RESUMEN
STUDY QUESTION: What is the functional significance of Tex13b in male germ cell development and differentiation? SUMMARY ANSWER: Tex13b regulates male germ cell differentiation by metabolic reprogramming during spermatogenesis. WHAT IS KNOWN ALREADY: Studies in mice and humans suggest that TEX13B is a transcription factor and is exclusively expressed in germ cells. STUDY DESIGN, SIZE, DURATION: We sequenced the coding regions of TEX13B in 628 infertile men and 427 ethnically matched fertile control men. Further, to identify the molecular function of Tex13b, we created a Tex13b knockout and conditional overexpression system in GC-1spg (hereafter, GC-1) cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Our recent exome sequencing study identified novel candidate genes for male infertility. TEX13B was found to be one of the potential candidates, hence we explored the role of TEX13B in male infertility within a large infertile case-control cohort. We performed functional analyses of Tex13b in a GC-1 cell line using CRISPR-Cas9. We differentially labelled the cell proteins by stable isotope labelling of amino acids in cell culture (SILAC) and performed mass spectrometry-based whole-cell proteomics to identify the differential protein regulation in knockout cells compared to wild-type cells. We found that Tex13b knockout leads to downregulation of the OXPHOS complexes and upregulation of glycolysis genes, which was further validated by western blotting. These results were further confirmed by respirometry analysis in Tex13b knockout cells. Further, we also performed a conditional overexpression of TEX13B in GC-1 cells and studied the expression of OXPHOS complex proteins by western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a rare variant, rs775429506 (p.Gly237Glu), exclusively in two non-obstructive-azoospermia (NOA) men, that may genetically predispose these men for infertility. Further, we demonstrated that Tex13b functions in the transcription regulation of OXPHOS complexes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We examined the function of Tex13b in GC-1 in vitro by knocking out and conditional overexpression, for understanding the function of Tex13b in germ cells. Unfortunately, this could not be replicated in either an animal model or in patient-derived tissue due to the non-availability of an animal model or patient's testis biopsies. WIDER IMPLICATIONS OF THE FINDINGS: This study identified that Tex13b plays an important role in male germ cell development and differentiation. The findings of this study would be useful for screening infertile males with spermatogenic failure and counselling them before the implementation of assisted reproduction technique(s). STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the Council of Scientific and Industrial Research (CSIR) under the network project (BSC0101 and MLP0113) and SERB, the Department of Science and Technology, Government of India (J C Bose Fellowship: JCB/2019/000027). The authors do not have any competing interest.
RESUMEN
BACKGROUND: Infertility affects around 15% of all couples worldwide and is increasingly linked to variants in genes specifically expressed in the testis. Well-established causes of male infertility include pathogenic variants in the genes TEX11, TEX14, and TEX15, while few studies have recently reported variants in TEX13B, TEX13C, FAM9A (TEX39A), and FAM9B (TEX39B). OBJECTIVES: We aimed at screening for novel potential candidate genes among the human TEX ("testis expressed") genes as well as verifying previously described disease associations in this set of genes. MATERIALS AND METHODS: To this end, we screened the exome sequencing data of 1305 men, including 1056 crypto- and azoospermic individuals, and determined cell-specific expression by analyzing testis-specific single-cell RNA sequencing data for genes with identified variants. To investigate the overarching role in male fertility, we generated testis-specific knockdown (KD) models of all 10 orthologous TEX genes in Drosophila melanogaster. RESULTS: We detected rare potential disease-causing variants in TEX10, TEX13A, TEX13B, TEX13C, TEX13D, ZFAND3 (TEX27), TEX33, FAM9A (TEX39A), and FAM9B (TEX39B), in 28 infertile men, of which 15 men carried variants in TEX10, TEX27, and TEX33. The KD of TEX2, TEX9, TEX10, TEX13, ZFAND3 (TEX27), TEX28, TEX30, NFX1 (TEX42), TEX261, and UTP4 (TEX292) in Drosophila resulted in normal fertility. DISCUSSION: Based on our findings, the autosomal dominant predicted genes TEX10 and ZFAND3 (TEX27) and the autosomal recessive predicted gene TEX33, which all three are conceivably required for germ cell maturation, were identified as novel potential candidate genes for human non-obstructive azoospermia. We additionally identified hemizygous loss-of-function (LoF) variants in TEX13B, TEX13C, and FAM9A (TEX39A) as unlikely monogenic culprits of male infertility as LoF variants were also found in control men. CONCLUSION: Our findings concerning the X-linked genes TEX13B, TEX13C, and FAM9A (TEX39A) contradict previous reports and will decrease false-positive reports in genetic diagnostics of azoospermic men.