ADAD2 functions in spermiogenesis and piRNA biogenesis in mice

Abstract

Background: 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.

Objectives: This study aimed to investigate the cause of male sterility in Adad2 mutant mice and to understand the molecular functions of ADAD2.

Materials and methods: 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.

Results: 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.

Discussion and conclusion: In collaboration with RNF17 and other RNA-binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.

Keywords: ADAD1; ADAD2; CRISPR/Cas9; RNA-binding proteins; infertility; male reproduction; piRNA biogenesis; sperm; spermatogenesis; testis.

Figure 1.. Adad2 is indispensable for male reproduction in mice.

(A) Multi-tissue RT-PCR analyses of mouse Adad1 and Adad2. La, ladder; He, heart; Li, liver; Sp, spleen; Lu, lung; Ki, kidney; Br, brain; St, stomach; In, intestine; Te, testis; Ov, ovary; Ut, uterus; Ep, epididymis. The expression of Hprt was analyzed in parallel as a loading control. (B) Postnatal testis RT-PCR analyses of mouse Adad1 and Adad2. The expression of Adad1 and Adad2 was analyzed in mouse testes from postnatal day 5 to 42. (C – D) Schematic representation of Adad2 null mouse generation by CRISPR/Cas9. Single guide RNAs (sgRNAs) #1 and #2, which target the first and the tenth exons, respectively, were used to delete the entire coding region of Adad2. Fw and Rv #1 primers were used to detect the wildtype allele, whereas Fw and Rv #2 primers were used to detect the null allele by genomic PCR. (E) Immunodetection of ADAD2 in wildtype and homozygous null testis lysates using mAb#19–8. BASIGIN was analyzed as a loading control. (F) Fertility tests of Adad2 null male mice. (G) Testis relative to body weight in wildtype and Adad2 null males. ns, no significant difference. (H) Observation of sperm extracted from cauda epididymis from wildtype and Adad2 null males.

Figure 2.. Adad2 deficient male mice show impaired spermiogenesis.

(A) Histological analyses on the testes and epididymides of Adad2^+/+, Adad2^–/–, and Adad2^Δ/Δ males. (B) Immunostaining of ADAD2 on the testis cryosection using the antibody against aa 1 – 93 of mouse ADAD2. Yellow arrowheads indicate pachytene spermatocytes with the ADAD2 signals. Yellow dotted lines represent the basement membrane of the seminiferous tubules.

Figure 3.. Ablation of ADAD2 causes dramatic transcriptomic changes in the male germline.

(A) An MA plot depicting differentially expressed genes in the testes from wildtype and Adad2^Δ/Δ male mice at 24 days postpartum. Each dot represents one gene; the x-axis and y-axis denotes the log2 value of mean expression level and fold change, respectively. Black, red, and blue indicates no significant difference, significant upregulation, and significant downregulation (more than 2-fold change), respectively. (B) RT-PCR analyses of Prm2, Stambp, and Tex101 in the spermatocytes (Sc) and spermatids (St) from Adad2^+/Δ and Adad2^Δ/Δ male mice. Gapdh was analyzed in parallel as a loading control. (C – D) RT-qPCR analyses of Stambp and Tex101 in the spermatocytes (Sc) and spermatids (St) from Adad2^+/Δ and Adad2^Δ/Δ male mice.

Figure 4.. ADAD2 interacts with RNA-binding proteins implicated in piRNA biogenesis.

(A) Co-IP/MS analysis of ADAD2-interacting proteins. Co-IP was performed using agarose resin crosslinked with the polyclonal antibody against aa 1 – 93 of mouse ADAD2. ADAD1, ADAD2, and RNF17 are detected with high specificity in wildtype testis lysate. (B) Representative RNA-binding proteins enriched in the ADAD2-associated proteome. Blue indicates the proteins potentially involved in both piRNA biogenesis and spermatogenesis, whereas green highlights the ones implicated in spermatogenesis. (C – E) Co-IP followed by Western blot analyses. Co-IP experiments were performed using magnetic beads conjugated with the antibodies against ADAD2 (pAb), MAEL, or MILI. The immunoprecipitated proteins were then analyzed by Western blot using antibodies against ADAD1, ADAD2 (mAb#19–8), MAEL, MILI, MIWI, RNF17, and YTHDC2. Red arrowheads indicate the protein bands specifically detected in the wildtype IP samples. (F) Immunohistochemistry analyses of ADAD2 and RNF17 in wildtype and Adad2^–/– testis cryosections. ADAD2 was immunodetected using mAb#19–8 and cell nuclei were visualized by Hoechst 33342.

Figure 5.. Depletion of ADAD2 leads to changes in the piRNA populations.

(A) Length distribution of small RNAs in Adad2^+/+ and Adad2^Δ/Δ testes. RPM, reads per million reads. (B) Distribution of the 1^st and 10^th nucleotides of piRNAs (24 – 35 nt in length) in Adad2^+/Δ and Adad2^Δ/Δ testes. (C) Expression of piRNAs in Adad2^+/+ and Adad2^Δ/Δ testes. LTR, long terminal repeat retrotransposons; LINE, non-LTR autonomous long interspersed element; SINE, non-LTR non-autonomous short-interspersed element. (D) Relative expression of cluster-derived and non-cluster-derived pachytene piRNAs bound to MILI or MIWI in Adad2^+/+ and Adad2^Δ/Δ testes.