The production of gametes in mammals is an elaborate process that begins during embryogenesis and continues during the reproductive life of the organism. Gametogenesis involves the production of highly specialized cells with unique organelles designed to accomplish the union of sperm and egg at fertilization. The timely production of developmental^ important products involves the regulated translation of mRNAs that have accumulated earlier during gametogenesis. Deviation from the wild-type timing of translation can cause cessation of gametogenesis and lead to sterility. The goals of this proposal are to further our understanding of the mechanisms of translational repression and activation during mammalian spermatogenesis. The studies in this proposal will focus on the regulation of posttranscriptional control in adult male germ line cells in mice.
In Aim 1, the function of the Y box proteins MSY2 and MSY4 will be investigated using conditional gene targeting.
In Aim 2, MSY2 and MSY4-interacting proteins will be identified by mass spectrometry analysis of ribonucleoprotein particles precipitated with MSY2 and MSY4 antibodies. Prm1-enriched mRNPs will be isolated using biotinylated antisense RNAs and streptavidin-coated immunomagnetic beads.
In Aim 3, proteins identified by mass spectrometry will be verified using complementary methods and functional studies will be pursued in cell culture and in vitro to determine how the associated proteins contribute to translational repression and mRNA stability. The knowledge gained from these studies may be useful in the genetic assessment of male infertility in humans, and lead to the development of male contraceptives designed to disrupt essential regulatory steps during normal spermatogenesis.
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