Loss-of-function analysis reveals distinct requirements of the translation initiation factors eIF4E, eIF4E-3, eIF4G and eIF4G2 in Drosophila spermatogenesis

Abstract

In eukaryotes, post-transcriptional regulation of gene expression has a key role in many cellular and developmental processes. Spermatogenesis involves a complex developmental program that includes changes in cell cycle dynamics and dramatic cellular remodeling. Translational control is critical for spermatogenesis in Drosophila as many mRNAs synthesized in the spermatocytes are translated only much later during spermatid differentiation. Testes-specific translation initiation factors eIF4E-3 and eIF4G2 are essential specifically for male fertility. However, details of their roles during different stages of spermatogenesis are unknown, and the role of canonical translation initiation factors in spermatogenesis remains unexplored. In this study, we addressed the functional role of eIF4E-1, eIF4E-3, eIF4G and eIF4G2 in testes development and formation of mature sperm. Using the UAS-Gal4 system and RNA interference, we systematically knocked down these four genes in different stages of germ cell development, and in the somatic cells. Our results show that eIF4E-1 function in early germ cells and the surrounding somatic cells is critical for spermatogenesis. Both eIF4E-1 and eIF4E-3 are required in spermatocytes for chromosome condensation and cytokinesis during the meiotic stages. Interestingly, we find that eIF4G knockdown did not affect male fertility while eIF4G2 has distinct functions during spermatogenesis; it is required in early germ cells for proper meiotic divisions and spermatid elongation while its abrogation in spermatocytes caused meiotic arrest. Double knockdown of eIF4G and eIF4G2 shows that these proteins act redundantly during the early stages of spermatogenesis. Taken together, our analysis reveals spatio-temporal roles of the canonical and testes-specific translation initiation factors in coordinating developmental programs during spermatogenesis.

Fig 1. Distribution of eIF4E-1, eIF4E-3, eIF4G and eIF4G2 in the wild-type testes.

(A) Schematic diagram showing the stages of spermatogenesis in Drosophila testes. The hub cells (HC) at the apical tip of the testes maintain the stem cells (SC) which include a germline stem cell and a somatic cyst stem cell. The germline stem cell differentiates into spermatogonia (SG) which divide mitotically to produce primary spermatocytes (PSC). The spermatocytes undergo rapid cellular growth to form mature spermatocytes (MSC) that undergo meiosis, producing haploid onion stage spermatids (OSS). Following cellular transformation and differentiation, they develop flagella with the nuclei (magenta) at the distal end of the testes. ES = Elongated spermatids. Whole-mount immunostaining of adult testes using antibodies that recognise eIF4E-1 (B-B'', green), eIF4E-3 (C-C'', red), eIF4G (D-D'', green) and eIF4G2 (E-E'', green). The top panel (B-E) shows the entire testes while the middle (B'-E') and lower panels (B''-E'') show the apical and distal end, respectively. DAPI is shown in magenta. Scale bar 100 μm.

Fig 2. Knockdown of eIF4E-1 and eIF4E-3 affects testes development and spermatid differentiation.

Wild-type testes (A-A''') and testes expressing eIF4E-1 shRNA with nos-Gal4:VP16 driver (B-B''') were stained with anti-eIF4E-3 (red, A, B) and anti-eIF4G antibody (green, A', A'' & B', B''). The small rudimentary testes are marked by arrows while the accessory glands are marked by arrowheads (B, B'). A magnified view of the apical tip of eIF4E-1 knockdown testes shows a mass of undifferentiated cells surrounded by a disorganised muscle sheath (B'', B''') as compared with the wild-type (A'', A'''). The DNA is stained with DAPI (magenta). Scale bars 100 μm (A, A', B, B') and 50 μm (A''', B'''). Expression of eIF4E-1 shRNA using c587-Gal4 driver results in a degenerate testes structure (C', arrows) as revealed by phase contrast microscopy. The arrowheads indicate the accessory glands. The wild-type testis is shown in C. Scale bar 100 μm. (D) The distal end of wild-type testes, stained with anti-eIF4E-1 (green) and anti-eIF4E3 (red) antibody shows well organized haploid nuclear bundles (magenta, arrows). A similar region of a bam-Gal4-eIF4E-1 RNAi testis (D', D'') co-stained with anti-eIF4E-1 (green) and anti-eIF4E-3 (red) antibody reveals severe defects in nuclear compaction and individualisation; the post-meiotic nuclei (stained with DAPI, magenta) are found along the elongated spermatids (arrows). Scale bar 100 μm. (D''', D'''') Knockdown of eIF4E-3 in spermatocytes using bam-Gal4:VP16 driver results in loss of nuclear condensation and bundling at the distal end of the testes (arrows). eIF4E-3 and eIF4E-1 immunostaining is shown in red and green, respectively. The DNA is stained with DAPI (magenta). Scale bar 100 μm.

Fig 3. Distribution of Don Juan-GFP and Orb in eIF4E-1 and eIF4E-3 knockdown testes.

GFP fluorescence of Don Juan-GFP (DJ-GFP) in the wild-type background (A) or testes expressing eIF4E-1 (B) and eIF4E-3 (C) shRNA under the bam-Gal4:VP16 driver. Whole mount anti-Orb staining (green) of wild-type (A') or eIF4E-1 (B') and eIF4E-3 (C') knockdown testes. All knockdowns were performed with shRNA driven by the bam-Gal4:VP16 driver. The testis outline is outlined with a dashed line in A-C. DAPI is shown in magenta. Scale bar 100 μm.

Fig 4. Effects of eIF4E-1 and eIF4G2 knockdown on spermatogenesis.

Phase-contrast micrographs of squash preparations (left panel) and the corresponding Hoescht staining (right panel). As compared with the wild-type (A-B'), testes expressing shRNA targeting eIF4E-1 (C-D') and eIF4G2 (G) in the spermatocytes using bam-Gal4:VP16 driver show defects in nuclear condensation and cytokinesis, while knockdown of eIF4G2 using nos-Gal4:VP16 driver (E-F') results in defective cytokinesis. The nuclei in prophase I, anaphase I and onion stage spermatids are marked by arrows in magenta, yellow and red, respectively. The arrowhead (yellow) shows the nebenkern in panels B & B', D & D' and E' & E' while the mitochondrial structure in elongated spermatids and spermatocytes is marked in F & F' and G & G', respectively. Note the three chromosome clumps in B', C' and D'.

Fig 5. eIF4G is required in the somatic cyst cells for normal spermatogenesis.

Wild-type testes (A-C') and testes expressing eIF4G shRNA with c587-Gal4 driver (A''-C''') were co-stained with anti-eIF4G (red) and anti-eIF4E-3 (green) antibodies. Loss of anti-eIF4G staining in the cyst cells at the apical tip of testis (B'') indicates efficient knockdown of the protein. The testis shows an increased number of abnormal germ cell cysts at its apical tip (B'') and reduced meiotic and post-meiotic cysts along its length (compare A' & C' with A''' & C''') as revealed by anti-eIF4E-3 staining (green). Although mature sperm nuclear bundles are found at the distal end of the testes, they appear disorganised (C''', arrows) and nuclei at various stages of condensation are also found at the caudal end of the elongating spermatids (B''', arrows).

Fig 6. Knockdown of eIF4G2 in early stages affects meiotic divisions and differentiation during spermatogenesis.

Wild-type testes (A-A'', C-C'') and testes expressing eIF4G2 shRNA under nos-Gal4:VP16 control (B-B'', D-D'') were co-stained with anti-eIF4E-3 (red) and anti-eIF4G (green). Panels A-B'' show entire testes while the distal end of the testes is shown C-D''. The merged images are shown in A''-D''. DAPI staining is shown in magenta. Note the absence of elongated flagellar axonemes and nuclear bundles in the eIF4G2 knockdown testes (D') as compared with the wild-type (C'). Scale bar 100 μm.

Fig 7. eIF4G2 function in the spermatocytes is essential for meiotic divisions during spermatogenesis.

Co-staining of wild-type testes (A-A'', C-C'') and testes expressing shRNA targeting eIF4G2 using bam-Gal4:VP16 driver (B-B'', D-D'') with anti-eIF4G2 (green) and anti-eIF4E-3 (red) antibody. Knockdown of eIF4G2 in the spermatocytes does not affect eIF4E-3 distribution at the apical tip of the testes (compare A' with B') but causes meiotic arrest and results in degenerating germ cells at the distal end (D-D''). The merged images are shown in A''-D''. DNA is stained with DAPI (magenta). Scale bar 100 μm.

Fig 8. eIF4G and eIF4G2 act redundantly during early spermatogenesis.

Knockdown of eIF4G and eIF4G2 using the nos-Gal4:VP16 driver results in a rudimentary testes structure (marked by arrows). Anti-eIF4E-1 (A) and anti-eIF4E-3 (B) immunostainings are shown in green and red, respectively, while the merged image is shown in C. Absence of eIF4E-3 positive cells indicates spermatogenesis arrest at the spermatogonia stage. The magnified image of the apical tip of the testis (arrow, D, E) shows a mass of cells. DNA is stained with DAPI (magenta). Scale bar 100 μm.