The goal of this project is to study the regulation of mRNA for proteins that are cytoskeletally associated in the rat testis Sertoli cell (SC), and other non-muscle cells. Calmodulin (CaM), myosin light chain kinase (MLCK), and glycogen phosphorylase (PHOS) regulate and are involved in cytoskeletal activity during secretion and other cell processes. The SC is an appropriate model in which to study regulation because there are at least 3 fold changes in protein/cell for each of these protein at specific stages in development, which correlate with morphological and functional changes in the SC. Differential protein expression between SC enriched testis and normal testis engaged in spermatogenesis suggest that the germinal and non-germinal cells regulate expression of these proteins differently. The number, identies, and amounts of mRNAs coding for each of these proteins will be assessed by hybridization to specific cDNAs. Developmental changes in mRNA will be correlated with changes in protein level to determine whether SC and germ cells regulate protein synthesis at the level of gene expression or post-transcriptionally. The influence of hormones on expression of these mRNAs and proteins will be assessed by hypophysectomizing animals and following testis regression and restimulation by FSH and/or testosterone. Because these proteins are ubiquitous in mammalian cells, data from this study may be applicable to a wide variety of cell types. Since CaM is a major protein in sperm and is necessary for sperm motility, understanding the mechanism of CaM expression in developing germ cells has applicability to both infertility and contraceptive studies. MLCK has also been implicated in sperm motility. Glycogen degradation by PHOS has been implicated as a major source of nutrients for developing germinal cells in human testis. The mRNA filters prepared during this study can be subsequently rehybridized to an unlimited number of specific cDNAs, therefore providing the potential to assess mRNA regulation of many other cytoskeletal and non-cytoskeletal proteins that may be important for testis function.
