Transcriptional Regulatory Role of NELL2 in Preproenkephalin Gene Expression.

Preproenkephalin (PPE) is a precursor molecule for multiple endogenous opioid peptides Leu-enkephalin (ENK) and Met-ENK, which are involved in a wide variety of modulatory functions in the nervous system. Despite the functional importance of ENK in the brain, the effect of brain-derived factor(s) on PPE expression is unknown. We report the dual effect of neural epidermal growth factor (EGF)-likelike 2 (NELL2) on PPE gene expression. In cultured NIH3T3 cells, transfection of NELL2 expression vectors induced an inhibition of PPE transcription intracellularly, in parallel with downregulation of protein kinase C signaling pathways and extracellular signal-regulated kinase. Interestingly, these phenomena were reversed when synthetic NELL2 was administered extracellularly. The in vivo disruption of NELL2 synthesis resulted in an increase in PPE mRNA level in the rat brain, suggesting that the inhibitory action of intracellular NELL2 predominates the activation effect of extracellular NELL2 on PPE gene expression in the brain. Biochemical and molecular studies with mutant NELL2 structures further demonstrated the critical role of EGF-like repeat domains in NELL2 for regulation of PPE transcription. These are the first results to reveal the spatio-specific role of NELL2 in the homeostatic regulation of PPE gene expression.

GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation.

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation.

Polycomb represses a gene network controlling puberty via modulation of histone demethylase Kdm6b expression.

Female puberty is subject to Polycomb Group (PcG)-dependent transcriptional repression. Kiss1, a puberty-activating gene, is a key target of this silencing mechanism. Using a gain-of-function approach and a systems biology strategy we now show that EED, an essential PcG component, acts in the arcuate nucleus of the hypothalamus to alter the functional organization of a gene network involved in the stimulatory control of puberty. A central node of this network is Kdm6b, which encodes an enzyme that erases the PcG-dependent histone modification H3K27me3. Kiss1 is a first neighbor in the network; genes encoding glutamatergic receptors and potassium channels are second neighbors. By repressing Kdm6b expression, EED increases H3K27me3 abundance at these gene promoters, reducing gene expression throughout a gene network controlling puberty activation. These results indicate that Kdm6b repression is a basic mechanism used by PcG to modulate the biological output of puberty-activating gene networks.

Adeno-associated virus-binding antibodies detected in cats living in the Northeastern United States lack neutralizing activity.

Cats are a critical pre-clinical model for studying adeno-associated virus (AAV) vector-mediated gene therapies. A recent study has described the high prevalence of anti-AAV neutralizing antibodies among domestic cats in Switzerland. However, our knowledge of pre-existing humoral immunity against various AAV serotypes in cats is still limited. Here, we show that, although antibodies binding known AAV serotypes (AAV1 to AAV11) are prevalent in cats living in the Northeastern United States, these antibodies do not necessarily neutralize AAV infectivity. We analyzed sera from 35 client-owned, 20 feral, and 30 specific pathogen-free (SPF) cats for pre-existing AAV-binding antibodies against the 11 serotypes. Antibody prevalence was 7 to 90% with an overall median of 50%. The AAV-binding antibodies showed broad reactivities with other serotypes. Of 44 selected antibodies binding AAV2, AAV6 or AAV9, none exhibited appreciable neutralizing activities. Instead, AAV6 or AAV9-binding antibodies showed a transduction-enhancing effect. AAV6-binding antibodies were highly prevalent in SPF cats (83%), but this was primarily due to cross-reactivity with preventive vaccine-induced anti-feline panleukopenia virus antibodies. These results indicate that prevalent pre-existing immunity in cats is not necessarily inhibitory to AAV and highlight a substantial difference in the nature of AAV-binding antibodies in cats living in geographically different regions.

EAP1 regulation of GnRH promoter activity is important for human pubertal timing.

The initiation of puberty is orchestrated by an augmentation of gonadotropin-releasing hormone (GnRH) secretion from a few thousand hypothalamic neurons. Recent findings have indicated that the neuroendocrine control of puberty may be regulated by a hierarchically organized network of transcriptional factors acting upstream of GnRH. These include enhanced at puberty 1 (EAP1), which contributes to the initiation of female puberty through transactivation of the GnRH promoter. However, no EAP1 mutations have been found in humans with disorders of pubertal timing. We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited delayed puberty (DP). Variants were analyzed for rare, potentially pathogenic variants enriched in case versus controls and relevant to the biological control of puberty. We identified one in-frame deletion (Ala221del) and one rare missense variant (Asn770His) in EAP1 in two unrelated families; these variants were highly conserved and potentially pathogenic. Expression studies revealed Eap1 mRNA abundance in peri-pubertal mouse hypothalamus. EAP1 binding to the GnRH1 promoter increased in monkey hypothalamus at the onset of puberty as determined by chromatin immunoprecipitation. Using a luciferase reporter assay, EAP1 mutants showed a reduced ability to trans-activate the GnRH promoter compared to wild-type EAP1, due to reduced protein levels caused by the Ala221del mutation and subcellular mislocation caused by the Asn770His mutation, as revealed by western blot and immunofluorescence, respectively. In conclusion, we have identified the first EAP1 mutations leading to reduced GnRH transcriptional activity resulting in a phenotype of self-limited DP.

Methylome profiling of healthy and central precocious puberty girls.

BACKGROUND: Recent studies demonstrated that changes in DNA methylation (DNAm) and inactivation of two imprinted genes (MKRN3 and DLK1) alter the onset of female puberty. We aimed to investigate the association of DNAm profiling with the timing of human puberty analyzing the genome-wide DNAm patterns of peripheral blood leukocytes from ten female patients with central precocious puberty (CPP) and 33 healthy girls (15 pre- and 18 post-pubertal). For this purpose, we performed comparisons between the groups: pre- versus post-pubertal, CPP versus pre-pubertal, and CPP versus post-pubertal. RESULTS: Analyzing the methylome changes associated with normal puberty, we identified 120 differentially methylated regions (DMRs) when comparing pre- and post-pubertal healthy girls. Most of these DMRs were hypermethylated in the pubertal group (99%) and located on the X chromosome (74%). Only one genomic region, containing the promoter of ZFP57, was hypomethylated in the pubertal group. ZFP57 is a transcriptional repressor required for both methylation and imprinting of multiple genomic loci. ZFP57 expression in the hypothalamus of female rhesus monkeys increased during peripubertal development, suggesting enhanced repression of downstream ZFP57 target genes. Fourteen other zinc finger (ZNF) genes were related to the hypermethylated DMRs at normal puberty. Analyzing the methylome changes associated with CPP, we demonstrated that the patients with CPP exhibited more hypermethylated CpG sites compared to both pre-pubertal (81%) and pubertal (89%) controls. Forty-eight ZNF genes were identified as having hypermethylated CpG sites in CPP. CONCLUSION: Methylome profiling of girls at normal and precocious puberty revealed a widespread pattern of DNA hypermethylation, indicating that the pubertal process in humans is associated with specific changes in epigenetically driven regulatory control. Moreover, changes in methylation of several ZNF genes appear to be a distinct epigenetic modification underlying the initiation of human puberty.

Correcting deregulated Fxyd1 expression rescues deficits in neuronal arborization and potassium homeostasis in MeCP2 deficient male mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the MECP2 gene. In the absence of MeCP2, expression of FXYD domain-containing transport regulator 1 (FXYD1) is deregulated in the frontal cortex (FC) of mice and humans. Because Fxyd1 is a membrane protein that controls cell excitability by modulating Na+, K+-ATPase activity (NKA), an excess of Fxyd1 may reduce NKA activity and contribute to the neuronal phenotype of Mecp2 deficient (KO) mice. To determine if Fxyd1 can rescue these RTT deficits, we studied the male progeny of Fxyd1 null males bred to heterozygous Mecp2 female mice. Maximal NKA enzymatic activity was not altered by the loss of MeCP2, but it increased in mice lacking one Fxyd1 allele, suggesting that NKA activity is under Fxyd1 inhibitory control. Deletion of one Fxyd1 allele also prevented the increased extracellular potassium (K+) accumulation observed in cerebro-cortical neurons from Mecp2 KO animals in response to the NKA inhibitor ouabain, and rescued the loss of dendritic arborization observed in FC neurons of Mecp2 KO mice. These effects were gene-dose dependent, because the absence of Fxyd1 failed to rescue the MeCP2-dependent deficits, and mimicked the effect of MeCP2 deficiency in wild-type animals. These results indicate that excess of Fxyd1 in the absence of MeCP2 results in deregulation of endogenous K+ conductances functionally associated with NKA and leads to stunted neuronal growth.

Elucidating the genetic architecture of reproductive ageing in the Japanese population.

Population studies elucidating the genetic architecture of reproductive ageing have been largely limited to European ancestries, restricting the generalizability of the findings and overlooking possible key genes poorly captured by common European genetic variation. Here, we report 26 loci (all P < 5 × 10-8) for reproductive ageing, i.e. puberty timing or age at menopause, in a non-European population (up to 67,029 women of Japanese ancestry). Highlighted genes for menopause include GNRH1, which supports a primary, rather than passive, role for hypothalamic-pituitary GnRH signalling in the timing of menopause. For puberty timing, we demonstrate an aetiological role for receptor-like protein tyrosine phosphatases by combining evidence across population genetics and pre- and peri-pubertal changes in hypothalamic gene expression in rodent and primate models. Furthermore, our findings demonstrate widespread differences in allele frequencies and effect estimates between Japanese and European associated variants, highlighting the benefits and challenges of large-scale trans-ethnic approaches.

Trithorax dependent changes in chromatin landscape at enhancer and promoter regions drive female puberty.

Polycomb group (PcG) proteins control the timing of puberty by repressing the Kiss1 gene in hypothalamic arcuate nucleus (ARC) neurons. Here we identify two members of the Trithorax group (TrxG) of modifiers, mixed-lineage leukemia 1 (MLL1), and 3 (MLL3), as central components of an activating epigenetic machinery that dynamically counteracts PcG repression. Preceding puberty, MLL1 changes the chromatin configuration at the promoters of Kiss1 and Tac3, two genes required for puberty to occur, from repressive to permissive. Concomitantly, MLL3 institutes a chromatin structure that changes the functional status of a Kiss1 enhancer from poised to active. RNAi-mediated, ARC-specific Mll1 knockdown reduced Kiss1 and Tac3 expression, whereas CRISPR-Cas9-directed epigenome silencing of the Kiss1 enhancer selectively reduced Kiss1 activity. Both interventions delay puberty and disrupt reproductive cyclicity. Our results demonstrate that an epigenetic switch from transcriptional repression to activation is crucial to the regulatory mechanism controlling the timing of mammalian puberty.

Seminal Plasma Induces Ovulation in Llamas in the Absence of a Copulatory Stimulus: Role of Nerve Growth Factor as an Ovulation-Inducing Factor.

Llamas are considered to be reflex ovulators. However, semen from these animals is reported to be rich in ovulation-inducing factor(s), one of which has been identified as nerve growth factor (NGF). These findings suggest that ovulation in llamas may be elicited by chemical signals contained in semen instead of being mediated by neural signals. The present study examines this notion. Llamas displaying a preovulatory follicle were assigned to four groups: group 1 received an intrauterine infusion (IUI) of PBS; group 2 received an IUI of seminal plasma; group 3 was mated to a male whose urethra had been surgically diverted (urethrostomized male); and group 4 was mated to an intact male. Ovulation (detected by ultrasonography) occurred only in llamas mated to an intact male or given an IUI of seminal plasma and was preceded by a surge in plasma LH levels initiated within an hour after coitus or IUI. In both ovulatory groups, circulating ß-NGF levels increased within 15 minutes after treatment, reaching values that were greater and more sustained in llamas mated with an intact male. These results demonstrate that llamas can be induced to ovulate by seminal plasma in the absence of copulation and that copulation alone cannot elicit ovulation in the absence of seminal plasma. In addition, our results implicate ß-NGF as an important mediator of seminal plasma-induced ovulation in llamas because ovulation does not occur if ß-NGF levels do not increase in the bloodstream, a change that occurs promptly after copulation with an intact male or IUI of seminal plasma.

The Emerging Role of Epigenetics in the Regulation of Female Puberty.

In recent years the pace of discovering the molecular and genetic underpinnings of the pubertal process has accelerated considerably. Genes required for human puberty to occur have been identified and evidence has been provided suggesting that the initiation of puberty requires coordinated changes in the output of a multiplicity of genes organized into functional networks. Recent evidence suggests that a dual mechanism of epigenetic regulation affecting the transcriptional activity of neurons involved in stimulating gonadotropin-releasing hormone release plays a fundamental role in the timing of puberty. The Polycomb group (PcG) of transcriptional silencers appears to be a major component of the repressive arm of this mechanism. PcG proteins prevent the premature initiation of female puberty by silencing the Kiss1 gene in kisspeptin neurons of the arcuate nucleus (ARC) of the hypothalamus. Because the abundance of histone marks either catalyzed by--or associated with--the Trithorax group (TrxG) of transcriptional activators increases at the time when PcG control subsides, it appears that the TrxG complex is the counteracting partner of PcG-mediated gene silencing. In this chapter, we discuss the concept that a switch from epigenetic repression to activation within ARC kisspeptin neurons is a core mechanism underlying the initiation of female puberty.

Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression.

In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty.

FBXW12, a novel F box protein-encoding gene, is deleted or methylated in some cases of epithelial ovarian cancer.

Epithelial ovarian cancer is one of the most lethal of gynecological malignancies. Due to its lack of early symptoms, detection usually occurs when the tumor is no longer confined to the ovary. We previously identified Fbxw15, a gene encoding an F-box protein in the mouse ovary, and showed that its expression is developmentally regulated. Here we report the molecular analysis of its human homologue, FBXW12 in epithelial ovarian tumors. To search for FBXW12 gene mutations, we PCR-amplified and sequenced the coding region of FBXW12, the gene's 5-untranslated region and the proximal promoter in each of 30 EOC tumors. Promoter methylation was determined by DNA bisulfite conversion, followed by methylation specific PCR. FBXW12 intracellular localization was identified by means of immunohistochemistry. A complete deletion of the gene's coding region, the 5'-UTR and the proximal promoter, was observed in 3 EOC samples. Eight of the remaining 27, had a deletion of the 5'-UTR, and the proximal promoter. FBXW12 mRNA was detected in 2 of the 19 samples without deletions. The methylation specific PCR results demonstrated CpGs methylation in the FBXW12 proximal promoter. Immunohistochemistry assay revealed that within the normal ovary, FBXW12 has an oocyte specific expression, whereas in EOC samples it is present in the ovarian surface epithelium. Our results indicate that the FBXW12 gene is deleted in approximately ten percent of the EOC cases studied; such deletions comprised either the FBXW12 promoter or the mRNA-encoding region. Moreover, FBXW12 could be epigenetically silenced by CpGs methylation in some of these EOC cases.

Pro-nerve growth factor in the ovary and human granulosa cells.

BACKGROUND: Pro-nerve growth factor must be cleaved to generate mature NGF, which was suggested to be a factor involved in ovarian physiology and pathology. Extracellular proNGF can induce cell death in many tissues. Whether extracellular proNGF exists in the ovary and may play a role in the death of follicular cells or atresia was unknown. MATERIALS AND METHODS: Immunohistochemistry of human and rhesus monkey ovarian sections was performed. IVF-derived follicular fluid and human granulosa cells were studied by RT-PCR, qPCR, Western blotting, ATP- and caspase-assays. RESULTS AND CONCLUSION: Immunohistochemistry of ovarian sections identified proNGF in granulosa cells and Western blotting of human isolated granulosa cells confirmed the presence of proNGF. Ovarian granulosa cells thus produce proNGF. Recombinant human proNGF even at high concentrations did not affect the levels of ATP or the activity of caspase 3/7, indicating that in granulosa cells proNGF does not induce death. In contrast, mature NGF, which was detected previously in follicular fluid, may be a trophic molecule for granulosa cells with unexpected functions. We found that in contrast to proNGF, NGF increased the levels of the transcription factor early growth response 1 and of the enzyme choline acetyl-transferase. A mechanism for the generation of mature NGF from proNGF in the follicular fluid may be extracellular enzymatic cleavage. The enzyme MMP7 is known to cleave proNGF and was identified in follicular fluid and as a product of granulosa cells. Thus the generation of NGF in the ovarian follicle may depend on MMP7.

Expression of the beta-2 adrenergic receptor (ADRB-2) in human and monkey ovarian follicles: a marker of growing follicles?

BACKGROUND: ADRB-2 was implicated in rodent ovarian functions, including initial follicular growth. In contrast, ADRB-2 expression and function in nonhuman primate and human ovary were not fully known but innervation and significant levels of norepinephrine (NE), which is a ligand at the ADRB-2, were reported in the ovary. METHODS: We studied expression of ADRB-2 in human and rhesus monkey ovary (RT-PCR, immunohistochemistry; laser micro dissection) and measured levels of norepinephrine (NE; ELISA) in monkey follicular fluid (FF). 3D cultures of monkey follicles (4 animals) were exposed to NE or the ADRB-2 agonist isoproterenol (ISO), and follicular development (size) was monitored. Upon termination expression of ADRB-2, FSH receptor and aromatase genes were examined. RESULTS: Immunohistochemistry and RT-PCR of either human follicular granulosa cells (GCs) obtained by laser micro dissection or isolated monkey follicles revealed ADRB-2 in GCs of primordial, primary, secondary and tertiary follicles. Staining of GCs in primordial and primary follicles was intense. In large preantral and antral follicles the staining was heterogeneous, with positive and negative GCs present but GCs lining the antrum of large follicles were generally strongly immunopositive. Theca, interstitial, and ovarian surface epithelial cells were also positive. NE was detected in FF of preovulatory antral monkey follicles (0.37 + 0.05 ng/ml; n = 7; ELISA) but not in serum. We examined preantral follicles ranging from 152 to 366 µm in diameter in a 3D culture in media supplemented with follicle stimulating hormone (FSH). Under these conditions, neither NE, nor ISO, influenced growth rate in a period lasting up to one month. Upon termination of the cultures, all surviving follicles expressed aromatase and FSH receptors, but only about half of them also co-expressed ADRB-2. The ADRB-2 expression was not correlated with the treatment but was positively correlated with the follicular size at the beginning and at the end of the culture period. Hence, expression of ADRB-2 was found in the largest and fastest-in vitro growing follicles. CONCLUSIONS: The results imply ADRB-2-mediated actions in the development of primate follicles. Drugs interfering with ADRB-2 are used to treat medical conditions and may have unexplored effects in the human ovary.

Epigenetic regulation of female puberty.

Substantial progress has been made in recent years toward deciphering the molecular and genetic underpinnings of the pubertal process. The availability of powerful new methods to interrogate the human genome has led to the identification of genes that are essential for puberty to occur. Evidence has also emerged suggesting that the initiation of puberty requires the coordinated activity of gene sets organized into functional networks. At a cellular level, it is currently thought that loss of transsynaptic inhibition, accompanied by an increase in excitatory inputs, results in the pubertal activation of GnRH release. This concept notwithstanding, a mechanism of epigenetic repression targeting genes required for the pubertal activation of GnRH neurons was recently identified as a core component of the molecular machinery underlying the central restraint of puberty. In this chapter we will discuss the potential contribution of various mechanisms of epigenetic regulation to the hypothalamic control of female puberty.

Excess of nerve growth factor in the ovary causes a polycystic ovary-like syndrome in mice, which closely resembles both reproductive and metabolic aspects of the human syndrome.

Polycystic ovarian syndrome (PCOS), the most common female endocrine disorder of unknown etiology, is characterized by reproductive abnormalities and associated metabolic conditions comprising insulin resistance, type 2 diabetes mellitus, and dyslipidemia. We previously reported that transgenic overexpression of nerve growth factor (NGF), a marker of sympathetic hyperactivity, directed to the ovary by the mouse 17α-hydroxylase/C17-20 lyase promoter (17NF mice), results in ovarian abnormalities similar to those seen in PCOS women. To investigate whether ovarian overproduction of NGF also induces common metabolic alterations of PCOS, we assessed glucose homeostasis by glucose tolerance test, plasma insulin levels, and body composition by dual-energy x-ray absorptiometry scan in young female 17NF mice and wild-type mice. 17NF mice exhibited increased body weight and alterations in body fat distribution with a greater accumulation of visceral fat compared with sc fat (P < .01). 17NF mice also displayed glucose intolerance (P < .01), decreased insulin-mediated glucose disposal (P < .01), and hyperinsulinemia (P < .05), which, similar to PCOS patients, occurred independently of body weight. Additionally, 17NF mice exhibited increased sympathetic outflow observed as increased interscapular brown adipose tissue temperature. This change was evident during the dark period (7 pm to 7 am) and occurred concomitant with increased interscapular brown adipose tissue uncoupling protein 1 expression. These findings suggest that overexpression of NGF in the ovary may suffice to cause both reproductive and metabolic alterations characteristic of PCOS and support the hypothesis that sympathetic hyperactivity may contribute to the development and/or progression of PCOS.

Loss-of-function mutations in PNPLA6 encoding neuropathy target esterase underlie pubertal failure and neurological deficits in Gordon Holmes syndrome.

CONTEXT: Gordon Holmes syndrome (GHS) is characterized by cerebellar ataxia/atrophy and normosmic hypogonadotropic hypogonadism (nHH). The underlying pathophysiology of this combined neurodegeneration and nHH remains unknown. OBJECTIVE: We aimed to provide insight into the disease mechanism in GHS. METHODS: We studied a cohort of 6 multiplex families with GHS through autozygosity mapping and whole-exome sequencing. RESULTS: We identified 6 patients from 3 independent families carrying loss-of-function mutations in PNPLA6, which encodes neuropathy target esterase (NTE), a lysophospholipase that maintains intracellular phospholipid homeostasis by converting lysophosphatidylcholine to glycerophosphocholine. Wild-type PNPLA6, but not PNPLA6 bearing these mutations, rescued a well-established Drosophila neurodegenerative phenotype caused by the absence of sws, the fly ortholog of mammalian PNPLA6. Inhibition of NTE activity in the LßT2 gonadotrope cell line diminished LH response to GnRH by reducing GnRH-stimulated LH exocytosis, without affecting GnRH receptor signaling or LHß synthesis. CONCLUSION: These results suggest that NTE-dependent alteration of phospholipid homeostasis in GHS causes both neurodegeneration and impaired LH release from pituitary gonadotropes, leading to nHH.

Loss of Ntrk2/Kiss1r signaling in oocytes causes premature ovarian failure.

Neurotrophins (NTs), once believed to be neural-specific trophic factors, are now known to also provide developmental cues to non-neural cells. In the ovary, NTs contribute to both the formation and development of follicles. Here we show that oocyte-specific deletion of the Ntrk2 gene that encodes the NTRK2 receptor (NTRK2) for neurotrophin-4/5 and brain-derived neurotrophic factor (BDNF) results in post-pubertal oocyte death, loss of follicular organization, and early adulthood infertility. Oocytes lacking NTRK2 do not respond to gonadotropins with activation of phosphatidylinositol 3-kinase (PI3K)-AKT-mediated signaling. Before puberty, oocytes only express a truncated NTRK2 form (NTRK2.T1), but at puberty full-length (NTRK2.FL) receptors are rapidly induced by the preovulatory gonadotropin surge. A cell line expressing both NTRK2.T1 and the kisspeptin receptor (KISS1R) responds to BDNF stimulation with activation of Ntrk2 expression only if kisspeptin is present. This suggests that BDNF and kisspeptin that are produced by granulosa cells (GCs) of periovulatory follicles act in concert to mediate the effect of gonadotropins on Ntrk2 expression in oocytes. In keeping with this finding, the oocytes of NTRK2-intact mice fail to respond to gonadotropins with increased Ntrk2 expression in the absence of KISS1R. Our results demonstrate that the preovulatory gonadotropin surge promotes oocyte survival at the onset of reproductive cyclicity by inducing oocyte expression of NTRK2.FL receptors that set in motion an AKT-mediated survival pathway. They also suggest that gonadotropins activate NTRK2.FL expression via a dual communication pathway involving BDNF and kisspeptin produced in GCs and their respective receptors NTRK2.T1 and KISS1R expressed in oocytes.

Kisspeptin receptor haplo-insufficiency causes premature ovarian failure despite preserved gonadotropin secretion.

Premature ovarian failure (POF) affects 1% of women in reproductive age, but its etiology remains uncertain. Whereas kisspeptins, the products of Kiss1 that act via Kiss1r (aka, Gpr54), are known to operate at the hypothalamus to control GnRH/gonadotropin secretion, additional actions at other reproductive organs, including the ovary, have been proposed. Yet, their physiological relevance is still unclear. We present here a series of studies in Kiss1r haplo-insufficient and null mice suggesting a direct role of kisspeptin signaling in the ovary, the defect of which precipitates a state of primary POF. Kiss1r hypomorph mice displayed a premature decline in ovulatory rate, followed by progressive loss of antral follicles, oocyte loss, and a reduction in all categories of preantral follicles. These alterations were accompanied by reduced fertility. Because of this precocious ovarian ageing, mice more than 48 weeks of age showed atrophic ovaries, lacking growing follicles and corpora lutea. This phenomenon was associated with a drop in ovarian Kiss1r mRNA expression, but took place in the absence of a decrease in circulating gonadotropins. In fact, FSH levels increased in aged hypomorph animals, reflecting loss of follicular function. In turn, Kiss1r-null mice, which do not spontaneously ovulate and have arrested follicular development, failed to show normal ovulatory responses to standard gonadotropin priming and required GnRH prestimulation during 1 week in order to display gonadotropin-induced ovulation. Yet, the magnitude of such ovulatory responses was approximately half of that seen in control immature wild-type animals. Altogether, our data are the first to demonstrate that Kiss1r haplo-insufficiency induces a state of POF, which is not attributable to defective gonadotropin secretion. We also show that the failure of follicular development and ovulation linked to the absence of Kiss1r cannot be fully rescued by (even extended) gonadotropin replacement. These findings suggest a direct ovarian role of kisspeptin signaling, the perturbation of which may contribute to the pathogenesis of POF.