born AVPV cells, daily BrdU injections were given to female rats on P28–56, and tissue was collected on P77 to assess colocalization of BrdU and markers for mature neurons or glia. Of the pubertally born AVPV cells, approximately 15% differentiated into neurons, approximately 19% into astrocytes, and approximately 23% into microglia. Thus, both neuro- and gliogenesis occur in the pubertal female rat AVPV and potentially contribute to maturation of female reproductive function.

In primates, despite the fact that GnRH neurons are mature at birth, a gonadal steroid independent central inhibition restrains the initiation of puberty. The neural substrates responsible for this central inhibition, however, are unclear. In this study, we tested the hypothesis that neuroestradiol release in the hypothalamus decreases prior to the pubertal increase in GnRH release. We found that in female monkeys at the prepubertal stage, when GnRH release was low, estradiol (E2) levels in the stalk-median eminence of the hypothalamus were higher than those in older, early pubertal females in which nocturnal GnRH release begins to increase. Furthermore, estrone (E1) levels were higher in the stalk-median eminence of prepubertal and early pubertal monkeys compared with midpubertal monkeys, which have the highest GnRH release. The elevated E2 and E1 levels at the prepubertal stage are likely hypothalamic in origin because circulating E2 and E1 levels in prepubertal and early pubertal monkeys were much lower than those in midpubertal monkeys. Heightened synthesis and release of neuroestradiol during the prepubertal period and subsequent reduction at puberty onset indicate possible roles for neuroestradiol in central inhibition of GnRH release. The mechanism governing the reduction in neuroestradiol synthesis at puberty onset remains to be determined.

Optimal fertility in humans and animals relies on the availability of sufficient metabolic fuels, information about which is communicated to the brain via levels of the hormones leptin and insulin. The circadian clock system is also critical; this input is especially evident in the precise timing of the female-specific surge of GnRH and LH secretion that triggers ovulation the next day. Chronodisruption and metabolic imbalance can both impair reproductive activity, and these two disruptions exacerbate each other, such that they often occur simultaneously. Kisspeptin neurons located in the anteroventral periventricular nucleus of the hypothalamus are able to integrate both circadian and metabolic afferent inputs and use this information to modulate the timing and magnitude of the preovulatory GnRH/LH surge. In an environment in which exposure to high caloric diets and chronodisruptors such as artificial night lighting, shift work, and transmeridian travel have become the norm, the implications of these factors for couples struggling to conceive deserve closer attention and more public education.

By mediating estrogen synthesis and follicular growth in response to FSH, the ovarian FSH receptor (FSHR) is essential for female fertility. Indeed, ovarian stimulation via administration of FSH to women with infertility is part of the primary therapeutic intervention used in assisted reproductive technology. In physiological and therapeutic contexts, current dogma dictates that once ovulation has occurred, FSH/FSHR signaling is no longer required for successful pregnancy outcomes. However, a continued role for FSH during pregnancy is suggested by recent studies demonstrating extraovarian FSHR in the female reproductive tract. Furthermore, functional roles for FSHR in placenta and in uterine myometrium have now been demonstrated. In placenta, vascular endothelial FSHR of fetal vessels within the chorionic villi (human) or labyrinth (mouse) mediate angiogenesis, and it has further been shown that deletion of placental Fshr in mice has deleterious effects on pregnancy. In uterine myometrium, changes in the densities of FSHR in muscle fiber and stroma in the nonpregnant state, early pregnancy, and term pregnancy differentially regulate contractile activity, suggesting that signaling through myometrial FSHR may contribute to the quieting of contractile activity required for successful implantation and that the temporal upregulation of the FSHR at term pregnancy may be required for the appropriate timing of parturition. In addition, extraovarian expression of mRNAs encoding the glycoprotein hormone α subunit and the FSH β subunit has been demonstrated, suggesting that these novel aspects of extraovarian FSH/FSHR signaling during pregnancy may be mediated by locally synthesized FSH.

Xenoestrogens from synthetic or natural origin represent an increasing risk of disrupted endocrine functions including the physiological activity of the hypothalamo-pituitary-gonad axis. Ethinyl estradiol (EE2) is a synthetic estrogen used in contraceptive pills, whereas zearalenone (ZEA) is a natural mycoestrogen found with increasing prevalence in various cereal crops. Both EE2 and ZEA are agonists of estrogen receptor-α and accelerate puberty. However, the neuroendocrine mechanisms that are responsible for this effect remain unknown. Immature female Wistar rats were treated with EE2 (10 μg/kg), ZEA (10 mg/kg), or vehicle for 10 days starting from postnatal day 18. As a marker of puberty, the vaginal opening was recorded and neuropeptide and related transcription factor mRNA levels were measured by quantitative real time PCR and in situ hybridization histochemistry. Both ZEA and EE2 accelerated the vaginal opening, increased the uterine weight and the number of antral follicles in the ovary, and resulted in the increased central expression of gnrh. These changes occurred in parallel with an earlier increase of kiss1 mRNA in the anteroventral and rostral periventricular hypothalamus and an increased kisspeptin (KP) fiber density and KP-GnRH appositions in the preoptic area. These changes are compatible with a mechanism in which xenoestrogens overstimulate the developmentally unprepared reproductive system, which results in an advanced vaginal opening and an enlargement of the uterus at the periphery. Within the hypothalamus, ZEA and EE2 directly activate anteroventral and periventricular KP neurons to stimulate GnRH mRNA. However, GnRH and gonadotropin release and ovulation are disrupted due to xenoestrogen-mediated inhibitory KP signaling in the arcuate nucleus.

Ovulation is one of the cornerstones of female fertility. Disruption of the ovulatory process results in infertility, which affects approximately 10% of couples. Using a unique model in which the dominant follicle is collected across the periovulatory period in women, we have identified a leukocyte chemoattractant, chemokine ligand 20 (CCL20), in the human ovary. CCL20 mRNA is massively induced after an in vivo human chorionic gonadotropin (hCG) stimulus in granulosa (>10 000-fold) and theca (>4000-fold) cells collected during the early ovulatory (12–18 h) and late ovulatory (18–34 h) periods after hCG administration. Because the LH surge sets in motion an inflammatory reaction characterized by an influx of leukocytes and CCL20 is known to recruit leukocytes in other systems, the composition of ovarian leukocytes (CD45+) containing the CCL20 receptor CCR6 was determined immediately prior to ovulation. CD45+/CCR6+ cells were primarily natural killer cells (41%) along with B cells (12%), T cells (11%), neutrophils (10%), and monocytes (9%). Importantly, exogenous CCL20 stimulated ovarian leukocyte migration 59% within 90 minutes. Due to the difficulties in obtaining human follicles, an in vitro model was developed using granulosa-lutein cells to explore CCL20 regulation. CCL20 expression increased 40-fold within 6 hours after hCG, was regulated partially by the epithelial growth factor pathway, and was positively correlated with progesterone production. These results demonstrate that hCG dramatically increases CCL20 expression in the human ovary, that ovarian leukocytes contain the CCL20 receptor, and that CCL20 stimulates leukocyte migration. Our findings raise the prospect that CCL20 may aid in the final ovulatory events and contribute to fertility in women.

Throughout most of the ovulatory cycle, estrogen negative feedback restrains the GnRH neuronal system. Just before ovulation, however, estrogen negative feedback is removed to permit stimulation of the preovulatory GnRH/LH surge (positive feedback) by the circadian clock in the suprachiasmatic nucleus (SCN). The mammalian ortholog of avian gonadotropin-inhibitory hormone, RFamide-related peptide 3 (RFRP-3), participates in the circadian-timed removal of estrogen negative feedback to permit the LH surge. The present study examined the specific neurochemical means by which the SCN controls RFRP-3 activity and explored whether the RFRP-3 system exhibits time-dependent responsiveness to SCN signaling to precisely time the LH surge. We found that RFRP-3 cells in female Syrian hamsters (Mesocricetus auratus) receive close appositions from SCN-derived vasopressin-ergic and vasoactive intestinal peptide (VIP)-ergic terminal fibers. Central VIP administration markedly suppressed RFRP-3 cellular activity in the evening, but not the morning, relative to saline controls, whereas vasopressin was without effect at either time point. Double-label in situ hybridization for Rfrp-3 and the VIP receptors VPAC1 and VPAC2 revealed that the majority of RFRP-3 cells do not coexpress either receptor in Syrian hamsters or mice, suggesting that SCN VIP-ergic signaling inhibits RFRP-3 cells indirectly. The timing of this VIP-mediated disinhibition is further coordinated via temporally gated responsiveness of RFRP-3 cells to circadian signaling. Together, these findings reveal a novel circadian hierarchy of control coordinating the preovulatory LH surge and ovulation.

Polycystic ovary syndrome (PCOS) is a frequent pathology that affects more than 5% of women of reproductive age. Among other heterogeneous symptoms, PCOS is characterized by abnormalities in angiogenesis. Metformin has been introduced in the treatment of PCOS to manage insulin resistance and hyperglycemia. Besides its metabolic effects, metformin has been shown to improve ovulation, pregnancy and live birth rates in PCOS patients. In the present study, we used a dehydroepiandrosterone-induced PCOS rat model to analyze the effect of metformin administration on ovarian angiogenesis. We found that metformin was able to restore the increased levels of vascular endothelial growth factor, angiopoietin (ANGPT)1, and ANGPT1/ANGPT2 ratio and the decreased levels of platelet-derived growth factor B and platelet-derived growth factor D observed in the dehydroepiandrosterone-treated rats. These effects could take place, at least in part, through a decrease in the levels of serum insulin. We also found an improvement in follicular development, with a lower percentage of small follicles and cysts and a higher percentage of antral follicles and corpora lutea after metformin administration. The improvement in ovarian angiogenesis is likely to restore the accumulation of small follicles observed in PCOS rats and to reduce cyst formation, thus improving follicular development and the percentage of corpora lutea. These results open new insights into the study of metformin action not only in glucose metabolism but also in ovarian dysfunction in PCOS women Fil: Di Pietro, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Parborell, Maria Fernanda Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Irusta, Griselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Pascuali, Natalia Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Bas, Diana Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Bianchi, Maria Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Tesone, Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Abramovich, Dalhia Nurit. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina

The hypothalamic-based kisspeptin-signaling system is a major positive regulator of the neuroendocrine-reproductive axis in mammals. During the last decade, major advances have been made in understanding how this signaling system is regulated and how it can be manipulated clinically to achieve beneficial outcomes in treating sex steroid-dependent disorders. Interestingly, kisspeptin was not first identified as a regulator of fertility. Instead, approximately 7 years earlier KISS1 was reported to be expressed in nonmetastatic melanoma cells and was subsequently demonstrated to act as a powerful suppressor of the metastatic potential of malignant melanoma cells. Since this discovery, numerous studies have demonstrated the expression of the kisspeptin-signaling system at several peripheral sites implicating it in biological processes such as the regulation of ovarian function, embryo implantation, placentation, angiogenesis, insulin secretion, and kidney development. Although much work remains to be done to assess how important kisspeptin signaling is in regulating some of these processes, for other processes recent studies have made tremendous strides toward such an assessment. Using mice lacking either Kiss1 or Kiss1r alleles, researchers have provided compelling evidence for kisspeptin playing a major role in regulating breast cancer metastasis, oocyte survival, follicular maturation, ovulation, and embryo implantation. This review critically discusses the findings from these as well as other studies which suggest roles for kisspeptin in regulating important physiological processes beyond the brain. It also discusses the challenges that lie ahead in determining whether findings made with animal models are relevant in humans.