The overall objective of this study is to determine whether the cAMP responsive transcriptional factor, CREB, is essential for spermatogenesis. Previous studies have determined that CREB is expressed in the Sertoli cells and that CREB levels vary in a cyclical manner as germ cells develop. The main model employed will be the selective knockout of CREB action in testis by an adenoviral vector expressing a mutant form of CREB (Ad F-CREBM1) which acts as a competitive inhibitor of wild type CREB. The cDNA for CREBM1 will be linked with a flag epitope, which will allow identification and quantitation of AdF-CREBM1 in infect primary Sertoli cells, and in the testis. Another virus containing a gene for beta galactosidase will be prepared to serve as a control for effects of viral injection. Initial studies will determine the toxicity and effectiveness of the viruses in vitro. TO better understand CREB action in vivo. virus preparations will be injected directly into the seminiferous tubules of rats to allow for infection of Sertoli cells. Testis will be taken from fats four days post infection to allow for quantitation of infection rate and evaluate the incidence of germ cell apoptosis in AdF-CREBM1 infected testes versus controls. From another group of infected rats, testes will be taken 14 days post infection and undergo morphological assessment of testis tissue sections. Five candidate proteins that are regulated by CREB and may potentially modulate germ apoptosis observed after AdF-cREBM1 infection. Five candidate proteins that are regulated by CREB and may potentially modulate germ cell apoptosis have been identified. The expression levels will be determined in primary Sertoli cells after AdF-CREBM1 infection and in testis tissue sections from AdF-CREBM1 infected rats.
