Identification and distribution of 14-3-3 protein isoforms in mouse oocytes and eggs

The 14-3-3 proteins are known regulators in important intracellular events including signal transduction, cell cycle control and embryonic development. 14-3-3 (also known as YWHA or tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein) plays a central role in mitosis in mammalian cells and meiosis in amphibians, but its role in mammalian meiosis has not been entirely defined. The mammalian isoforms of 14-3-3 are beta, eta, gamma, epsilon, tau/theta, zeta, and sigma (the official protein designation for 14-3-3 sigma is SFN). To begin an examination of the role of 14-3-3 in mammalian meiosis and oocyte maturation, we have determined which isoforms are present in oocytes and eggs and examined their subcellular distribution. 14-3-3 isoforms in protein extracts from oocytes and eggs were detected by Western blotting using isoform-specific antibodies. All isoforms were identified by Western blots of oocyte and egg proteins, except sigma. Immunofluorescence confocal microscopy confirmed the presence of all of the isoforms in both oocytes and eggs with characteristic differences in some of their intracellular localizations. for example, 14-3-3 epsilon is found throughout the oocyte with some cortical accumulation, while it is notably absent in the interior of the egg. on the other hand, 14-3-3 eta is diffusely dispersed in the oocyte and attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. 14-3-3 tau, while distributed throughout oocytes and eggs, shows selective absence along the inner nuclear membrane of all oocytes examined. Some of the isoforms (beta, zeta and gamma) express prominent cortical accumulation in oocytes, but are uniformly dispersed within eggs. The identification and study of differential subcellular distribution of 14-3-3 isoforms in female germ cells will enable characterization of potential 14-3-3 isoform-specific interactions with other key proteins involved in meiosis and oocyte maturation. This will lead to a better understanding of the individual functional roles of the 14-3-3 protein isoforms in mammalian oogenesis.