Kevin O'Connell in our laboratory has isolated a collection of mutant animals exhibiting a conditional sterile uncoordinated phenotype. His analysis of
oj29ts mutants grown at 25 degrees has shown that these mutants arrest as one cell embryos and lack a functional microtubule organizing center during interphase. The nuclei break down upon entering mitosis, however no mitotic spindle forms. These mutants exhibit other defects consistent with the absence of a centrosome. For example, polarity defects are apparent, as cytoplasmic streaming is absent, and the ruffling of the membrane is not asymmetric. There are also defects in pronuclear migration, as both pronuclei migrate to the center of the egg, instead of meeting at the posterior side of the egg. At 25 degrees,
oj29ts homozygous hermaphrodites lay 100% dead eggs, which failed to divide.
oj29ts mutants outcrossed with wild-type males also lay 100% dead eggs, with an early embryonic phenotype identical to those produced through self fertilization. Hence, the contribution of a functional centrosome by the wild-type sperm is not sufficient to rescue cell division in
oj29ts mutant animals. The self progeny of heterogeneous
oj29ts hermaphrodites all live, suggesting that the maternal contribution is sufficient to rescue the cell division defect; however, these animals are sterile and uncoordinated, presumably from a zygotic requirement during post-embryonic gonad and ventral nerve cord development. We are currently using transmission electron microscopy to examine
oj29ts sperm in order to determine ultrastructural defects in the centrosome.
oj29ts L4 hermaphrodites were shifted to 25 degrees overnight, and were then fixed in OsO4, dehydrated in acetone and embedded in epon resin. The hermaphrodites were sectioned, and sperm within the spermatheca are being analyzed via transmission electron microscopy for centrosomal defects.