Cell lineages of the early human gonad commit to one of the two mutually antagonistic organogenetic fates, the testis or the ovary. Some individuals with a 46,XX karyotype develop testes or ovotestes (testicular or ovotesticular disorder of sex development; TDSD/OTDSD), due to the presence of the testis-determining gene, SRY. Other rare complex syndromic forms of TDSD/OTDSD are associated with mutations in pro-ovarian genes that repress testis development (e.g. WNT4); however, the genetic cause of the more common non-syndromic forms is unknown. Steroidogenic factor-1 (known as NR5A1) is a key regulator of reproductive development and function. Loss-of-function changes in NR5A1 in 46,XY individuals are associated with a spectrum of phenotypes in humans ranging from a lack of testis formation to male infertility. Mutations in NR5A1 in 46,XX women are associated with primary ovarian insufficiency, which includes a lack of ovary formation, primary and secondary amenorrhoea as well as early menopause. Here, we show that a specific recurrent heterozygous missense mutation (p.Arg92Trp) in the accessory DNA-binding region of NR5A1 is associated with variable degree of testis development in 46,XX children and adults from four unrelated families. Remarkably, in one family a sibling raised as a girl and carrying this NR5A1 mutation was found to have a 46,XY karyotype with partial testicular dysgenesis. These unique findings highlight how a specific variant in a developmental transcription factor can switch organ fate from the ovary to testis in mammals and represents the first missense mutation causing isolated, non-syndromic 46,XX testicular/ovotesticular DSD in humans.

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Mammalian spermatogenesis is a complex and highly orchestrated combination of processes in which male germline proliferation and differentiation result in the production of mature spermatozoa. If recent genome-wide studies have contributed to the in-depth analysis of the male germline protein-encoding transcriptome, little effort has yet been devoted to the systematic identification of novel unannotated transcribed regions expressed during mammalian spermatogenesis. We report high-resolution expression profiling of male germ cells in rat, using next-generation sequencing technology and highly enriched testicular cell populations. Among 20 424 high-confidence transcripts reconstructed, we defined a stringent set of 1419 long multi-exonic unannotated transcripts expressed in the testis (testis-expressed unannotated transcripts [TUTs]). TUTs were divided into 7 groups with different expression patterns. Most TUTs share many of the characteristics of vertebrate long noncoding RNAs (lncRNAs). We also markedly reinforced the finding that TUTs and known lncRNAs accumulate during the meiotic and postmeiotic stages of spermatogenesis in mammals and that X-linked meiotic TUTs do not escape the silencing effects of meiotic sex chromosome inactivation. Importantly, we discovered that TUTs and known lncRNAs with a peak expression during meiosis define a distinct class of noncoding transcripts that exhibit exons twice as long as those of other transcripts. Our study provides new insights in transcriptional profiling of the male germline and represents a high-quality resource for novel loci expressed during spermatogenesis that significantly contributes to rat genome annotation.

Background:The sperm DNA methylation landscape is unique and critical for offspring health. If gamete-derived DNA methylation escapes reprograming in early embryos, epigenetic defects in sperm may be transmitted to the next generation. Current techniques to assess sperm DNA methylation show bias toward CpG-dense regions and do not target areas of dynamic methylation, those predicted to be environmentally sensitive and tunable regulatory elements.Objectives:Our goal was to assess variation in human sperm DNA methylation and design a targeted capture panel to interrogate the human sperm methylome.Methods:To characterize variation in sperm DNA methylation, we performed whole genome bisulfite sequencing (WGBS) on an equimolar pool of sperm DNA from a wide cross section of 30 men varying in age, fertility status, methylenetetrahydrofolate reductase (MTHFR) genotype, and exposures. With our targeted capture panel, in individual samples, we examined the effect of MTHFR genotype (n=13 677CC, n=8 677TT), as well as high-dose folic acid supplementation (n=6, per genotype, before and after supplementation).Results:Through WGBS we discovered nearly 1 million CpGs possessing intermediate methylation levels (20–80%), termed dynamic sperm CpGs. These dynamic CpGs, along with 2 million commonly assessed CpGs, were used to customize a capture panel for targeted interrogation of the human sperm methylome and test its ability to detect effects of altered folate metabolism. As compared with MTHFR 677CC men, those with the 677TT genotype (50% decreased MTHFR activity) had both hyper- and hypomethylation in their sperm. High-dose folic acid supplement treatment exacerbated hypomethylation in MTHFR 677TT men compared with 677CC. In both cases, >80% of altered methylation was found in dynamic sperm CpGs, uniquely measured by our assay.Discussion:Our sperm panel allowed the discovery of differential methylation following conditions affecting folate metabolism in novel dynamic sperm CpGs. Improved ability to examine variation in sperm DNA methylation can facilitate comprehensive studies of environment–epigenome interactions. https://doi.org/10.1289/EHP4812

SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n = 274) and two independent cohorts of women with primary ovarian insufficiency (POI; n = 153 and n = 104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P = 4.5 × 10−5) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.

The human APOBEC3A and APOBEC3B genes (A3A and A3B) encode DNA mutator enzymes that deaminate cytidine and 5-methylcytidine residues in single-stranded DNA (ssDNA). They are important sources of mutations in many cancer genomes which show a preponderance of CG->TA transitions. Although both enzymes can hypermutate chromosomal DNA in an experimental setting, only A3A can induce double strand DNA breaks, even though the catalytic domains of A3B and A3A differ by only 9% at the protein level. Accordingly we sought the molecular basis underlying A3B attenuation through the generation of A3A-A3B chimeras and mutants. It transpires that the N-terminal domain facilitates A3B activity while a handful of substitutions in the catalytic C-terminal domain impacting ssDNA binding serve to attenuate A3B compared to A3A. Interestingly, functional attenuation is also observed for the rhesus monkey rhA3B enzyme compared to rhA3A indicating that this genotoxic dichotomy has been selected for and maintained for some 38 million years. Expression of all human ssDNA cytidine deaminase genes is absent in mature sperm indicating they contribute to somatic mutation and cancer but not human diversity.

Objective: Steroidogenic factor 1 (SF1, NR5A1) is a key transcriptional regulator of genes involved in the hypothalamicpituitary-gonadal axis. Recently, SF1 mutations were found to be a frequent cause of 46,XY disorders of sex development (DSD) in humans. We investigate the frequency of NR5A1 mutations in an Egyptian cohort of XY DSD. Design: Clinical assessment, endocrine evaluation and genetic analysis of 50 Egyptian XY DSD patients (without adrenal insufficiency) with a wide phenotypic spectrum. Methods: Molecular analysis of NR5A1 gene by direct sequencing followed by in vitro functional analysis of the two novel missense mutations detected. Results: Three novel heterozygous mutations of the coding region in patients with hypospadias were detected. p.Glu121AlafsX25 results in severely truncated protein, p.Arg62Cys lies in DNA-binding zinc finger, whereas p.Ala154Thr lies in the hinge region of SF1 protein. Transactivation assays using reporter constructs carrying promoters of anti-Mü llerian hormone (AMH), CYP11A1 and TESCO core enhancer of Sox9 showed that p.Ala154Thr and p.Arg62Cys mutations result in aberrant biological activity of NR5A1. A total of 17 patients (34%) harboured the p.Gly146Ala polymorphism. Conclusion: We identified two novel NR5A1 mutations showing impaired function in 23 Egyptian XY DSD patients with hypospadias (8.5%). This is the first study searching for NR5A1 mutations in oriental patients from the Middle East and Arab region with XY DSD and no adrenal insufficiency, revealing a frequency similar to that in European patients (6.5-15%). We recommend screening of NR5A1 in patients with hypospadias and gonadal dysgenesis. Yearly follow-ups of gonadal function and early cryoconservation of sperms should be performed in XY DSD patients with NR5A1 mutations given the risk of future fertility problems due to early gonadal failure.

BackgroundThe persistent organochlorine dichlorodiphenyltrichloroethane (DDT) is banned world-wide due to its negative health effects and persistence in the environment. It is exceptionally used as an insecticide for malaria control. Exposure occurs in regions where DDT is applied, as well as in the arctic where it’s endocrine disrupting metabolite, p,p’-dichlorodiphenyldichloroethylene (p,p’-DDE) accumulates in marine mammals and fish. DDT and p,p’-DDE exposures are linked to birth defects, infertility, cancer, and neurodevelopmental delays. Of particular concern is the potential of DDT use to impact the health of generations to come. Generational effects of toxicant exposures have been described in animal models and implicated germline epigenetic factors. Similar generational effects have been shown in epidemiological studies. Although advances in understanding the molecular mechanisms mediating this epigenetic inheritance have been made, there remain major knowledge gaps in how this occurs in humans. In animal and human models, DNA methylation (DNAme) has been implicated in paternal epigenetic effects. In animal models, histone H3K4 trimethylation (H3K4me3) has been shown to be responsive to the paternal environment and linked with epigenetic transmission to the embryo. Our objectives were to define the associations between p,p’-DDE serum levels and alterations in the sperm methylome and H3K4me3 enrichment using next generation sequencing. We aimed to compare regions of epigenomic sensitivity between geographically diverse populations with different routes and levels of exposures, and to identify interactions between altered DNAme and H3K4me3 regions. The potential for p,p’-DDE to impact the health of the next generation was explored by examining the functions of the genomic regions impacted, their roles during embryo development, and in health and disease.MethodsIn the Limpopo Province of South Africa, we recruited 247 VhaVenda South African men from 12 villages that either used indoor residual spraying with DDT for malaria control or not. We selected 49 paired blood and semen samples, from men that ranged from 18 to 32 years of age (mean 25 years). Sample inclusion was based on normal sperm counts (> 15 million/ml), normal sperm DNA fragmentation index, and testing a range of p,p’-DDE exposure levels (mean 10,462.228 ± 1,792.298 ng/ml). From a total of 193 samples, 47 Greenlandic Inuit blood and semen paired samples were selected from the biobank of the INUENDO cohort. The subjects ranged from 20 to 44 years of age (mean 31 years), were born in Greenland, and all had proven fertility. Sample selection was based on obtaining a range of p,p’-DDE exposure levels (mean 870.734 ± 134.030 ng/ml). Here we determined the molecular responses at the level of the sperm epigenome to serum p,p’-DDE levels using MethylC-Capture-seq (MCC-seq) and chromatin-immunoprecipitation followed by sequencing (ChIP-seq). We identified genomic regions with altered DNA methylation (DNAme) and differential enrichment of histone H3 lysine 4 trimethylation (H3K4me3) in sperm. We used in silico analyses to discover regions of differential methylation associated with p,p’-DDE levels that were predicted to be transmitted and persist in the embryo.ResultsAlterations in DNAme and H3K4me3 enrichment followed dose response-like trends, and we identified overlapping genomic regions with DNAme sensitivities in both populations. Altered DNAme and H3K4me3 in sperm occurred at transposable elements and regulatory regions involved in fertility, disease, development, and neurofunction. A subset of regions with altered sperm DNAme and H3K4me3 were predicted to persist in the pre-implantation embryo and were associated with embryonic gene expression.LimitationsThe samples were collected from remote areas of the world thus sample size is relatively small. The populations differed in the routes of exposure, timing of collection, mean age (mean of 25 versus 31 years of age in South African and Greenlandic populations respectively) and in the timing of p,p’-DDE measurement. Moreover, the Greenlandic Inuit men were proven fertile whereas the fertility status of the South African men was unknown. Confounding factors such as other environmental exposures and selection bias cannot be ruled out.ConclusionsThese findings suggest that in men, DDT and p,p’-DDE exposure impacts the sperm epigenome in a dose-responsive manner and may negatively impact the health of future generations through epigenetic mechanisms.