Fig. 1. Deficiency of X-linked TENT5D causes male infertility by disrupting mRNA stability during spermatogenesis.

a Pedigree of family HX001 affected by a stop-gain TENT5D variant. The black-filled square indicates the male case with oligoasthenoteratozoospermia. Sanger sequencing confirmed the hemizygous TENT5D variant in proband HX001 II-1. Schematic representation of TENT5D and phylogenetic conservation of the variant location. b, c SEM (b) and TEM (c) analyses of the spermatozoa from a male control individual and the TENT5D-associated proband. Scale bars, 5 μm. d Expression of Tent5d was investigated by reverse-transcription PCR in various tissues from adult male mice. Gapdh was used as an internal control. e, f The sperm count (e) and percentages of motile sperm (f) in WT (Tent5d⁺/Y) and Tent5d-mutated (Tent5d⁻/Y) male mice (**P < 0.01, ***P < 0.001). Error bars represent the standard error of the mean. Two-tailed Student’s paired or unpaired t-tests were used as appropriate. g H&E staining performed on testis sections of Tent5d⁺/Y and Tent5d⁻/Y male mice. Scale bars, 20 μm. h PAS staining and immunostaining assays were performed on adult mouse testis sections. Scale bars, 20 μm. i Volcano plots of the transcriptome analysis showing the expression changes of mRNAs in spermatocytes and round spermatids of MU and WT male mice. j PCR-based poly(A) test of Clu, Cst9, Cst12, Defb19 and Gkap1 mRNAs in the testes of MU and WT male mice. The downregulated genes were marked in pink. k Schematic model showing that TENT5D stabilizes mRNAs by extending the length of poly(A) tails in spermatocytes and round spermatids. l Representative development of mouse embryos. The offspring was obtained by “FACS + ROSI” using round spermatids from Tent5d⁺/Y and Tent5d⁻/Y male mice.