Nine genes abundantly expressed in the epididymis are not essential for male fecundity in mice

Abstract

Background: Spermatozoa become competent for fertilization during transit through the epididymis. As spermatozoa from the proximal caudal epididymis can fertilize eggs, proteins from the caput and corpus epididymis are required for sperm maturation.

Objectives: Microarray analysis identified that more than 17,000 genes are expressed in the epididymis; however, few of these genes demonstrate expression restricted to the epididymis. To analyze epididymis-enriched gene function in vivo, we generated knockout (KO) mutations in nine genes that are abundantly expressed in the caput and corpus region of the epididymis.

Materials and methods: KO mice were generated using the CRISPR/Cas9 system. The histology of the epididymis was observed with hematoxylin and eosin staining. KO males were caged with wild-type females for 3-6 months to check fertility.

Results: We generated individual mutant mouse lines having indel mutations in Pate1, Pate2, or Pate3. We also deleted the coding regions of Clpsl2, Epp13, and Rnase13, independently. Finally, the 150 kb region encoding Gm1110, Glb1l2, and Glb1l3 was deleted to generate a triple KO mouse line. Histology of the epididymis and sperm morphology of all KO lines were comparable to control males. The females mated with these KO males delivered pups at comparable numbers as control males.

Discussion and conclusion: We revealed that nine genes abundantly expressed in the caput and corpus epididymis are dispensable for sperm function and male fecundity. CRISPR/Cas9-mediated KO mice generation accelerates the screening of epididymis-enriched genes for potential functions in reproduction.

Keywords: genetically modified mice; genome editing; sperm maturation.

Figure 1

Tissue expression analysis and conservation of target genes between some mammalian species. (A) RT‐PCR analysis. Actin beta (Actb) was used as the control CG: coagulating gland, SV: seminal vesicle. (B) Phylogenetic trees. The values under branches and parentheses show the distances and the lengths of the amino acid sequences, respectively.

Figure 2

Fecundity of KO males of Pate family genes. (A) Pate family genes within murine genomic locus |chromosome 9qA4|. (B) Genome editing efficiency of injecting gRNA/Cas9‐expressing plasmids into eggs. (C) DNA sequencing of KO mice of Pate family genes. Enzyme mutation (em) 1 for Pate1: 1 base ‘T’ insertion; em1 for Pate2: 4 base ‘TAGA’ deletion; em1 and em2 for Pate3: 1 base ‘T’ and 5 base ‘CTTCT’ deletion. Black boxes: coding region, Blue colored letters: initial methionine. (D) Sperm morphology observed under phase contrast. (E) Male fecundity. There was no difference in average litter size between control and KO males of each gene (Pate1: p = 0.57, Pate2: p = 0.80, Pate3: p = 0.76). Heterozygous males of each gene were used as controls. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Fecundity of Clpsl2 KO males. (A) Genome editing efficiency with gRNA/Cas9 RNPs. Black arrows: primers for genotyping, Black boxes: coding region, EP: electroporation. (B) Genotyping with PCR and DNA sequencing. Three primers (Fw, Rv#1, and Rv#2) were used for the PCR (also see panel A). em1: 2962 bp deletion. (C) Histological analysis with H&E staining. Dashed areas in left panels were enlarged (right panels). Scale bars on the left and right panels are 200 μm and 50 μm, respectively. (D) Sperm morphology observed under phase contrast. Scale bars: 50 μm. (E) Male fecundity. There was no difference in average litter size between WT and KO males (p = 0.20). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Fecundity of Epp13 KO males. (A) Genome editing efficiency with gRNA/Cas9 RNPs. Black arrows: primers for genotyping, Black boxes: coding region, (B) Genotyping with PCR and DNA sequencing. Three primers (Fw, Rv#1, and Rv#2) were used for the PCR (also see panel A). em1: 28,607 bp deletion. (C) Histological analysis with H&E staining. Dashed areas in left panels were enlarged (right panels). Scale bars on the left and right panels are 200 μm and 50 μm, respectively. (D) Sperm morphology observed under phase contrast. Scale bars: 50 μm. (E) Male fecundity. There was no difference in average litter size between WT and KO males (p = 0.28). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Fecundity of Rnase13 KO males. (A) Genome editing efficiency with gRNA/Cas9 RNPs. Black arrows: primers for genotyping, Black boxes: coding region. (B) Genotyping with PCR and DNA sequencing. Two primers were used for PCR (also see panel A). em1: 587 bp deletion, (C) Histological analysis with H&E staining. Dashed areas in left panels were enlarged (right panels). Scale bars on the left and right panels are 200 μm and 50 μm, respectively. (D) Sperm morphology observed under phase contrast. Scale bars: 50 μm. (E) Male fecundity. There was no difference in average litter size between WT and KO males (p = 0.51). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

Fecundity of Glb1l2‐Gm1110 KO mice. (A) Genome editing efficiency with gRNA/Cas9 RNPs. Black arrows: primers for genotyping, Black boxes: coding region. (B) Genotyping with PCR and DNA sequencing. Three primers (Fw#1, Fw#2, and Rv) were used for the PCR (also see panel A). em1: 156,758 bp deletion. (C) Histological analysis with H&E staining. Dashed areas in left panels were enlarged (right panels). Scale bars on the left and right panels are 200 μm and 50 μm, respectively. (D) Sperm morphology observed under phase contrast. Scale bars: 50 μm. (E) Male fecundity. There was no difference in average litter size between WT and KO males (p = 0.83). [Colour figure can be viewed at wileyonlinelibrary.com]