Molecular survey of myosins in zebrafish oocytes

Abstract

Actin and myosin are cytoskeletal proteins that are known to mediate several cellular activities, including the movement of cells, secretion, retrieval of membrane by exocytosis and muscle contraction. Based on studies with somatic cells, there is support for the notion that myosins may play a role in early events of vertebrate gametogenesis and fertilization. Two strategies were employed to survey myosin expression in zebrafish ovary tissue and activated eggs: one dimensional electrophoresis and Western blotting of homogenates; and synthesis of cDNA clones using reverse-transcriptase and Polymerase Chain Reaction methods with degenerate primers coding for sequences in the myosin head domain.

Gels of ovary and egg homogenates displayed a single band whose molecular weight was estimated to be 205 kDa (egg) and 210-215 kDa (ovary). Anti-human platelet myosin heavy chain antibody recognized this band in both samples as well as the rabbit myosin protein on Western blots. Standard methods were used to synthesize single and double-stranded cDNA from ovary total RNA. Degenerate primers prepared against the myosin head domain were used. PCR product was successfully ligated into a P-GEM vector.