Mutation of Eif4g3, encoding a eukaryotic translation initiation factor, causes male infertility and meiotic arrest of mouse spermatocytes

Abstract

The ENU-induced repro8 mutation was identified in a screen to uncover genes that control mouse gametogenesis. repro8 causes male-limited infertility, with failure of spermatocytes to exit meiotic prophase via the G2/MI transition. The repro8 mutation is in the Eif4g3 gene, encoding eukaryotic translation initiation factor 4, gamma 3. Mutant germ cells appear to execute events of meiotic prophase normally, and many proteins characteristic of the prophase-to-metaphase transition are not obviously depleted. However, activity of CDC2A (CDK1) kinase is dramatically reduced in mutant spermatocytes. Strikingly, HSPA2, a chaperone protein for CDC2A kinase, is absent in mutant spermatocytes in spite of the presence of Hspa2 transcript, consistent with the observation that the repro8 phenotype is markedly similar to the phenotype of the Hspa2 knockout. Thus, EIF4G3 is required for HSPA2 translation in spermatocytes, a finding that provides the first genetic evidence for selective translational control of meiotic exit in mammalian spermatocytes.

Fig. 1.

PAS-stained testis sections at different developmental stages reveal meiotic arrest in repro8 mutants. (A,B) In adult males, spermatogonia, spermatocytes and spermatids are present in wild-type testes (A), whereas in mutant testes (B), only spermatogonia and spermatocytes were found. (C,D) At P14, no major differences were observed in spermatogenic cell content between wild-type (C) and repro8 mutant (D) testes. (E,F) By P17, repro8 mutant testes (F) exhibited more spermatocytes with highly condensed chromatin (arrow) compared with wild-type testes (E). (G,H) At P21, metaphase spermatocytes were present (G, arrow) in wild-type testes; however, no metaphase I spermatocytes were found in repro8 mutant testes (H) and many spermatocytes exhibited highly condensed chromatin. Scale bar: 100 μm.

Fig. 2.

Essential events in the prophase of meiosis I appear normal in repro8 mutants. (A,B) The extent of synapsis, reflected by the pattern of antibody labeling of SYCP1 (green), a central element protein of the synaptonemal complex (SC), and SYCP3 (red), a protein of the lateral element of the SC, was indistinguishable between wild-type (A) and repro8 mutant (B) spermatocytes. (C-F) The pattern of phosphorylated histone H2AFX (γH2AX) labeling, a marker of DNA double-strand breaks and repair, was identical between wild-type (C,E) and repro8 mutant (D,F) spermatocytes early in prophase (C,D) and at the mid-to-late pachytene stage (E,F). (G-L) Progress of recombination events was similar between wild-type and repro8 mutant spermatocytes. Labeling for RAD51, which accumulates at sites of double-strand breaks, was intense in early prophase (G,H), largely restricted to the X and Y chromosomes by the mid-pachytene stage (I,J), and similar in wild-type (G,I) and repro8 mutant (H,J) spermatocytes. MLH1 foci, marking sites of reciprocal recombination, were observed in similar numbers in both wild-type (K) and repro8 mutant (L) spermatocytes. Scale bar: 10 μm.

Fig. 3.

A mutation in Eif4g3 causes the repro8 phenotype. (A) The G-to-C mutation is marked by an open box. This mutation results in an amino acid change from alanine to proline. The sequencing result was confirmed by sequencing DNA from each of three repro8 mutant, repro8/+ heterozygous and B6 mice. (B) Alignment of conserved protein sequence domains of mouse and human EIF4G3 and fly eIF4G2; note that the fly eIF4G2 does not contain the W2 domain. The red arrow shows the position of the repro8 mutation. (C) A diagram of exons 1-4 and 33-36 of Eif4g3 transcript ENSMUST00000105831 showing the approximate locations of the gene trap (GT) construct in XC431 and the repro8 mutation (exons and introns in this panel not drawn to scale). (D) Representative testis cross-sections from repro8/+ and compound heterozygous repro8/Eif4g3^Gt males, revealing meiotic arrest in the latter, and thus demonstrating allelism of repro8 and Eif4g3. Scale bar: 100 μm.

Fig. 4.

RT-PCR reveals testis Eif4g3 transcripts. (A) RNA from the W2 domain affected by the repro8 mutation was amplified by RT-PCR at different developmental stages. m, Eif4g3^repro8 mutant; P, postnatal day; w, wild type; Wv, germ-cell-free Kit^w/Kit^wv testis. Size of the RT-PCR product is 192 bp. The internal control was Actb (actin B), with a PCR product of 240 bp. (B) Diagram of variant Eif4g3 transcripts annotated by Ensembl showing the position of primers for the RT-PCR presented in C, and identifying transcripts a and b displayed in C. (C) Evidence for testis-specific Eif4g3 transcripts; testis 1, adult testis; testis 2, P14 testis (B6); testis 3, P14 testis (C3H). Sizes of RT-PCR products: a, 313 bp; b, 280 bp; c, 184 bp; d, 151 bp.

Fig. 5.

CDC2A kinase activity is absent and Eif4g3^repro8 mutant spermatocytes are unable to undergo meiotic prophase I to metaphase I transition. Lysate for immunoprecipitation with anti-CDC2 was prepared from whole testes at P14, before obvious germ-cell loss in Eif4g3^repro8 mutant testes. (A) Results of the immunoprecipitation and kinase activity assay, with and without olumoucine, a selective inhibitor of CDC2A kinase. CDC2A kinase activity was present in the wild-type immunoprecipitate but absent in the Eif4g3^repro8 mutant immunoprecipitate and both olomoucine-treated controls. IP, immunoprecipitation; repro8, Eif4g3^repro8 mutant testes; WT, wild type. (B) After okadaic acid (OA) treatment to induce the prophase I to metaphase I transition, pachytene spermatocytes from wild-type spermatocytes form highly condensed and individualized metaphase I chromosomes, whereas Eif4g3^repro8 mutants (repro8) do not.

Fig. 6.

Protein components and activators of MPF are present in Eif4g3^repro8 mutant spermatocytes. (A-J) Immunohistochemistry (A,B,D,E,G,H) and western blotting (C,F,I,J) revealed that CDC2A (A-C), CCNB1 (D-F) and the MPF activator CDC25B (G-I) were present in both wild-type (A,D,G) and Eif4g3^repro8 mutant (B,E,H) spermatocytes. TUBA (tubulin alpha) was detected as a loading control for western blots (J). Scale bar: 100 μm.

Fig. 7.

HSPA2 is absent, but Hspa2 transcript present, in Eif4g3^repro8 mutant spermatocytes. (A-F) Both immunohistochemistry (A, wild type; B, Eif4g3^repro8 mutant) and western blotting (C, wild type and Eif4g3^repro8 mutant; E, repro8/+ heterozygote and repro8/Eif4g3^Gt heterozygote) revealed the presence of HSPA2 in wild-type spermatocytes, but its absence in Eif4g3^repro8 mutant and repro8/Eif4g3^Gt heterozygous spermatocytes. TUBA was used as a loading control (D,F). (G) Hspa2 transcript was detected by RT-PCR at different developmental stages. P, postnatal day; m, Eif4g3^repro8 mutant; w, wild type). The size of the Hspa2 PCR product is 575 bp. The internal control was Actb (actin B), with a PCR product of 240 bp. Scale bar: 100 μm