Fig. 7: Mutations in NKAPL contribute to human non-obstructive azoospermia.

a NKAPL mutations identified in four patients with azoospermia. Three heterozygous missense mutations and a frameshift mutation were identified: heterozygous missense mutations and a frameshift mutation (c.1046_1047TGdel [p.M349fs, referred to as NKAPL^M349fs]). b NKAPL protein has an N-terminal arginine/serine-rich (RS) domain, repetitive basic sequences (the basic domain) and a C-terminal DUF926 domain of unknown functions. The deletion of TG base in NKAPL gene converts arginine 355 to a stop codon (R355*), thus causes a premature termination. c Sanger sequencing confirmed three heterozygous missense mutations and a frameshift mutation in azoospermia patients. d Western blot showing the presence of wild-type and truncated NKAPL proteins in testes from wild-type, Nkapl^+/M349fs heterozygotes and Nkapl^M349fs homozygotes at P21. Experiments were performed with biological duplicates. e, f Testicular atrophy in Nkapl^M349fs mice at 8-week-old. Testis sizes (e), testis weights (f) of Nkapl^+/+, Nkapl^+/M349fs and Nkapl^M349fs mice. n = 4 for Nkapl^+/+ and Nkapl^+/M349fs, n = 3 for Nkapl^M349fs. Data are presented as mean ± SD. p < 0.001, two-tailed unpaired Student’s t test. g Absence of sperm in Nkapl^M349fs males. n = 3, 8-week-old males. Data are presented as mean ± SD. * p < 0.05, **** p < 0.0001, two-tailed unpaired Student’s t test. h Histological analysis of Nkapl^+/+ and Nkapl^M349fs testes at 8 week-old. Apoptotic late stages of spermatocytes in stage XII tubules of Nkapl^M349fs are marked by black arrows. Nkapl^M349fs tubules were arrested either at late stages of spermatocytes (black arrows) or at the round spermatid stage (black lines). Scale bars, 20 μm. i RNA-seq analysis of differentially expressed genes in Nkapl^M349fs homozygotes compared with their wild-types at P21. Blue dots represent significantly downregulated transcripts, and red dots indicate upregulated transcripts (fold change ≥ 1.5, p < 0.05). DESeq2 with the Wald test method was used. j Venn diagram depicting the overlap between downregulated genes in Nkapl KO and Nkapl^M349fs homozygotes. k Downregulated genes in Nkapl KO (fold change ≥ 1.5, p < 0.05) with DESeq2 were used to produce a heatmap depicting their corresponding expression levels in Nkapl^M349fs homozygotes.