biostudies-literatureLu YEunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNICHD NIH HHSTakeda Science FoundationBill and Melinda Gates Foundation698-709https://www.ebi.ac.uk/biostudies/studies/S-EPMC10073342biostudies-literatureUnknown11(4)<h4>Background</h4>Adenosine deaminase domain containing 2 (ADAD2) is a testis-specific protein composed of a double-stranded RNA binding domain and a non-catalytic adenosine deaminase domain. A recent study showed that ADAD2 is indispensable for the male reproduction in mice. However, the detailed functions of ADAD2 remain elusive.<h4>Objectives</h4>This study aimed to investigate the cause of male sterility in Adad2 mutant mice and to understand the molecular functions of ADAD2.<h4>Materials and methods</h4>Adad2 homozygous mutant mouse lines, Adad2^-/- and Adad2^Δ/Δ , were generated by CRISPR/Cas9. Western blotting and immunohistochemistry were used to reveal the expression and subcellular localization of ADAD2. Co-immunoprecipitation tandem mass spectrometry was employed to determine the ADAD2-interacting proteins in mouse testes. RNA-sequencing analyses were carried out to analyze the transcriptome and PIWI-interacting RNA (piRNA) populations in wildtype and Adad2 mutant testes.<h4>Results</h4>Adad2^-/- and Adad2^Δ/Δ mice exhibit male-specific sterility because of abnormal spermiogenesis. ADAD2 interacts with multiple RNA-binding proteins involved in piRNA biogenesis, including MILI, MIWI, RNF17, and YTHDC2. ADAD2 co-localizes and forms novel granules with RNF17 in spermatocytes. Ablation of ADAD2 impairs the formation of RNF17 granules, decreases the number of cluster-derived pachytene piRNAs, and increases expression of ping-pong-derived piRNAs.<h4>Discussion and conclusion</h4>In collaboration with RNF17 and other RNA-binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.AndrologyADAD2 functions in spermiogenesis and piRNA biogenesis in mice.PMC10073342R01HD088412R01 HD088412INV‐001902P01 HD087157P01HD087157Castaneda JMLu YChang HYKobayashi HKojima-Kita KShirane KMatsuyama MKuramochi-Miyagawa SKoyano TNagamori IMatzuk MMYu ZIkawa MNishimura TfalseADAD2 functions in spermiogenesis and piRNA biogenesis in mice.<h4>Background</h4>Adenosine deaminase domain containing 2 (ADAD2) is a testis-specific protein composed of a double-stranded RNA binding domain and a non-catalytic adenosine deaminase domain. A recent study showed that ADAD2 is indispensable for the male reproduction in mice. However, the detailed functions of ADAD2 remain elusive.<h4>Objectives</h4>This study aimed to investigate the cause of male sterility in Adad2 mutant mice and to understand the molecular functions of ADAD2.<h4>Materials and methods</h4>Adad2 homozygous mutant mouse lines, Adad2^-/- and Adad2^Δ/Δ , were generated by CRISPR/Cas9. Western blotting and immunohistochemistry were used to reveal the expression and subcellular localization of ADAD2. Co-immunoprecipitation tandem mass spectrometry was employed to determine the ADAD2-interacting proteins in mouse testes. RNA-sequencing analyses were carried out to analyze the transcriptome and PIWI-interacting RNA (piRNA) populations in wildtype and Adad2 mutant testes.<h4>Results</h4>Adad2^-/- and Adad2^Δ/Δ mice exhibit male-specific sterility because of abnormal spermiogenesis. ADAD2 interacts with multiple RNA-binding proteins involved in piRNA biogenesis, including MILI, MIWI, RNF17, and YTHDC2. ADAD2 co-localizes and forms novel granules with RNF17 in spermatocytes. Ablation of ADAD2 impairs the formation of RNF17 granules, decreases the number of cluster-derived pachytene piRNAs, and increases expression of ping-pong-derived piRNAs.<h4>Discussion and conclusion</h4>In collaboration with RNF17 and other RNA-binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.2023-01-01T00:00:00Z2023 May2026-04-21T03:18:27.922Z2026-04-21T03:13:51.92ZS-EPMC100733423669824910.1111/andr.13400