Biallelic mutations in RNA-binding protein ADAD2 cause spermiogenic failure and non-obstructive azoospermia in humans

• Published in: Human reproduction open 2023, Vol.2023 (3), p.hoad022-hoad022
• Main Authors: Shi, Baolu, Shah, Wasim, Liu, Li, Gong, Chenjia, Zhou, Jianteng, Abbas, Tanveer, Ma, Hui, Zhang, Huan, Yang, Menglei, Zhang, Yuanwei, Ullah, Nadeem, Mahammad, Zubair, Khan, Mazhar, Murtaza, Ghulam, Ali, Asim, Khan, Ranjha, Sha, Jiahao, Yuan, Yan, Shi, Qinghua
• Format: Article
• Language: English
• Subjects: Binding proteins; Genetic aspects; Health aspects; Infertility, Male; Mutation (Biology); Original
• ISSN: 2399-3529; 2399-3529
• DOI: 10.1093/hropen/hoad022

Abstract STUDY QUESTION What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis? SUMMARY ANSWER Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice. WHAT IS KNOWN ALREADY NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification. STUDY DESIGN, SIZE, DURATION Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2Mut/Mut mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and Adad2Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2WT/Mut male mice and six Adad2WT/Mut female mice. A total of 120 Adad2Mut/Mut, Adad2WT/Mut, and WT mice were used in this study. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS, SETTING, METHODS Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages. MAIN RESULTS AND