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Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although its genetic etiology has been extensively investigated, most POI cases remain unexplained. Using whole-exome sequencing, we identified a homozygous variant in RAD51B –(c.92delT) in two sisters with POI. In vitro studies revealed that this variant leads to translation reinitiation at methionine 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, Rad51b-c.92delT variant reduced replication fork progression of patient-derived lymphoblastoid cell lines and pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability., This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Grant 2014/14231-0 (to MMF); FAPESP Grant 2013/02162-8, Nucleo de Estudos e Terapia Celular e Molecular (NETCEM), Conselho Nacional de Desenvolvimento Científico e Tecnológico Grant 303002/2016- 6 (to BBM); and FAPESP Grant 2014/50137-5 (to SELA). This work was supported by MINECO (PID2020-120326RB-I00) and by Junta de Castilla y León (CSI239P18 and CSI148P20). NFM, FSS, and MRMH are supported by European Social Fund/JCyLe grants (EDU/310/2015, EDU/556/2019 and EDU/1992/2020). YBC and RSU are funded by a grant from MINECO (BS-2015–073993 and BFU2017-89408-R). Experiments performed at CNIO were supported by grant PID2019-106707-RB to JM, co-sponsored by EU ERDF funds. SM was supported by an international postdoctoral contract “CNIO Friends”. The proteomic analysis was performed in the Proteomics Facility of Centro de Investigación del Cáncer, Salamanca, Grant PRB3(IPT17/0019 -ISCIII-SGEFI/ERDF). CIC-IBMCC is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia cofunded by the Castilla–León autonomous government and the European Regional Development Fund (CLC–2017–01). Veitia’s Lab is supported by the University of Paris and the Centre National de la Recherche Scientifique.

International audience; In 46,XY men, testis is determined by a genetic network(s) that both promotes testis formation and represses ovarian development. Disruption of this process results in a lack of testis-determination and affected individuals present with 46,XY gonadal dysgenesis (GD), a part of the spectrum of Disorders/Differences of Sex Development/Determination (DSD). A minority of all cases of GD are associated with pathogenic variants in key players of testis-determination, SRY, SOX9, MAP3K1 and NR5A1. However, most of the cases remain unexplained. Recently, unbiased exome sequencing approaches have revealed new genes and loci that may cause 46,XY GD. We critically evaluate the evidence to support causality of these factors and describe how functional studies are continuing to improve our understanding of genotype-phenotype relationships in genes that are established causes of GD. As genomic data continues to be generated from DSD cohorts, we propose several recommendations to help interpret the data and establish causality.

International audience; In humans, pathogenic variants in the DHH gene underlie cases of 46,XY gonadal dysgenesis. DHH is part of the Hedgehog family of proteins, which require extensive processing, including self-cleavage of the precursor for efficient signalling. In our work, we have assessed the effect of several human DHH pathogenic variants involved in recessive complete or partial gonadal dysgenesis, on protein processing and sub-cellular localization. We found that a subset of variants was unable to perform self-cleavage, which correlated albeit not perfectly with an altered subcellular localization of the resulting proteins. For the processing-proficient variants, we used structural modelling tools and molecular dynamic (MD) simulations to predict the potential impact of the variants on protein conformation and/or interaction with partners. Our study contributes to a better understanding of the molecular mechanisms involved in DHH dysfunction leading to 46,XY disorders of sex development.

The PI3K/AKT signaling pathway is known to regulate a broad range of cellular processes, and it is often altered in several types of cancers. Recently, somatic AKT1 mutations leading to a strong activation of this kinase have been reported in juvenile granulosa cell tumors. However, the molecular role of AKT1 in the supporting cell lineage of the ovary is still poorly understood. To get insights into its function in such cells, we depleted Akt1 in murine primary granulosa cells and assessed the molecular consequences at both the transcript and protein levels. We were able to corroborate the involvement of AKT1 in the regulation of metabolism, apoptosis, cell cycle, or cytoskeleton dynamics in this ovarian cell type. Consistently, we showed in established granulosa cells that depletion of Akt1 provoked altered directional persistent migration and increased its velocity. This study also allowed us to put forward new direct and indirect targets of the kinase. Indeed, a series of proteins involved in intracellular transport and mitochondrial physiology were significantly affected by Akt1 depletion. Using in silico analyses, we also propose a set of kinases and transcription factors that can mediate the action of AKT1 on the deregulated transcripts and proteins. Taken altogether, our results provide a resource of direct and indirect AKT1 targets in granulosa cells and may help understand its roles in this ovarian cell type.

Gonad differentiation is a crucial step conditioning the future fertility of individuals and most of the master genes involved in this process have been investigated in detail. However, transcriptomic analyses of developing gonads from different animal models have revealed that hundreds of genes present sexually dimorphic expression patterns. DMXL2 was one of these genes and its function in mammalian gonads was unknown. We therefore investigated the phenotypes of total and gonad-specific Dmxl2 knockout mouse lines. The total loss-of-function of Dmxl2 was lethal in neonates, with death occurring within 12 hours of birth. Dmxl2-knockout neonates were weak and did not feed. They also presented defects of olfactory information transmission and severe hypoglycemia, suggesting that their premature death might be due to global neuronal and/or metabolic deficiencies. Dmxl2 expression in the gonads increased after birth, during follicle formation in females and spermatogenesis in males. DMXL2 was detected in both the supporting and germinal cells of both sexes. As Dmxl2 loss-of-function was lethal, only limited investigations of the gonads of Dmxl2 KO pups were possible. They revealed no major defects at birth. The gonadal function of Dmxl2 was then assessed by conditional deletions of the gene in gonadal supporting cells, germinal cells, or both. Conditional Dmxl2 ablation in the gonads did not impair fertility in males or females. By contrast, male mice with Dmxl2 deletions, either throughout the testes or exclusively in germ cells, presented a subtle testicular phenotype during the first wave of spermatogenesis that was clearly detectable at puberty. Indeed, Dmxl2 loss-of-function throughout the testes or in germ cells only, led to sperm counts more than 60% lower than normal and defective seminiferous tubule architecture. Transcriptomic and immunohistochemichal analyses on these abnormal testes revealed a deregulation of Sertoli cell phagocytic activity related to germ cell apoptosis augmentation. In conclusion, we show that Dmxl2 exerts its principal function in the testes at the onset of puberty, although its absence does not compromise male fertility in mice., Author summary DMXL2 gene dysfunction underlies various human diseases, including breast cancer, non-syndromic hearing loss, and polyendocrine-polyneuropathy syndrome, demonstrating the large range of potential actions of DMXL2. We show here that Dmxl2 expression is crucial for survival in mice, as neonates die within hours of birth when this gene is inactivated. The transmission of olfactory information is affected, leading to an absence of suckling and impaired feeding. Severe hypoglycemia is also observed in male neonates. We observed Dmxl2 expression in several organs, including the brain, heart and adrenal glands, potentially corresponding to some of the phenotypes observed in Dmxl2-deficient pups. We also described Dmxl2 expression in the reproductive tracts and gonads and showed that Dmxl2 inactivation specifically in the testes has a significant effect on the initial waves of spermatogenesis, resulting in very low levels of sperm production at puberty. Our results suggest that DMXL2 deficits should be considered in men with impaired fertility, as pathogenic variants of this gene may be associated with male infertility in humans.

In a 46, XY individual, the presence of the Y chromosome harboring the testis-determining factor (SRY) triggers testis determination and differentiation. In a 46, XX individual, the absence of SRY along with the activation of genes associated with the female pathway leads to ovarian development. The latter process has long been considered as a default pathway. However, recent studies have cast doubts on this dogma. Here, after a brief overview of the main steps of ovarian development, we focus on three genes WNT4, RSPO1 and FOXL2 that are essential for ovarian determination, differentiation and/or maintenance. Especial attention is paid to FOXL2 whose mutations are responsible for the blepharophimosis syndrome, often associated with female infertility, and for cancer. We highlight the cooperation of WNT4, RSPO1 and FOXL2 within a regulatory network and the need for further research to better understand their role in defining and maintaining ovarian identity.

Premature ovarian insufficiency (POI) is a frequent pathology that affects women under 40 years of age, characterized by an early cessation of menses and high FSH levels. Despite recent progresses in molecular diagnosis, the etiology of POI remains idiopathic in most cases. Whole-exome sequencing of members of a Colombian family affected by POI allowed us to identify a novel homozygous donor splice-site mutation in the meiotic gene MSH4 (MutS Homolog 4). The variant followed a strict mendelian segregation within the family and was absent in a cohort of 135 women over 50 years of age without history of infertility, from the same geographical region as the affected family. Exon trapping experiments showed that the splice-site mutation induced skipping of exon 17. At the protein level, the mutation p.Ile743_Lys785del is predicted to lead to the ablation of the highly conserved Walker B motif of the ATP-binding domain, thus inactivating MSH4. Our study describes the first MSH4 mutation associated with POI and increases the number of meiotic/DNA repair genes formally implicated as being responsible for this condition. © The Author 2017. Published by Oxford University Press. All rights reserved.