FSH and androgens act on Sertoli cells to regulate the secretion of proteins that function in the control of spermatogenesis. However, the mechanisms by which these hormones regulate Sertoli gene expression remain incompletely understood. One of the secretory proteins, androgen binding protein (ABP), has been used extensively as a marker for studies on the regulation of Sertoli cell function. Although the role of ABP in spermatogenesis and sperm maturation is not known, the hormonal control of ABP secretion closely parallels the control of spermatogenesis. Our goal is to develop the rat ABP gene as a model system to determine the mechanisms of hormone mediated regulation of Sertoli cell secretory processes. Toward these goals, we have prepared a rat testis cDNA library in Lambda gt11 and have isolated recombinants that contain cDNA complementary to ABP mRNA. Recombinants were identified with ABP antiserum and their identity was confirmed by hybrid-arrested translation and epitope selection. The total nucleotide sequence of ABP cDNA isolates has revealed the amino acid sequence of the ABP precursor and its signal peptide. Other studies suggest that both protomers of ABP are coded for by one mRNA species and one gene. With the ABP cDNA we propose to isolate the gene for ABP, map and sequence intron-exon regions, identify the transcription initiation site and locate regulatory DNA sequences. A particular emphasis will be placed on examination of the 5'-upstream region of the gene which may contain hormone regulatory sequences. ABP cDNA will also be used as a probe to study hormone regulated gene expression. These studies will include in vivo experiments with hypophysectomized rats and Tfm rats as well as in vitro experiments with isolated seminiferous tubules and cultured Sertoli Cells. The levels of ABP mRNA will be analyzed by northern blot hybridization and further quantitated by solution hybridization with RNA probes. We will also attempt to measure transcription rates of the ABP gene by hybrid selection assay and translation rates of ABP mRNA by immune precipitation. This work will develop a system to study the mechanisms of FSH and androgen action using a gene expressed specifically in Sertoli Cells.
Showing the most recent 10 out of 31 publications
