A homozygous loss-of-function mutation in the C-terminal of Tdrd5 causing early spermiogenesis arrest and azoospermia in mice

TDRD5 is a germ cell-specific RNA granule component that plays a crucial role in chromatoid body organization, piRNA biogenesis, and spermiogenesis. We previously identified a homozygous C-terminal nonsense mutation in TDRD5 in an infertile patient. While in vitro studies suggested the production of a stable truncated protein, the physiological consequences of this specific C-terminal truncation in vivo remain unknown.

This study aimed to elucidate the in vivo pathogenicity of the TDRD5 C-terminal nonsense mutation and its underlying mechanism using a mouse model.

We generated a Tdrd5 knock-in mouse carrying a premature termination mutation (Q764X) at the corresponding C-terminal region of murine TDRD5 using CRISPR/Cas9 gene editing. The reproductive phenotype was assessed through fertility testing, histological analysis (H&E, PAS), and evaluation of apoptosis (TUNEL). The expression of TDRD5 protein was assessed by immunoblot analysis, and retrotransposon activity was examined by co-staining for LINE1 and the DNA damage marker γH2AX.

Homozygous Tdrd5^Q764X/Q764X male mice exhibited infertility, reduced testis size, and azoospermia. Histological examination revealed spermiogenic arrest at the round spermatid stage with defective acrosome biogenesis. Contrary to in vitro findings, immunoblot analysis demonstrated the complete absence of both full-length and truncated TDRD5 proteins in Tdrd5^Q764X/Q764X testes. In addition, the mutant testes exhibited aberrant LINE1 upregulation, increased γH2AX signals in spermatocytes, and elevated germ cell apoptosis, thereby phenocopying the defects observed in Tdrd5^-/- mice.

Our findings demonstrated that the C-terminal nonsense mutation causes a loss-of-function phenotype in vivo. This study highlighted the indispensable role of the TDRD5 C-terminus in maintaining protein stability and ensuring successful spermiogenesis in mice.