Nuclear localization of EIF4G3 suggests a role for the XY body in translational regulation during spermatogenesis in mice - PubMed (original) (raw)

Nuclear localization of EIF4G3 suggests a role for the XY body in translational regulation during spermatogenesis in mice

Jianjun Hu et al. Biol Reprod. 2018.

Abstract

Eukaryotic translation initiation factor 4G (EIF4G) is an important scaffold protein in the translation initiation complex. In mice, mutation of the Eif4g3 gene causes male infertility, with arrest of meiosis at the end of meiotic prophase. This study documents features of the developmental expression and subcellular localization of EIF4G3 that might contribute to its highly specific role in meiosis and spermatogenesis. Quite unexpectedly, EIF4G3 is located in the nucleus of spermatocytes, where it is highly enriched in the XY body, the chromatin domain formed by the transcriptionally inactive sex chromosomes. Moreover, many other, but not all, translation-related proteins are also localized in the XY body. These unanticipated observations implicate roles for the XY body in controlling mRNA metabolism and/or "poising" protein translation complexes before the meiotic division phase in spermatocytes.

Keywords: meiosis; nucleus; sex chromosomes; spermatocyte; translational regulation.

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Figures

Figure 1.

Figure 1.

Expression of Eif4g1 and Eif4g3 and their cellular localization during spermatogenesis. (A) Quantitative RT-PCR analysis of mRNA levels of Eif4g1 and Eif4g3 in testes from 7 to 17 dpp. Each analysis was performed twice, and representative results are shown. Gapdh was used as an internal control. (B) Western-blot analysis of protein levels of EIF4G1 and EIF4G3 in testis during the first wave of spermatogenesis. Total protein extracts were prepared from whole testes of mice at age of 8–45 dpp. The loading control is TUBA1A; position of molecular weight markers (kDa) is shown to the right. (C) The top two panels show immunohistochemical labeling of EIF4G1 and EIF4G3 of seminiferous tubule cross sections, with boxed area in higher magnification in the lower two panels, showing details of cellular localization of EIF4G1 and EIF4G3. E = elongating spermatids; R = round spermatids; P = pachytene spermatocytes, S = spermatogonia. Bars = 40 μm.

Figure 2.

Figure 2.

Cap-analog pull-down and co-immunoprecipitation (Co-IP) analyses of interaction of EIF4G3 with other proteins. (A) Cap-analog pull-down analysis with whole testis protein extracts from adult wild-type (+/+) and Eif4g3repro8/repro8 mutant (–/–) mice. The γ-aminophenyl m7GTP cap analog was used as bait to pull-down cap-binding protein complexes. Sepharose 4B was used as a negative control. The pull-down samples were analyzed by western blotting with mixture of EIF4G3, EIF4A2 and EIF4E antibodies. (B) Co-IP using anti-EIF4E and anti-EIF4G3 antibodies with whole testis protein extracts from adult wild-type (+/+) and _Eif4g3repro8/repro8_mutant (–/–) mice followed by western blot analysis with EIF4G3, EIF4A2, and EIF4E antibodies. (C-D) Reciprocal co-IP using EIF4G3 (C) and ELAVL1 (D) antibodies with whole testis protein extracts from adult wild-type (+/+, at two concentrations) and Eif4g3repro8/repro8 (–/–) mutant mice, followed by western blot analysis with EIF4G3 and ELAVL1 antibodies. (E) IP analysis using anti-EIF4G3 antibody for immunoprecipitation from testis extracts of adult wild type (+/+), followed by western blotting with anti-PABPC1. (F) IP analysis using anti-EIF4G3 antibody for immunoprecipitation from testis extracts of adult wild-type (+/+), Eif4g3repro8/repro8 mutant (–/–) and heterozygous (+/–) mice, followed by western blotting with DAZL antibody. Molecular weights (kDa), inferred from markers co-electrophoresed, are shown to the right of each blot.

Figure 3.

Figure 3.

Immunofluorescence labeling of EIF4G3, EIF4G1, and meiotic prophase markers in spermatocytes. (A) EIF4G3 labeling in meiotic prophase cells also labeled with antibody to SYCP3 (synaptonemal complex protein) and DAPI (DNA) to identify meiotic prophase substages. EIF4G3 is highly enriched in the XY body (circled) during pachynema. (B) EIF4G1 labeling in meiotic prophase cells also labeled with antibody to SYCP3 and with DAPI. EIF4G1 is not reliably detected in the nucleus, or in the XY body (circled in far right panel). (C) Triple labeling of pachytene spermatocytes for EIF4G3, SYCP3, and HIST1H1T (histone H1t, a protein marker of mid-to-late pachynema). EIF4G3 is strong in mid-to-late pachytene spermatocytes (HIST1H1T high) and is negative or weak in early pachytene spermatocytes (HIST1H1T negative or weak). (D) Double labeling of pachytene spermatocytes for EIF4G3 and pH2AFX (phosphorylated histone H2AFX) and ATR (ATM-related protein), proteins known to localize to the XY body. (E) Double labeling of pachytene spermatocytes for EIF4G3 and HIST1H1T and POLR2A (RNA polymerase 2A), proteins known to not specifically localize to the XY body during pachynema. (F) Immunolabeling of EIF4G3 in late prophase spermatocytes isolated from Eif4g3repro8/repro8 mutant and heterozygous testes. (G) Immunolabeling with anti-EIF4G3 (Bethyl) in late prophase spermatocytes isolated from Eif4g3exon5/exon5 mutant lacking the conserved and testis-specific exon 5 and labeling of EIF4G3 in late prophase spermatocytes isolated from wild-type mice using the EIF4G3 exon5-specific antibody (Handel Lab/Thermo). (H) Co-immunolabeling of EIF4G3 and pH2AFX in Spo11tm1Mjn/J heterozygous and mutant spermatocytes. Left: anti-EIF4G3 staining co-localizes with anti-pH2AFX in the XY body in late spermatocytes isolated from Spo11tm1Mjn/J heterozygous. Right: anti-EIF4G3 staining is not co-localized with the pH2AFX-labeled pseudo-sex body or with any other domain in the Spo11tm1Mjn/J mutant. Bars = 5 μm.

Figure 4.

Figure 4.

Some translational factors are relatively enriched in the XY body of pachytene spermatocytes. (A-D) EIF4F complex members EIF4A1, EIF4E, EIF4E2, and phosphorylated EIF4E were found in the XY body. (E-H) Small ribosomal subunit protein RPS6 and large ribosomal subunit protein RPL10L, and RNA-binding proteins MSI1 and TSN were relatively enriched in the XY body. (I-L) Translational activators MKNK1 and MKNK2, and repressors EIF4EBP1 and EIF2C4 (AGO4), were found in the XY body. Bars = 5 μm.

Figure 5.

Figure 5.

Relative enrichment of HSPA2 and cell-cycle regulators in the XY body. (A) HSPA2, a target of Eif4g3repro8 mutant, was detected on chromosomal axes, including the X-Y axes, in spermatocyte nuclei. (B-E) Relative enrichment of cell-cycle regulators in the XY body. Phosphorylated CDK1, CDK2, CHEK1, and CHEK2 were all relatively enriched in the XY body and/or XY synaptonemal complex of spermatocytes. The boxed regions of these color-merged panels are shown in single color of red and green in the bottom row. Bars = 5 μm.

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