P-066 Testis expressed 44 is essential for spermatogenesis and male fertility

Abstract

Abstract Study question Dose Testis expressed 44 (TEX44) gene associated with spermatogenesis and TEX44 mutations required for male infertility ? Summary answer TEX44 is expressed in testis and participated in spermatogenesis. And mutations of TEX44 may be involved in risk of asthenozoospermia and lead to subfertile. What is known already The World Health Organization estimates that 7-9 % of male population worldwide struggle with infertility. Asthenoteratozoospermia is one of the major factors for male infertility, whereas the causes of large numbers of cases are still unknown.TEX44 is a protein coding gene, which is specifically expressed in male germ cells. However, the expression, localization pattern and precise underlying mechanisms of TEX44 in mouse and human have not been clearly during spermatogenesis and risk of asthenozoospermia. Study design, size, duration This study was an analysis of 120 Han Chinese men with asthenoteratozoospermia by whole-exome sequencing（WES）, at the Reproductive Medical Center of Peking University Third Hospital between January 2018 and January 2022.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committees of Peking University Third Hospital and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Participants/materials, setting, methods Real-time polymerase chain reaction (Q-PCR) and western blot assays (WT) were used to investigate the mRNA and protein levels of TEX44 in testes. Immunofluorescent analysis was performed on testis sections by TEX44 antibodies during the spermatogenesis. TEX44 knockout mice were generated by CRISPR/Cas9 gene-editing. Computer-assisted sperm analysis (CASA) and fertility testing were also carried out to evaluate the effect of TEX44 on spermatogenesis and male reproduction.TEX44 mutation was an analysis of asthenozoospermia patients using WES. Main results and the role of chance TEX44 mRNA and protein were found to be expressed at the highest level in mouse testis by Q-PCR and WT. Immunohistochemical results revealed TEX44 was located in the cytoplasm of elongating spermatids and exhibited specific localization to the flagellum and manchette during spermiogenesis. Tex44 knockout mice presented no detectable difference in testis-to-body weight ratios when compared with wild-type mice. However, gross testis morphology shows reduction of testis size in TEX44 knockout mice compared wild-type after 12 weeks old. Tex44(-/-) males are subfertile because of abnormal sperm movement and reduced motility.Proportions of motile and progressive movement showing spermatozoa of TEX44 knockout mice were always 30–50% lower than that in controls using CASA.Tex44-/- mice are fertile despite a significant reduction in sperm motility. But the average total number of mouses born of Tex44-/- mice were decreased (P < 0.01). In addition, WES analysis revealed two novel frameshite point mutation patients in TEX44 gene during asthenozoospermia patients. Of these missense mutations, patients with asthenozoospermia have abnormal sperm movement and reduced motility. Fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Limitations, reasons for caution Additional cases are in need of study, especially with TEX44 mutation patients from only a small number of asthenozoospermia patients. Protein analysis and function were limited. In future investigations, a larger sample size should be used and the role of the interaction proteins with TEX44 should be analyzed. Wider implications of the findings TEX44 was validated as spermatids-essential gene could play important roles in spermatogenesis. The identification and characterization of TEX44 enhances our understanding of the molecular mechanisms of spermatids differentiation and provides insight into its potential role in the asthenozoospermia.These found will provide important guidance for genetic counseling and assisted reproduction treatments. Trial registration number This study was supported by the National Natural Science Foundation of China (NO.81671513) and Beijing Natural Science Foundation (NO.7172236).