TEX29 is a novel acrosome marker dispensable for spermatogenesis and fertilization in mice

Spermatogenesis is precisely regulated by an intricate genetic network, but the biological roles of numerous testis-enriched genes remain unelucidated. This study aimed to systematically investigate the expression pattern, subcellular localization and functional significance of Testis-Expressed Gene 29 (Tex29) in mice and male fertility.

Integrated molecular, cellular, and animal model approaches were employed. Tex29 mRNA and protein expression were analyzed by molecular and immunofluorescence staining techniques. CRISPR/Cas9-mediated genome editing was used to generate Tex29-knockout (KO) mice. Fertility assessment, histological examination of testes, sperm quality analysis, and transmission/scanning electron microscopy were performed on Tex29-KO mice. Additionally, whole-exome sequencing was conducted in 165 infertile men to identify Tex29 variants.

Tex29 mRNA was specifically expressed in testes, first detectable on postnatal day 18 and gradually upregulated during testicular maturation. TEX29 protein was specifically localized to the acrosome of spermatids and mature sperm throughout spermiogenesis. Tex29-KO males exhibited normal fertility with litter sizes comparable to wild-type (WT) controls, and their seminiferous tubules retained intact structure with all spermatogenic stages. No significant differences in sperm concentration, viability, or motility were observed between Tex29-KO and WT mice. Although Tex29-KO sperm maintained normal overall morphology and canonical "9 + 2" axonemal structure in the flagellum, a subset showed acrosomal membrane abnormalities in the apical region. In vitro fertilization (IVF) rates and blastocyst development were uncompromised in Tex29-KO mice. Two synonymous TEX29 variants (c.66C > T, p.Asp22Asp; c.207C > A, p.Ile69Ile) were identified in 7 of 165 infertile men, and four couples carrying these variants achieved live births.

TEX29 is a novel testis-specific acrosomal marker protein essential for maintaining normal acrosomal membrane integrity during murine spermiogenesis. Notably, Tex29 is dispensable for spermatogenesis and male fertility in mice. The functional role of TEX29 in human spermatogenesis and fertility remains to be fully determined due to limited clinical evidence. These findings provide valuable insights for basic research on acrosome biogenesis and male infertility associated with acrosomal abnormalities.