Translational control of meiotic cell cycle progression and spermatid differentiation in male germ cells by a novel eIF4G homolog

Abstract

Translational control is crucial for proper timing of developmental events that take place in the absence of transcription, as in meiotic activation in oocytes, early embryogenesis in many organisms, and spermatogenesis. Here we show that a novel form of the translation initiation complex component eIF4G in Drosophila, eIF4G2, is required specifically for male germ cells to undergo meiotic division and proper spermatid differentiation. Flies mutant for eIF4G2 are viable and female fertile but male sterile. Spermatocytes form, but the germ cells in mutant males skip the major events of the meiotic divisions and form aberrant spermatids with large nuclei. Consistent with the failure to undergo the meiotic divisions, function of eIF4G2 is required post-transcriptionally for normal accumulation of the core cell cycle regulatory proteins Twine and CycB in mature spermatocytes. Loss of eIF4G2 function also causes widespread defects in spermatid differentiation. Although differentiation markers Dj and Fzo are expressed in late-stage eIF4G2 mutant germ cells, several key steps of spermatid differentiation fail, including formation of a compact mitochondrial derivative and full elongation. Our results suggest that an alternate form of the translation initiation machinery may be required for regulation and execution of key steps in male germ cell differentiation.

Fig. 1. eIF4G2 encodes a novel eIF4G homolog in Drosophila

(A) Alignments of Drosophila eIF4G and eIF4G2 proteins with human eIF4GI (EIF4G1 – HUGO) and eIF4GII (EIF4G3 – HUGO). Black sections indicate recognized domains, with percentage identical to the eIF4GI middle domain marked. (B) Myc-eIF4E1 co-immunoprecipitates with HA-eIF4G2 from S2 cells. Immunoprecipitation with anti-HA. Western blot probed with anti-Myc and anti-HA, as indicated. Crude extract is 1/10 of each pre-immunoprecipitation (IP) sample.

Fig. 2. Loss-of-function mutations in eIF4G2 result in male sterility

(A) Diagram of eIF4G2 intron/exon structure and four strong loss-of-function alleles. Gray, protein coding sequence; black, untranslated regions; diagonal lines, region encoding conserved middle domain. (B) Apical third of a wild-type testis. (C) Apical third of a testis from an eIF4G2 homozygote. (D) Part of testis from eIF4G2 homozygote. (E) Part of testis from eIF4G2 homozygote carrying a single copy of the genomic rescue transgene. eIF4G2 mutant flies were eIF4G2^BR21-37/eIF4G2^Z3-3283. Scale bars: 100 μm.

Fig. 3. eIF4G2 is expressed in a stage-specific pattern within the testis

(A–D) In situ hybridization on wild-type testes; (A) eIF4G2 antisense, (B) eIF4G2 sense, (C) eIF4G antisense, (D) eIF4G sense. (E,F) Anti-HA immunostaining on testes from flies carrying the HA-eIF4G2-rescuing transgene. (E) Spermatocytes (arrow) and elongated spermatids (arrowhead). (F) Early post-meiotic spermatids (arrowhead). (G) Anti-HA staining on a non-transgenic testis (yw). Scale bars: 100 μm.

Fig. 4. eIF4G2 is required in the testis for meiotic division

Live squashes of wild-type and mutant germ cells undergoing meiosis. (A,C,E,G,I) Phase contrast. (B,D,F,H,J) Hoechst staining. (A,B) Wild-type and (C,D) eIF4G2 mature spermatocytes. (E,F) Wild-type spermatocytes with condensed chromosomes. (G,H) eIF4G2 spermatocytes with partially condensed chromosomes. (I,J) Wild-type cells in metaphase (arrows) and anaphase (arrowheads). All images are at the same magnification. Scale bar: 20 μm. eIF4G2 mutant flies were eIF4G2^BR21-37/eIF4G2^Z3-3283.

Fig. 5. eIF4G2 is required for translation of twine and cycB in spermatocytes

(A,B) Anti-CycB immunofluorescence on (A) wild-type and (B) eIF4G2 mutant testes. Bracket indicates expression of CycB in mature spermatocytes in wild type. (C,D) X-gal staining of (C) wild-type and (D) eIF4G2 mutant testes expressing a twine-lacZ reporter. (E,F) In situ hybridization on wild-type (E) and eIF4G2 (F) testes with cycB antisense probe. (G,H) In situ hybridization on wild-type (G) and eIF4G2 (H) testes with twine antisense probe. Scale bars: 100 μm. eIF4G2 mutant flies were eIF4G2^BR21-37/eIF4G2^Z3-3283.

Fig. 6. eIF4G2 is required for proper spermatid differentiation

Live squashes of wild-type and mutant germ cells initiating spermatid differentiation. (A,C,E,G,I,K) Phase contrast. (B,D,F,H,J,L) Hoechst staining. (A,B) Wild-type early round spermatids with haploid nuclei (arrow) and mitochondrial derivative (arrowhead). (C,D) Elongating spermatids in wild type. (E,F) Late-stage eIF4G2 cells with large nuclei (arrow) and aggregating mitochondria (arrowhead). (G,H) Terminal cells in eIF4G2 with aggregated mitochondria (arrowhead), large nuclei (arrow) and aberrant partial cellular elongation. (I,J) twine early spermatids. (K,L) twine elongating spermatids. All images are at the same magnification. Scale bar: 20 μm. eIF4G2 mutant flies were eIF4G2^BR21-37/eIF4G2^Z3-3283. twine mutant flies were twe^HB5/twe^K08310.

Fig. 7. eIF4G2 is not required for the translation of the spermatid differentiation markers fzo and dj

(A–D) Anti-tubulin immunostaining of squashed preparations of (A,B) wild-type and (C,D) eIF4G2 testes. (A,C) DAPI; (B,D) anti-Tub. (E–H) Anti-Fzo immunostaining (F,H) and DAPI (E,G) on squashed preparations of (E,F) wild-type and (G,H) eIF4G2 testes. Arrows indicate location of nuclei; arrowheads mark mitochondrial derivatives (F) or mitochondrial aggregates (H). (I,J) Dj-GFP reporter in (I) wild-type and (J) eIF4G2 testes. Arrowheads indicate Dj-GFP in J. Scale bars: 20 μm. eIF4G2 mutant flies were eIF4G2^BR21-37/eIF4G2^Z3-3283.