Figure 6.

Phenotypes resulting from dsRNA knockdown of odCDK1a. A) Significant (**P < 0.01) knockdown of the targeted odCDK1a was verified by qRT-PCR. No significant off-target effects on other odCDK1 paralogs were detected. B) Oocytes spawned from females where ovaries had been co-injected with dsRNA (GFP-positive) against odCDK1a and capped mRNA coding for histone H2B-eGFP (selection marker for successful injection), failed to develop after exposure to sperm from wild type males. Oocytes spawned from wild-type non-injected females (Control) exposed to the same pool of wild type sperm developed normally. Oocytes derived from females whose ovaries had been co-injected as above, but failed to exhibit histone H2B-eGFP fluorescence (GFP-negative) also developed normally. The number of oocytes/embryos assessed is given across the top of the histogram bars. Legend: UF, UnFertilized; CA, early embryo Cleavage Arrest; D, Developed normally. C) Oocytes that were spawned from ovaries where odCDK1a had been knocked down were considerably smaller (***P < 0.001) compared to oocytes produced from wild-type and H2B-eGFP negative females. A similar effect in reducing spawned oocyte size was observed upon treatment of day 6 females with 200 nM calyculin A (Calyc.; **P < 0.01) or 20 μM roscovitine (Rosc.; ***P < 0.001) as compared to control females incubated in the presence of the DMSO solute alone. Scale bars = 20 μm. D) Ring canal diameters of growing oocytes were not affected by dsRNA knockdown of odCDK1a or by inhibiting PP1 phosphatase activity with 200 nM calyculin. E) Knockdown of odCDK1a did not significantly alter the proportions of selected vs. non-selected meiotic nuclei as determined by H3-pS28 staining (P = 0.19). F) Females injected with dsRNA targeting odCDK1a often released clumps of unresolved oocytes embedded in excess cytoplasm. This was never observed in wild type females, or in females injected with dsRNA targeting odCDK1d or odCDK1e.