The long term objective of this proposal is the delineation of cellular factors affecting testicular development and spermatogenesis. The immediate goal is to use the rat as an animal model for examining the biological role of the pregnancy-specific beta1-glycoprotein (PSG) in the testis. Multiple species of PSG are present in human placenta. Recent studies showed that PSG is also present in high abundance in the testis. PSG derived from human placenta has been shown to possess immunosuppressive and growth promoting activities. the presence of PSG in testis raises the question about its functional role in testis metabolism. To address this question, the first step is to establish the presence of PSG in rat testis. This will be achieved by molecular cloning of rat testicular PSG cDNA. Availability of PSG cDNAs will allow generation of gene-specific oligonucleotide probes for further investigation as well as preparation of pure proteins by in vitro expression of the cloned cDNAs. Molecular cloning of PSG cDNA will be achieved by screening of rat testicular cDNA library using human PSG cDNA as probe. Nucleotide structure of cloned cDNAs will be determined by Sanger's dideoxy chain termination methods. Proteins encoded by these cDNAs will be produced by using mammalian cell in vitro expression systems. Purified recombinant proteins will be used to raise monoclonal antibodies for subsequent studies. Acquisition of both cDNAs and antibodies will permit the precise cellular localization of PSG in rat testis. cDNAs will be used for in situ hybridization studies while antibodies will be used for immunohistochemical studies to define the cell types in which PSG mRNA and protein are present. The presence of PSG in spermatids at different stages of development will also be further studied. If multiple species of PSG are present in rat testis, the occurrence of the different PSG will be examined with gene-specific probes. Expression of PSG at different stage of testicular development will be determined in rats of different ages at both the mRNA and protein levels. Initial functional analysis of PSG will include identification of cellular binding target and examination of effects of PSG on germ cells and other testicular cells in culture. Cell binding experiments will be done with labeled proteins and Scatchard analysis. Effects of PSG on growth and morphology of these cells will be studied. The proposed project will shed light on the physiological functions of the PSG in testicular development and spermatogenesis in the rat and should provide a model system for further studies of these proteins in the human.
Showing the most recent 10 out of 15 publications
