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Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205
Address all correspondence and requests for reprints to: Dr. William W. Wright, Division of Reproductive Biology, School of Hygiene and Public Health, The Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205. E-mail: bwright{at}jhmi.edu
It is well known that male germ cells regulate the steady state levels of numerous transcripts expressed by Sertoli cells. To date, however, there has been no direct test of whether this regulation reflects changes in gene transcription and/or transcript stability. This study used two experimental approaches to test the hypothesis that germ cells regulate transcription of the cathepsin L gene by rat Sertoli cells. We examined this gene because, in vivo, steady state levels of cath L messenger RNA in Sertoli cells change in a stage-specific manner as the surrounding germ cells progress through the 14 stages of the cycle of the seminiferous epithelium. In the first experimental approach, seminiferous tubules at stages VI–VII and stages IX–XII were incubated for 1 h in 4-thiouridine, and the amount of metabolically labeled cath L messenger RNA was quantified. The results demonstrate that transcription of the cath L gene by Sertoli cells is 7-fold higher at stages VI–VII than at stages IX–XII. The second experimental approach examined the ability of germ cells to regulate the activity of cath L reporter constructs in mature Sertoli cells. Before these studies, we isolated a cath L genomic clone and demonstrated that this clone contains the transcription start site of the cath L gene expressed by Sertoli cells. Transient transfection analysis then demonstrated that two reporter constructs, containing 244 and about 2.1 kb of sequence upstream from the transcription start site, had similar activities in mature Sertoli cells. However, germ cells only affected the activity of the larger construct in Sertoli cells, which was reduced by 30%. We conclude that germ cells regulate transcription of the cath L gene by Sertoli cells and that repressive effects of germ cells are mediated by elements upstream from nucleotide -244 of this gene.
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