TALEN-Mediated Gene Disruption on Y Chromosome Reveals Critical Role of EIF2S3Y in Mouse Spermatogenesis

Abstract

The Y chromosome plays a critical role in spermatogenesis. Formerly, it had been difficult to generate knockout mice with specific Y chromosome mutations using conventional gene-targeting strategies. Recently, a transcription activator-like effector nuclease (TALEN) was successfully used for editing a mouse Y chromosome-linked gene. Here, we report the generation of a mouse model with a mutation in EIF2S3Y, a Y chromosome-linked gene, and analysis of its phenotype. The mouse carrying a targeted mutation of EIF2S3Y was infertile and had hypoplastic testes. Histological and electron microscopic analyses showed that differentiation of spermatogonia was arrested at the stage of spermatogonial stem cells (undifferentiated spermatogonia) and that the progression of spermatogenesis was interrupted, resulting in azoospermia. Using TALEN, we verified that EIF2S3Y performs a key function in differentiation of spermatogonial stem cells.

FIG. 1.

Generation of a transcription activator-like effector nuclease (TALEN)-mediated gene knockout in mice. (A) The Eif2s3y TALEN recognition sequence. Light gray boxes in the schematic indicate DNA-binding domains of the TALEN and deep gray boxes indicate the N- and C-terminal domains. The FokI endonuclease is fused with the C-terminal domain. The DNA sequence to which TALENs bind is underlined. The start codon is in boldface. (B) Sequences of Eif2s3y obtained in the region of the TALEN-mediated Eif2s3y knockout in mice. Deleted nucleotides are indicated by hyphens. Mutant 1 is an inframe mutant, mutant 2 is a frameshift mutant, and mutant 3 as a mosaic of two distinct frameshift mutations (single-nucleotide deletion and 4-bp deletion). The ratio of mosaicism is shown after the genotype. Δ, deletion; bp, base pairs. (C) Sequences of Eif2s3x obtained in the region of the TALEN-mediated Eif2s3y knockout in mice. No off-target mutations were found in the genomic regions of Eif2s3x surrounding the sequence homologous to the TALEN target site. The DNA sequences homologous to the Eif2s3y TALEN-binding regions are underlined. The start codon is in boldface.

FIG. 2.

Testes of the Eif2s3y knockout mice. (A) Macrostructure of a testis. The testes of mutants 2 and 3 were smaller compared to other fertile male testes. The testis weight was approximately one-sixth of the weight in the other strains. The scale bar is 2 mm. (B) Histological analysis [hematoxylin and eosin (H&E) staining] of the testis. Testes of mutants 2 and 3 exhibited spermatogonial proliferation block. The scale bar is 50 μm. (C) Histological analysis (H&E stain) of the epididymis. Histological features of the epididymis in mutants 2 and 3 were similar to those of wild-type mice, but no sperm was observed in mutants 2 and 3. The scale bar is 50 μm.

FIG. 3.

Immunohistochemistry and transmission electron microscopy (TEM) of the Eif2s3y knockout mice. (A) Immunohistochemical analyses using staining for PH3 (red)/GFRα1 (green)/DAPI (blue), TRA98, MCA, or TRA54. Mutant 2 showed an increased GFRα1 signal, a lesser TRA98 signal, an abnormal MCA signal, and no TRA54 signal, suggesting that the differentiation of undifferentiated spermatogonia was arrested. GFRα1, TRA98, MCA, and TRA54 are markers of undifferentiated spermatogonia, testicular germ cells, spermatocytes, and spermatids, respectively. The scale bar is 50 μm. (B) TEM images. Mutant 3 showed the presence of undifferentiated spermatogonia and Sertoli cells. Undifferentiated spermatogonia had a nucleus with a mottled appearance of the heterochromatin. The scale bar is 2 μm.