Multiple genes in the Pate5-13 genomic region contribute to ADAM3 processing†

Abstract

Sperm proteins undergo post-translational modifications during sperm transit through the epididymis to acquire fertilizing ability. We previously reported that the genomic region coding Pate family genes is key to the proteolytic processing of the sperm membrane protein ADAM3 and male fertility. This region contains nine Pate family genes (Pate5-13), and two protein-coding genes (Gm27235 and Gm5916), with a domain structure similar to Pate family genes. Therefore, in this study, we aimed to identify key factors by narrowing the genomic region. We generated three knockout (KO) mouse lines using CRISPR/Cas9: single KO mice of Pate10 expressed in the caput epididymis; deletion KO mice of six caput epididymis-enriched genes (Pate5-7, 13, Gm27235, and Gm5916) (Pate7-Gm5916 KO); and deletion KO mice of four genes expressed in the placenta and epididymis (Pate8, 9, 11, and 12) (Pate8-12 KO). We observed that the fertility of only Pate7-Gm5916 KO males was reduced, whereas the rest remained unaffected. Furthermore, when the caput epididymis-enriched genes, Pate8 and Pate10 remained in Pate7-Gm5916 KO mice were independently deleted, both KO males displayed more severe subfertility due to a decrease in mature ADAM3 and a defect in sperm migration to the oviduct. Thus, our data showed that multiple caput epididymis-enriched genes within the region coding Pate5-13 cooperatively function to ensure male fertility in mice.

Keywords: UTJ migration; genome editing; knockout mice.

Graphical Abstract

Figure 1

Pate10 single KO males are fertile. (A) gRNA design. Black boxes, arrow, and red arrowhead indicate the protein-coding regions, primers for genotyping, and a guide RNA (gRNA), respectively. (B) Genotyping PCR. The primers shown in Panel A were used for genotyping. WT: wild type, Het: heterozygous, KO: knockout. (C) DNA sequencing. The KO allele disrupted 294 bases containing exon1 of Pate10 and introns before and after exon1. Underlined letters show the first Met in exon1. (D) Observation of Pate10 transcripts. Pate10 transcript between exons 2 and 3 was amplified. Genomic DNA was used to check for contamination of genome DNA in the epididymal cDNA. Actin beta (Actb) was used as a loading control. (E) Male fertility. ns: not significant (Mann–Whitney test).

Figure 2

Deletion KO males lacking six caput epididymis-enriched genes in the Pate7-Gm5916 region are subfertile. (A) gRNA design. Protein-coding genes within the murine genomic region between Pate10 and Pate8 are listed. Gm27235 and Gm5916 have domain structures similar to Pate family genes. Gm3434 and Gm48391 are a non-coding gene after Gm5916 and a pseudogene before Pate12, respectively. Black boxes, arrows, and red arrowheads show protein-coding regions, primers for genotyping, and gRNAs, respectively. (B) Genotyping PCR. Primers shown in Panel A were used for genotyping. (C) DNA sequencing. Pate7-Gm5916 KO alleles lacked 359 128 bases (chr 9, 35 687 794–36 046 921), leading to the disruption of six caput epididymis-enriched genes in the Pate7-Gm5916 region. Pate8-Pate12 KO alleles lacked 333 086 bases (chr 9, 36 161 130–36 494 215), leading to the disruption of four genes expressed in the placenta (Pate11 and 12) and epididymis (Pate8 and 9) in the Pate8–12 region. The underlined letters show the substituted bases. (D) Male fertility. Pate7-Gm5916 Het and Pate8-Pate12 Het mice were used as controls (ctrl). Statistical analyses between control and mutant mice were performed (^*P< 0.05, ns: not significant) (Kruskal–Wallis test). The fertility of Pate7-Gm5916 KO males was significantly reduced. (E) Southern blot hybridization. The probe design was shown in Supplementary Figure S3. Bands of the predicted size were detected in Pate7-Gm5916 mutants. (F) Detection of ADAM3. Sperm protein from each genotype (10 μg) was used for western blotting. Mature ADAM3 decreased in Pate7-Gm5916 KO sperm. IZUMO1, a membrane protein of the sperm head, was used as the loading control. TGC: testicular germ cells.

Figure 3

Deletion KO males lacking both Pate8 (or Pate10) and the Pate7-Gm5916 regions exhibited more severe subfertility. (A) gRNA design. To delete the remaining Pate8 and 10, predominantly expressed in the caput epididymis, from the Pate7-Gm5916 KO allele, we designed new gRNAs. Black boxes, arrows, and arrowheads indicate protein-coding regions, primers for genotyping, and gRNAs, respectively. (B) Genotyping PCR. Primers shown in Panel A were used for genotyping. (C) DNA sequencing. We additionally deleted 1389 bases (chr 9, 35 740 915–35 742 303) and 1411 bases (chr 9, 36 492 536–36 493 946) in Pate10 and Pate8 at Pate7-Gm5916 KO allele, respectively. The lower-case letters show the inserted bases. (D) Male fertility. Pate10 Het + Pate7-Gm5916 Het and Pate8 Het + Pate7-Gm5916 Het males were used as the ctrl. Statistical analysis between ctrl and mutant mice was performed (^**P < 0.01, Kruskal–Wallis test). Deletion KO males lacking both Pate8 (or Pate10) and the Pate7-Gm5916 regions were almost infertile. (E) Detection of ADAM3. Sperm protein from each genotype (20 μg) was used for western blotting. Mature ADAM3 almost disappeared from the cauda epididymal sperm of both KO males. IZUMO1, a membrane protein of the sperm head, was used as the loading control. TGC: testicular germ cells.

Figure 4

Quantitative comparison of ADAM3 in Pate mutant sperm and observation of sperm migration to an oviduct. (A) Detection of ADAM3 and IZUMO1. Sperm proteins from each genotype (10 μg) were transferred onto the same membrane, and then ADAM3 and IZUMO1 were detected. (B) Quantification of ADAM3. The signal intensity of each band in panel A was measured using ImageJ. ADAM3 in Pate8 (or Pate10) KO + Pate7 − Gm5916 KO sperm was further decreased compared to that in Pate7 − Gm5916 KO sperm. (C) Observation of sperm migration in the female reproductive tract. Male mutant mice with fluorescent-tagged sperm were mated with WT females. The right panels show enlarged photographs of the yellow dotted box in the left panels. Few sperm from Pate8 (or Pate10) KO + Pate7-Gm5916 KO males were observed in the oviduct (yellow arrows), compared to the control.