O-GlcNAc transferase governs spermatogenic mitotic-to-meiotic transition and progression by coordinating...

O-GlcNAcylation is a post-translational modification (PTM) uniquely catalyzed by O-GlcNAc transferase (OGT), which has been linked to tumorigenesis and neurodegeneration. However, its roles in mammalian spermatogenesis remain unexplored. This study aims to elucidate the functional mechanisms of OGT in spermatogenesis and male fertility.

We employed immunoprecipitation-mass spectrometry (IP-MS) to identify candidate O-GlcNAcylated substrates of OGT in juvenile mouse testes. To explore the physiological roles of OGT and O-GlcNAcylation, we constructed a mouse model with postnatal germ cell-specific deletion of Ogt via Stra8-Cre. In addition, we performed integrated bulk and single-cell RNA sequencing analyses to investigate the potential mechanisms by which OGT and O-GlcNAcylation deficiency impairs spermatogenesis.

The results showed stage-specific OGT enrichment and O-GlcNAcylation in mouse testicular spermatogonia and early spermatocytes. Furthermore, OGT was found to interact with and O-GlcNAcylate transcription factors (e.g., HCFC1) as well as splicing regulators (e.g., SRSF1 and SF3B3) in mouse testes. Postnatal germ cell-specific Ogt deletion impaired spermatogonial differentiation, disrupted meiotic initiation and progression, and induced apoptosis, ultimately leading to male infertility. Mechanistically, Bulk RNA sequencing (RNA-seq) analysis revealed that OGT deficiency dysregulated transcriptional and alternative splicing programs, affecting genes critical for the mitotic-meiotic transition (e.g., Ythdc2 and Rbm46) and meiotic progression (e.g., Stra8, Stag3, and Syce2) in the testes. Single-cell RNA sequencing further uncovered aberrant retention of mitotic transcripts (e.g., Ccna2 and Ccnb1) in spermatocytes and impaired mRNA metabolism during spermatogonial differentiation. In addition, OGT deficiency caused cytoplasmic mislocalization and reduced expression of core transcription factors and splicing regulators in spermatocytes.

These findings establish that OGT and its mediated O-GlcNAcylation coordinate essential gene expression and mRNA metabolism during mitotic-to-meiotic transition and meiotic progression. Moreover, our study provides mechanistic insights into the pathogenesis of male infertility associated with O-GlcNAcylation dysregulation.