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First published online 21 April 2009
doi: 10.1242/jcs.046771

Journal of Cell Science 122, 1529-1539 (2009)
Published by The Company of Biologists 2009
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A germline-specific isoform of eIF4E (IFE-1) is required for efficient translation of stored mRNAs and maturation of both oocytes and sperm

Melissa A. Henderson1, Elizabeth Cronland1, Steve Dunkelbarger2, Vince Contreras1, Susan Strome2 and Brett D. Keiper1,*

1 Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27834, USA
2 Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz, CA 95064, USA


Figure 1
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  		Fig. 1. Characterization of ife-1(bn127) mutant worms. (A) Depiction of ife-1 gene and location of the 590 bp bn127 deletion. Exons are shown as colored boxes with scale bar indicating 100 bp. (B) Whole worm genomic PCR of three individual ife-1 clones, one heterozygous ife-1/mT1, and one wild-type hermaphrodite using primers –523s and 827a. The wild-type ife-1 gene produces a PCR product of 1359 bp. The bn127 deletion produces a shorter 769 bp product. (C-E) Brood size and stage of offspring after 96 hours at 15°C (C), 20°C (D) or 25°C (E) for ife-1 and wild-type worms. Progeny were scored as oocytes (ooc), unhatched embryos (emb), early (L1/L2) or late (L3/L4) larvae or adults (Ad). Numbers are averages per adult worm (n=9). Error bars indicate standard deviation. At 15°C and 20°C, ife-1 mutant worms produced a small number of viable offspring. At 25°C ife-1 mutants produced only oocytes.

 

Figure 2
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  		Fig. 2. Nuclear and cellular morphology of wild-type, ife-1 and fem-2 hermaphrodite gonads. Whole worms were analyzed during their maximal fertile period at 3 days (wt and fem-2) or 4 days (ife-1; as a result of prolonged L4 stage) post hatching at 25°C. (A) DAPI staining of whole worms indicated the presence of sperm (sp) in wild-type spermatheca. Sperm were not present in the spermatheca of ife-1 (B) and fem-2 (C) hermaphrodites. The spermatheca is outlined by a dashed line. Arrowheads (^) indicate post-pachytene oocytes; e, fertilized embryos in the uterus of wild-type hermaphrodite; asterisks, unfertilized oocytes undergoing endomitosis. (D-F) Wild-type (D), ife-1 (E) and fem-2 (F) gonads with mature oocytes prior to fertilization. fem-2 oocytes stacked up in the absence of sperm for fertilization. Stacking of oocytes was not observed in the ife-1 gonad which also lacks sperm.

 

Figure 3
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  		Fig. 3. Oocyte development in ife-1 hermaphrodite gonads. Whole gonads were dissected from worms grown at 20°C (A-D) and 25°C (E-J). At 20°C, in the wild-type gonad there is normal late oocyte progression in the proximal region (A, DIC image; B, Hoechst stained). There are ten sequential oocytes with condensed chromosomes (post-pachytene) arranged in linear array (arrowheads; ^). ife-1 gonads grown at 20°C showed somewhat elongated, but otherwise normal oocytes in the proximal region (C,D). In contrast to wild type, only seven post-pachytene oocytes were aligned in the ife-1 gonad. Nuclear staining indicated that most ife-1 oocytes contained aligned bivalent chromosomes (^); a few failed to condense (d). Wild-type worms raised at 25°C (E,F) showed similar characteristics to those raised at 20°C with eight post-pachytene oocytes aligned in the proximal region. The gonad from ife-1 worms raised at 25°C (G,H) also showed similar characteristics to the ife-1 gonad at 20°C, with five oocytes with condensed bivalents (^). Fertilization did not occur in ife-1 worms at 25°C, and four unfertilized oocytes were observed in the uterus undergoing endomitosis (*). The feminized fem-2 gonad (I,J), in which no fertilization occurs, showed normal oocyte progression in the proximal region and accumulated 14 post-pachytene oocytes.

 

Figure 4
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  		Fig. 4. Morphology of ife-1 male gonads, spermatocytes and sperm. Squashed dissected male gonads from wild-type (wt) and ife-1 males grown at 20°C (A-D) and 25°C (E-H) depict progression through spermatogenesis. Wild-type testes stained for DNA with Hoechst (A) or visualized by DIC microscopy (B) showed highly condensed DNA in individual spermatids (sp) at the proximal (mature) end and immature germ cells in pachytene stages in the distal portion of the testis (t). The gut is indicated by g. ife-1 testes show a similar progression but fewer spermatids (C,D). Testes from male worms grown at 25°C were similarly prepared and analyzed (E-H). Wild-type testes (E,F) look similar to those at 20°C, with the highly condensed haploid nuclei in distinct individual spermatids. (Insets) Fourfold magnification of a selected field containing secondary spermatocytes and mature spermatids. The latter were identified by their rough appearance and a central `dimple'. By contrast, ife-1 testes at 25°C (G,H) contained many secondary spermatocytes with multiple nuclei (2-4; ^), but lacked mature spermatids. Terminal multinucleated spermatocytes failed in cytokinesis during residual body formation.

 

Figure 5
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  		Fig. 5. Effect of temperature on ife-1 fertility. Brood size from wild-type (A) and ife-1 (B) hermaphrodites shifted from 20°C to 25°C (blue) and 25°C to 20°C (pink) at 12 hour intervals. Wild-type hermaphrodites showed no temperature-induced sterility (A). By the L4 -young adult transition (60-72 hours), temperature downshifts no longer rescued fertility for ife-1 hermaphrodites, and temperature upshifts no longer induced complete sterility (B). Temperature-shifted wild-type hermaphrodites had similar brood sizes (120-220). Fertile ife-1 worms had much smaller broods (5-25). Staging of worms by morphology at time of shift demonstrated the prolonged ife-1 L4 stage (C). Points between stages indicate roughly equal percentages of worms at both stage. (D,E) Mating experiments with three males and one hermaphrodite of each of the indicated genotypes. Total bar height indicates brood size over 96 hours. Male offspring are shown in purple, and hermaphrodites shown in blue at 20°C (D) and 25°C (E). The mean number of offspring (#F1 worms) of triplicate matings were plotted. Error bars indicate the standard deviation from the mean. For clarity, only the `positive error' is displayed for purple bars, and the `negative error' is displayed for blue bars. (F) The number of inviable progeny (unfertilized oocytes and arrested embryos) of wild-type or ife-1 hermaphrodites mated with wild-type males. Percentage values above the bars indicate inviable offspring normalized to respective brood size. Error bars indicate standard deviation.

 

Figure 6
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  		Fig. 6. Analysis of maternal mRNA translation efficiency by polysome fractionation. Absorbance (A254) profiles for continuous gradient fractionation of wild-type (A) and ife-1 (B) mixed staged hermaphrodite worm extracts depict the polysome content of each strain. The highest resolved peak is the monosome (80S) and each peak to the right represents the addition of one ribosome (polysomes). (C-H) Sedimentation of individual maternal mRNAs. mRNA distribution of oma-1 (C), mex-1 (D), pos-1 (E), pal-1 (F), glp-1 (G) and gpd-3 (H) throughout the gradients was quantified by qRT-PCR. mRNA signal was normalized to RNA content in each fraction. Normalization corrects for slight differences in polysome yield, allowing direct comparison of the translational efficiency of each mRNA. Error bars indicate the standard deviation of triplicate qRT-PCR determinations.

 

Figure 7
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  		Fig. 7. Accumulation of MEX-1 protein in the absence of IFE-1. Dissected hermaphrodite gonads were fixed and immunostained for MEX-1 protein and costained with DAPI to visualize oocyte nuclei. (A) In the wild type, MEX-1 was first detected in the late pachytene stage of meiosis and progressively accumulates to the –1 oocyte. (B,C) In ife-1 gonads, the MEX-1 protein appeared in late pachytene, but then remained relatively constant (B) or decreased (C) in later oocytes. MEX-1 signal was normalized to exposure time using identical incident light and filter settings to allow direct comparisons.

 

Figure 8
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  		Fig. 8. Expression of pro-apoptotic proteins in hermaphrodite and male gonads. (A,B,E) Germ cell apoptotic corpses were detected by expression of the CED-1::GFP engulfment reporter. Only two germ cell corpses (*) were evident in the wild-type hermaphrodite gonad (A). The ife-1 gonad similarly showed two apoptotic corpses decorated with CED-1:GFP (B). (C,D) DAPI-staining verified the progression of oocyte nuclei through pachytene and diakinesis stages. As a positive control, depletion of IFG-1 p170 by ifg-1(RNAi) significantly increased germline apoptosis in the hermaphrodite gonad (E). Asterisks and brackets indicate the position of a cluster (six to eight) of cell corpses. (F-I) Expression of CED-4 was detected by immunostaining of dissected male testes. (F) Wild-type male gonad shows CED-4 accumulation in apoptosome-like structures only in late spermatocytes and spermatids. (G) Gonad from an ife-1 male showed CED-4 accumulation throughout spermatogenesis that eventually also resided in apoptosome-like structures, suggesting that CED-4 synthesis began earlier in spermatogenesis in the ife-1 male. (H,I) DAPI staining to show the nuclear morphology of early meiotic germ cells, primary spermatocytes (ps) and secondary spermatocytes and spermatids (sp).

 

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© The Company of Biologists Ltd 2009