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First published online 16 October 2008
doi: 10.1242/dev.025114

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-Endosulfine is a conserved protein required for oocyte meiotic maturation in Drosophila

¹ Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
² Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
³ Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

View larger version (97K): [in this window] [in a new window]   Fig. 1. endos00003 oocytes fail to undergo meiotic maturation. (A) DAPI-stained egg chambers in different stages. nc, nurse cells; oo, oocyte; da, dorsal appendages. Asterisks indicate position of the oocyte nucleus. Scale bar: 100 µm. (B-F) Control oocytes in prophase I (B-D) and in metaphase I (E,F). Arrows indicate non-exchange fourth chromosomes. (G-K) endos00003 oocytes have a prolonged prophase I (G-J) and abnormal DNA morphology at stage 14 (K). (L-P) twine1 oocytes show similar phenotypes to endos00003 oocytes. (Q-U) elgi1 oocytes in prophase I (Q,R) and in premature metaphase I (S-U). Scale bar: 5 µm. Control (F') and elgi1 (U') have dehydrated stage 14 oocytes. endos00003 (K') and twine1 (P') oocytes are not dehydrated and have abnormal yolk morphology. Scale bar: 100 µm. (V) Quantification of DNA morphology. The number of oocytes analyzed is shown above the bars. *P<0.001.

View larger version (98K): [in this window] [in a new window]   Fig. 2. endos00003 mutants have spindle defects. Stage 14 oocytes (A-F) and 0- to 3-hour embryos (G-O) stained with propidium iodide (DNA, red) and anti--tubulin (microtubules, green). (A) Control oocytes in metaphase I. (B) Typical endos00003 oocyte showing dispersed DNA without a spindle. (C) Rare endos00003 oocyte showing abnormal DNA associated with large spindle masses. (D,E) Similar twine1 phenotypes. (F) elgi1 oocyte in metaphase I. (G) Control embryos exhibiting typical embryonic mitoses. (H-J) endos00003-derived embryos showing dispersed DNA with no spindle (H), dispersed DNA with spindle masses (I), and tripolar spindles (J). twine1-derived embryos showing dispersed DNA (K), abnormal spindle masses (L), spindle asters with no DNA (M), and long, thin spindles (N). (O) Embryos derived from elgi1 females showing normal spindles and DNA. Scale bars: 10 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 3. Endos regulates Twine, Polo and MPM2 phosphoepitopes. (A) Anti-β-gal western blot, showing reduced expression of Twine::β-gal in endos00003 and endos00003 elgi1 stage 14 oocytes (30 stage 14 oocytes per lane). (B) X-gal staining (blue) of control, endos00003, elgi1 and endos00003 elgi1 oocytes reflecting Twine::β-gal levels at stages 13 and 14. Scale bar: 100 µm. (C) Polo western analysis of control, twine1, endos00003, elgi1 and endos00003 elgi1 stage 14 oocytes showing a strong reduction of Polo levels in endos00003 and endos00003 elgi1 (50 stage 14 oocytes per lane). (D) Cyclin B western analysis showing normal expression in endos00003 and elgi1 mutants, and slightly elevated expression in twine1 mutants (30 stage 14 oocytes per lane). (E) In vitro Cdk1 kinase assay using anti-Cdk1 or anti-Cyclin B immunoprecipitates (IP) from control, twine1, endos00003 and elgi1 stage 14 extracts. Mock immunoprecipitates were performed without antibodies. Immunoprecipitates were either immunoblotted using anti-Cdk1 or anti-Cyclin B antibodies, or subjected to a kinase assay using [32P]ATP and histone H1 as substrate. (F) Western blotting using MPM2 antibodies. Wild-type and elgi1 oocytes show high MPM2 levels, whereas endos00003, twine1 and endos00003 elgi1 oocytes have drastically reduced MPM2 phosphoepitopes (50 stage 14 oocytes per lane). Actin was used as a control.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Endos binds to a putative E3 ubiquitin ligase encoded by elgi. (A) DIVEC screen used to identify Endos interactors. (B) Autoradiogram showing that the GST::Endos fusion protein specifically binds to the closely related CG9014 and Elgi E3 ubiquitin ligases, but not to more distant E3s, such as Nopo and CG10916. (C) Elgi shares 81%, 80%, 57%, 57%, 56% and 54% amino acid identity with the A. aegypti, A. gambiae, X. laevis, D. rerio, M. musculus and H. sapiens homologs, respectively. Red box marks RING domain; identical amino acid residues are shaded. Asterisk indicates the first methionine for the predicted Elgi2 protein. (D) elgi alleles generated by imprecise excision of EY10782. Thick bars represent elgi exons (coding region in black). Gaps in black lines indicate deleted regions in elgi1 and elgi2. (E) Western blot showing normal Endos levels in elgi1 ovaries. Actin was used as a loading control. (F) Model for the role of Endos in oocyte meiotic maturation. Endos controls Polo kinase and, perhaps indirectly, Twine levels, leading to the activation of Cyclin B/Cdk1. By binding and inhibiting Elgi, Endos may have a parallel role in refining the timing of meiotic maturation.

View larger version (66K): [in this window] [in a new window]   Fig. 5. The function of -endosulfine might be evolutionarily conserved. (A-D) DAPI-stained stage 14 oocytes from control and endos00003 females expressing Endos or human -endosulfine (ENSA). (A) Control oocytes arrested in metaphase I. (B) endos mutant oocytes with dispersed DNA. (C,D) nanos-Gal4::VP16-driven germline expression of UAS-endos (C) or UAS-ENSA (D) transgenes showing endos rescue. Arrows indicate fourth chromosomes. Scale bar: 5 µm. (E) Western analysis showing ovarian expression of Endos and ENSA in rescued females. Actin was used as a loading control. Arrowheads indicate ENSA-specific bands (lower bands are probably degradation products), which are absent in the `No rescue' control. (F) Quantification of endos00003 rescue. The number of stage 14 oocytes (metaphase I arrest and dehydration) or eggs (hatch rate) analyzed is shown above the bars. *P<0.001. (G,H) Mouse ovary immunohistochemistry showing that anti-Endos antibodies strongly label the cytoplasm of oocytes (H), whereas no signal is detected by pre-immune serum (G). o, oocyte (arrows); c, cumulus cells; g, granulosa cells; a, antrum; i, interstitial cells. Scale bar: 200 µm.

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