BPA disrupts meiosis I in oogonia by acting on pathways including cell cycle regulation, meiosis initiation and spindle assembly.

The negative in utero effects of bisphenol A (BPA) on female reproduction are of concern since the ovarian reserve of primordial follicles is constituted during the fetal period. This time-window is difficult to access, particularly in humans. Animal models and explant culture systems are, therefore, vital tools for investigating EDC impacts on primordial germ cells (PGCs). Here, we investigated the effects of BPA on prophase I meiosis in the fetal sheep ovary. We established an in vitro model of early gametogenesis through retinoic acid (RA)-induced differentiation of sheep PGCs that progressed through meiosis. Using this system, we demonstrated that BPA (3 ×10-7 M & 3 ×10-5 M) exposure for 20 days disrupted meiotic initiation and completion in sheep oogonia and induced transcriptomic modifications of exposed explants. After exposure to the lowest concentrations of BPA (3 ×10-7 M), only 2 probes were significantly up-regulated corresponding to NR2F1 and TMEM167A transcripts. In contrast, after exposure to 3 × 10-5 M BPA, 446 probes were deregulated, 225 were down- and 221 were up-regulated following microarray analysis. Gene Ontology (GO) annotations of differentially expressed genes revealed that pathways mainly affected were involved in cell-cycle phase transition, meiosis and spindle assembly. Differences in key gene expression within each pathway were validated by qRT-PCR. This study provides a novel model for direct examination of the molecular pathways of environmental toxicants on early female gametogenesis and novel insights into the mechanisms by which BPA affects meiosis I. BPA exposure could thereby disrupt ovarian reserve formation by inhibiting meiotic progression of oocytes I and consequently by increasing atresia of primordial follicles containing defective oocytes.

Ovine fetal testis stage-specific sensitivity to environmental chemical mixtures.

Exposure of the fetal testis to numerous individual environmental chemicals (ECs) is frequently associated with dysregulated development, leading to impaired adult reproductive competence. However, 'real-life' exposure involves complex mixtures of ECs. Here we test the consequences, for the male fetus, of exposing pregnant ewes to EC mixtures derived from pastures treated with biosolids fertiliser (processed human sewage). Fetal testes from continuously exposed ewes were either unaffected at day 80 or exhibited a reduced area of testis immunostained for CYP17A1 protein at day 140. Fetal testes from day 140 pregnant ewes that were exposed transiently for 80-day periods during early (0-80 days), mid (30-110 days), or late (60-140 days) pregnancy had fewer Sertoli cells and reduced testicular area stained for CYP17A1. Male fetuses from ewes exposed during late pregnancy also exhibited reduced fetal body, adrenal and testis mass, anogenital distance, and lowered testosterone; collectively indicative of an anti-androgenic effect. Exposure limited to early gestation induced more testis transcriptome changes than observed for continuously exposed day 140 fetuses. These data suggest that a short period of EC exposure does not allow sufficient time for the testis to adapt. Consequently, testicular transcriptomic changes induced during the first 80 days of gestation may equate with phenotypic effects observed at day 140. In contrast, relatively fewer changes in the testis transcriptome in fetuses exposed continuously to ECs throughout gestation are associated with less severe consequences. Unless corrected by or during puberty, these differential effects would predictably have adverse outcomes for adult testicular function and fertility.

Long-term exposure to chemicals in sewage sludge fertilizer alters liver lipid content in females and cancer marker expression in males.

BACKGROUND: The increased incidence of diseases, including metabolic syndrome and infertility, may be related to exposure to the mixture of chemicals, which are ubiquitous in the modern environment (environmental chemicals, ECs). Xeno-detoxification occurs within the liver which is also the source of many plasma proteins and growth factors and plays an important role in the regulation of homeostasis. OBJECTIVES: The objective of this study was to investigate the effects of ECs on aspects of liver function, in a well characterized ovine model of exposure to a real-life EC mixture. METHODS: Four groups of sheep (n = 10-12/sex/treatment) were maintained long-term on control or sewage sludge-fertilized pastures: from conception to culling at 19 months of age in females and from conception to 7 months of age and thereafter in control plots until culling at 19 months of age in males. Environmental chemicals were measured in sheep livers and RNA and protein extracts were assessed for exposure markers. Liver proteins were resolved using 2D differential in-gel electrophoresis and differentially expressed protein spots were identified by liquid chromatography/tandem mass spectroscopy. RESULTS: Higher levels of polycyclic aromatic hydrocarbons (PAHs) and lower levels of polychlorinated biphenyls (PCBs) in the livers of control males compared to control females indicated sexually dimorphic EC body burdens. Increased levels of the PAHs Benzo[a]anthracene and chrysene and reduced levels of PCB 153 and PCB 180 were observed in the livers of continuously exposed females. EC exposure affected xenobiotic and detoxification responses and the liver proteome in both sexes and included major plasma-secreted and blood proteins, and metabolic enzymes whose pathway analysis predicted dysregulation of cancer-related pathways and altered lipid dynamics. The latter were confirmed by a reduction in total lipids in female livers and up-regulation of cancer-related transcript markers in male livers respectively by sewage sludge exposure. CONCLUSIONS: Our results demonstrate that chronic exposure to ECs causes major physiological changes in the liver, likely to affect multiple systems in the body and which may predispose individuals to increased disease risks.

The fetal ovary exhibits temporal sensitivity to a 'real-life' mixture of environmental chemicals.

The development of fetal ovarian follicles is a critical determinant of adult female reproductive competence. Prolonged exposure to environmental chemicals (ECs) can perturb this process with detrimental consequences for offspring. Here we report on the exposure of pregnant ewes to an environmental mixture of ECs derived from pastures fertilized with sewage sludge (biosolids): a common global agricultural practice. Exposure of pregnant ewes to ECs over 80 day periods during early, mid or late gestation reduced the proportion of healthy early stage fetal follicles comprising the ovarian reserve. Mid and late gestation EC exposures had the most marked effects, disturbing maternal and fetal liver chemical profiles, masculinising fetal anogenital distance and greatly increasing the number of altered fetal ovarian genes and proteins. In conclusion, differential temporal sensitivity of the fetus and its ovaries to EC mixtures has implications for adult ovarian function following adverse exposures during pregnancy.

TOPAZ1, a germ cell specific factor, is essential for male meiotic progression.

Topaz1 (Testis and Ovary-specific PAZ domain gene 1) is a germ cell specific gene highly conserved in vertebrates. The putative protein TOPAZ1 contains a PAZ domain, specifically found in PIWI, Argonaute and Zwille proteins. Consequently, Topaz1 is supposed to have a role during gametogenesis and may be involved in the piRNA pathway and contribute to silencing of transposable elements and maintenance of genome integrity. Here we report Topaz1 inactivation in mouse. Female fertility was not affected, but male sterility appeared exclusively in homozygous mutants in accordance with the high expression of Topaz1 in male germ cells. Pachytene Topaz1--deficient spermatocytes progress through meiosis without either derepression of retrotransposons or MSCI dysfunction, but become arrested before the post-meiotic round spermatid stage with extensive apoptosis. Consequently, an absence of spermatids and spermatozoa was observed in Topaz1(-/-) testis. Histological analysis also revealed that disturbances of spermatogenesis take place between post natal days 15 and 20, during the first wave of male meiosis and before the generation of haploid germ cells. Transcriptomic analysis at these two stages showed that TOPAZ1 influences the expression of one hundred transcripts, most of which are up-regulated in mutant testis at post natal day 20. Our results also showed that 10% of these transcripts are long non-coding RNA. This suggests that a highly regulated balance of lncRNAs seems to be essential during spermatogenesis for induction of appropriate male gamete production.

FOXL2 is a female sex-determining gene in the goat.

The origin of sex reversal in XX goats homozygous for the polled intersex syndrome (PIS) mutation was unclear because of the complexity of the mutation that affects the transcription of both FOXL2 and several long noncoding RNAs (lncRNAs). Accumulating evidence suggested that FOXL2 could be the sole gene of the PIS locus responsible for XX sex reversal, the lncRNAs being involved in transcriptional regulation of FOXL2. In this study, using zinc-finger nuclease-directed mutagenesis, we generated several fetuses, of which one XX individual bears biallelic mutations of FOXL2. Our analysis demonstrates that FOXL2 loss of function dissociated from loss of lncRNA expression is sufficient to cause an XX female-to-male sex reversal in the goat model and, as in the mouse model, an agenesis of eyelids. Both developmental defects were reproduced in two newborn animals cloned from the XX FOXL2(-/-) fibroblasts. These results therefore identify FOXL2 as a bona fide female sex-determining gene in the goat. They also highlight a stage-dependent role of FOXL2 in the ovary, different between goats and mice, being important for fetal development in the former but for postnatal maintenance in the latter.

Exposure to chemical cocktails before or after conception--- the effect of timing on ovarian development.

Exposure of female fetuses to environmental chemicals (ECs) during pregnancy results in a disturbed ovarian adult phenotype. We investigated the influence of pre- and/or post-conception exposure to low-level mixtures of ECs on the structure and function of the fetal ovine ovary. We examined ovarian morphology, expression of oocyte and granulosa cell-specific genes and proteome. Female fetuses were collected at day 110 of gestation, from dams exposed continuously until, and after mating, by grazing in pastures treated with sewage sludge as a fertiliser (TT) or in control fields treated with inorganic fertiliser (CC). In addition, in a cross-over design, fetal ovaries were collected from dams maintained on sludge pastures up to the time of mating but then transferred to control pastures (TC) and, reciprocally, those transferred from control to treated pastures at mating (CT). On examination, the proportion of type 1a follicles (activating primordial follicles) was significantly lower in animals from the CT groups compared with CC and TT groups (P<0.05). Of the 23 ovarian gene transcripts studied, 14 were altered in the ovaries of exposed fetuses (CT, TC, and TT) relative to controls, with the largest number of changes observed in cross-exposure pattern groups (CT or TC). Continuous EC exposure (TT) produced fewer transcript alterations and only two genes (INHBA and GSN) presented differential profiles between CC and TT. Fetal ovarian proteome analysis (2-DE gels) showed, across all exposure groups, 86 differentially expressed protein spots compared to controls. Animals in the CT group exhibited the highest number (53) while TC and TT presented the same number of affected protein spots (42). Fetal ovarian proteins with altered expression included MVP (major vault protein) and several members of the heat-shock family (HSPA4L, HSP90AA1 and HSF1). The present findings indicate that continuous maternal EC exposure before and during gestation, are less deleterious for fetal ovarian development than a change in maternal EC exposure between pre and post-conception. The pathways by which the ovary responds to this chemical stress were common in TT, CT, TC exposed foetuses. In addition to the period of pregnancy, the pre-conception period appears also as crucial for conditioning long-term effects of EC exposure on ovarian development and primordial follicle reserve and hence future fertility.

Peri-conceptional changes in maternal exposure to sewage sludge chemicals disturbs fetal thyroid gland development in sheep.

Ewes were exposed to sewage sludge-fertilized pastures in a study designed investigate pre-conceptual and/or gestational exposure to environmental chemicals. The in utero impact on fetal thyroid morphology and function at day 110 (of 145) of pregnancy was then determined. Pre-conceptual exposure increased the relative thyroid organ weights in male fetuses. The number of thyroid follicles in thyroids of fetuses after pre-conceptual or gestational exposure was reduced. This correlated with an increase in Ki67 positive cells. Pre-conceptual exposure to sewage sludge reduced small blood vessels in fetal thyroids. Thyroid tissues of exposed fetuses contained regions where mature angio-follicular units were reduced exhibiting decreased immunostaining for sodium-iodide symporter (NIS). Fetal plasma levels of fT3 and fT4 in exposed animals, however, were not different from controls suggesting compensatory changes in the thyroid gland to maintain homeostasis in exposed fetuses. The regional aberrations in thyroid morphology may impact on the post-natal life of the exposed offspring.

TOPAZ1, a novel germ cell-specific expressed gene conserved during evolution across vertebrates.

BACKGROUND: We had previously reported that the Suppression Subtractive Hybridization (SSH) approach was relevant for the isolation of new mammalian genes involved in oogenesis and early follicle development. Some of these transcripts might be potential new oocyte and granulosa cell markers. We have now characterized one of them, named TOPAZ1 for the Testis and Ovary-specific PAZ domain gene. PRINCIPAL FINDINGS: Sheep and mouse TOPAZ1 mRNA have 4,803 bp and 4,962 bp open reading frames (20 exons), respectively, and encode putative TOPAZ1 proteins containing 1,600 and 1653 amino acids. They possess PAZ and CCCH domains. In sheep, TOPAZ1 mRNA is preferentially expressed in females during fetal life with a peak during prophase I of meiosis, and in males during adulthood. In the mouse, Topaz1 is a germ cell-specific gene. TOPAZ1 protein is highly conserved in vertebrates and specifically expressed in mouse and sheep gonads. It is localized in the cytoplasm of germ cells from the sheep fetal ovary and mouse adult testis. CONCLUSIONS: We have identified a novel PAZ-domain protein that is abundantly expressed in the gonads during germ cell meiosis. The expression pattern of TOPAZ1, and its high degree of conservation, suggests that it may play an important role in germ cell development. Further characterization of TOPAZ1 may elucidate the mechanisms involved in gametogenesis, and particularly in the RNA silencing process in the germ line.

Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by laser capture microdissection.

BACKGROUND: Successful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult.The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA.Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis. RESULTS: We developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and three granulosa cell-specific genes (KL, GATA4, AMH).A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte.Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA. CONCLUSIONS: The ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.

[Genetics of early ovarian differentiation: recent data]. / La différenciation ovarienne précoce et son contrôle génétique.

Early ovarian development has long been thought of as a default pathway switched on passively by the absence of SRY gene. Recent genetic and transcriptomic studies challenge this view and show that two master pathways simultaneously repress male-specific genes and activate female-specific genetic cascades. This antagonistic action is maintained from embryonic stages to adulthood. The differentiation of the ovarian somatic component is regulated by both the forkhead transcription factor FOXL2 (alone or in combination with oestrogens according to the species) and ß-catenin pathway activated by Wnt4 and Rspo1. The sex-specific change in the fate of primordial germ cells depends on the gonad environment. Female gonocytes actively proliferate by mitosis then enter meiosis I until the diplotene stage. Primordial follicle formation occurs when oocytes are individually surrounded with pre-granulosa cells. In mammals, the population of primordial follicles serves as a resting and finite pool of oocytes available during the female reproductive life span. Recent data on factors controlling these molecular processes will be presented in this review.

Exposure to a complex cocktail of environmental endocrine-disrupting compounds disturbs the kisspeptin/GPR54 system in ovine hypothalamus and pituitary gland.

BACKGROUND: Ubiquitous environmental chemicals, including endocrine-disrupting chemicals (EDCs), are associated with declining human reproductive health, as well as an increasing incidence of cancers of the reproductive system. Verifying such links requires animal models exposed to "real-life," environmentally relevant concentrations/mixtures of EDC, particularly in utero, when sensitivity to EDC exposure is maximal. OBJECTIVES: We evaluated the effects of maternal exposure to a pollutant cocktail (sewage sludge) on the ovine fetal reproductive neuroendocrine axes, particularly the kisspeptin (KiSS-1)/GPR54 (G-protein-coupled receptor 54) system. METHODS: KiSS-1, GPR54, and ERalpha (estrogen receptor alpha) mRNA expression was quantified in control (C) and treated (T) maternal and fetal (110-day) hypothalami and pituitary glands using semiquantitative reverse transcription polymerase chain reaction, and colocalization of kisspeptin with LHbeta (luteinizing hormone beta) and ERalpha in C and T fetal pituitary glands quantified using dual-labeling immunohistochemistry. RESULTS: Fetuses exposed in utero to the EDC mixture showed reduced KiSS-1 mRNA expression across three hypothalamic regions examined (rostral, mid, and caudal) and had fewer kisspetin immunopositive cells colocalized with both LHbeta and ERalpha in the pituitary gland. In contrast, treatment had no effect on parameters measured in the adult ewe hypothalamus or pituitary. CONCLUSIONS: This study demonstrates that the developing fetus is sensitive to real-world mixtures of environmental chemicals, which cause significant neuroendocrine alterations. The important role of kisspeptin/GPR54 in regulating puberty and adult reproduction means that in utero disruption of this system is likely to have long-term consequences in adulthood and represents a novel, additional pathway through which environmental chemicals perturb human reproduction.

Identification of transcripts involved in meiosis and follicle formation during ovine ovary development.

BACKGROUND: The key steps in germ cell survival during ovarian development are the entry into meiosis of oogonies and the formation of primordial follicles, which then determine the reproductive lifespan of the ovary. In sheep, these steps occur during fetal life, between 55 and 80 days of gestation, respectively. The aim of this study was to identify differentially expressed ovarian genes during prophase I meiosis and early folliculogenesis in sheep. RESULTS: In order to elucidate the molecular events associated with early ovarian differentiation, we generated two ovary stage-specific subtracted cDNA libraries using SSH. Large-scale sequencing of these SSH libraries identified 6,080 ESTs representing 2,535 contigs. Clustering and assembly of these ESTs resulted in a total of 2,101 unique sequences depicted in 1,305 singleton (62.11%) and 796 contigs (37.9%) ESTs (clusters). BLASTX evaluation indicated that 99% of the ESTs were homologous to various known genes/proteins in a broad range of organisms, especially ovine, bovine and human species. The remaining 1% which exhibited any homology to known gene sequences was considered as novel. Detailed study of the expression patterns of some of these genes using RT-PCR revealed new promising candidates for ovary differentiation genes in sheep. CONCLUSION: We showed that the SSH approach was relevant to determining new mammalian genes which might be involved in oogenesis and early follicle development, and enabled the discovery of new potential oocyte and granulosa cell markers for future studies. These genes may have significant implications regarding our understanding of ovarian function in molecular terms, and for the development of innovative strategies to both promote and control fertility.

In utero exposure to low doses of environmental pollutants disrupts fetal ovarian development in sheep.

Epidemiological studies of the impact of environmental chemicals on reproductive health demonstrate consequences of exposure but establishing causative links requires animal models using 'real life' in utero exposures. We aimed to determine whether prolonged, low-dose, exposure of pregnant sheep to a mixture of environmental chemicals affects fetal ovarian development. Exposure of treated ewes (n = 7) to pollutants was maximized by surface application of processed sewage sludge to pasture. Control ewes (n = 10) were reared on pasture treated with inorganic fertilizer. Ovaries and blood were collected from fetuses (n = 15 control and n = 8 treated) on Day 110 of gestation for investigation of fetal endocrinology, ovarian follicle/oocyte numbers and ovarian proteome. Treated fetuses were 14% lighter than controls but fetal ovary weights were unchanged. Prolactin (48% lower) was the only measured hormone significantly affected by treatment. Treatment reduced numbers of growth differentiation factor (GDF9) and induced myeloid leukaemia cell differentiation protein (MCL1) positive oocytes by 25-26% and increased pro-apoptotic BAX by 65% and 42% of protein spots in the treated ovarian proteome were differently expressed compared with controls. Nineteen spots were identified and included proteins involved in gene expression/transcription, protein synthesis, phosphorylation and receptor activity. Fetal exposure to environmental chemicals, via the mother, significantly perturbs fetal ovarian development. If such effects are replicated in humans, premature menopause could be an outcome.

Genetic investigation of four meiotic genes in women with premature ovarian failure.

OBJECTIVE: The goal of this study was to determine whether mutations of meiotic genes, such as disrupted meiotic cDNA (DMC1), MutS homolog (MSH4), MSH5, and S. cerevisiae homolog (SPO11), were associated with premature ovarian failure (POF). DESIGN: Case-control study. METHODS: Blood sampling, karyotype, hormonal dosage, ultrasound, and ovarian biopsy were carried out on most patients. However, the main outcome measure was the sequencing of genomic DNA from peripheral blood samples of 41 women with POF and 36 fertile women (controls). RESULTS: A single heterozygous missense mutation, substitution of a cytosine residue with thymidine in exon 2 of MSH5, was found in two Caucasian women in whom POF developed at 18 and 36 years of age. This mutation resulted in replacement of a non-polar amino acid (proline) with a polar amino acid (serine) at position 29 (P29S). Neither 36 control women nor 39 other patients with POF possessed this genetic perturbation. Another POF patient of African origin showed a homozygous nucleotide change in the tenth of DMC1 gene that led to an alteration of the amino acid composition of the protein (M200V). CONCLUSIONS: The symptoms of infertility observed in the DMC1 homozygote mutation carrier and in both patients with a heterozygous substitution in exon 2 of the MSH5 gene provide indirect evidence of the role of genes involved in meiotic recombination in the regulation of ovarian function. MSH5 and DMC1 mutations may be one explanation for POF, albeit uncommon.

FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development.

Previous studies have equated FOXL2 as a crucial actor in the ovarian differentiation process in different vertebrate species. Its transcriptional extinction in the polled intersex syndrome (PIS) leads primarily to a drastic decrease of aromatase (CYP19) expression in the first steps of goat ovarian development. In this study, we provide a better characterization of early ovarian development in goat, and we provide experimental evidence demonstrating that FOXL2 represents a direct transcriptional activator of the CYP19 gene through its ovarian-specific promoter 2. Moreover, the ovarian location of FOXL2 and CYP19 proteins, together with their expression profiles in the female gonads, stress the involvement of FOXL2 co-factor(s) for regulating CYP19 transcription. Expressional analyses show that activin-betaA can be considered as a strong candidate for being one of these FOXL2 co-factors. Finally, we discuss evidence for a role of activin and estrogens in somatic and germinal cell proliferation occurring before germ cell meiosis. This period, of 20 days in goat, seems to have no equivalent in mouse. This species-specific difference could explain the phenotype discrepancy observed between XX goat PIS(-/-) and XX mouse Foxl2(-/-).

Foxl2 gene and the development of the ovary: a story about goat, mouse, fish and woman.

In this review, we describe recent results concerning the genetics of sex determination in mammals. Particularly, we developed the study of the FOXL2 gene and its implication in genetic anomalies in goats (PIS mutation) and humans (BPES). We present the expression of FOXL2 in the ovaries of different species.

Ovarian-specific expression of a new gene regulated by the goat PIS region and transcribed by a FOXL2 bidirectional promoter.

Studies on XX sex reversal in polled goats (PIS mutation: polled intersex syndrome) have led to the discovery of a female-specific locus crucial for ovarian differentiation. This genomic region is composed of at least two genes, FOXL2 and PISRT1, sharing a common transcriptional regulatory region, PIS. In this paper, we describe a third gene, PFOXic (promoter FOXL2 inverse complementary), located near FOXL2 in the opposite orientation. This gene composed of five exons encodes a 1723-bp cDNA, enclosing two repetitive elements in its 3' end. PFOXic mRNA encodes a putative protein of 163 amino acids with no homologies in any of the databases tested. The transcriptional expression of PFOXic is driven by a bidirectional promoter also enhancing FOXL2 transcription. In goats, PFOXic is expressed in developing ovaries, from 36 days postcoitum until adulthood. Ovarian-specific expression of PFOXic is regulated by the PIS region. PFOXic is found conserved only in Bovidae. But, a human gene located in the opposite orientation relative to FOXL2 can be considered a human PFOXic. Finally, we discuss evidence arguing for regulation of the level of FOXL2 transcription via the bidirectional promoter and the level of transcription of PFOXic.

Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation.

In goats, the PIS (polled intersex syndrome) mutation is responsible for both the absence of horns in males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. Nevertheless, a delay of 4-5 days is observed in comparison with normal testis differentiation in XY males. By positional cloning, we demonstrate that the PIS mutation is an 11.7-kb regulatory-deletion affecting the expression of two genes, PISRT1 and FOXL2 which could act synergistically to promote ovarian differentiation. The transcriptional extinction of these two genes leads, very early, to testis-formation in XX homozygous PIS-/- mutants. According to their expression profiles and bibliographic data, we propose that FOXL2 may be an ovary-differentiating gene, and the non-coding RNA PISRT1, an anti-testis factor repressing SOX9, a key regulator of testis differentiation. Under this hypothesis, SRY, the testis-determining factor would inhibit these two genes in the gonads of XY males, to ensure testis differentiation.

Isolation of chicken homolog of the FOXL2 gene and comparison of its expression patterns with those of aromatase during ovarian development.

Mutations in the forkhead transcription factor gene FOXL2 are involved in ovarian failure, which occurs in human BPES syndrome. This syndrome presents a sexually dimorphic expression, specific to the ovary in several vertebrates. We cloned the open reading frame of chicken FOXL2 (cFoxL2) and studied cFoxL2 expression in developing gonads and during adulthood to examine the role of FOXL2 in ovarian differentiation and function in birds. The spatial and temporal dynamics of cFoxL2 and aromatase expression were analyzed in parallel by using real-time quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry in attempt to investigate the possible role of cFoxL2 in the regulation of aromatase. The expression patterns of cFoxL2 and aromatase transcripts were highly correlated during the sex-differentiation period (4.7-12.7 days of incubation). Aromatase and cFoxL2 proteins were colocalized in the medullar part of female gonads on embryonic day 14. Fourteen days after hatching, cFoxL2 protein was mainly detected in granulosa cells of developing follicles. In adult ovary follicular envelopes, apart from granulosa cells, cFoxL2 transcript and protein were detected at lower levels in theca cells where aromatase was present. A high level of cFoxL2 transcription was also observed in maturing and ovulated oocytes. Our results confirm that FoxL2 is an early regulator of ovarian development in birds and may be involved in aromatase transcription regulation.