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in Rat Testicular Sertoli Cells1
a Center for Reproductive Biology, Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 99163-4231
Sertoli cell differentiation is initiated in the embryo to promote testicular development and male sex determination. In the adult, Sertoli cells are critical for maintenance of the spermatogenic process. Previously, Sertoli cell differentiation has been shown to be regulated in part by basic helix-loop-helix (bHLH) transcription factors. This was based on the observation that promoters of a number of Sertoli cell genes contained bHLH-responsive E-box response elements and that overexpression of Id, a negatively acting HLH protein, down-regulates Sertoli cell differentiated functions. Analysis of Sertoli cell bHLH proteins demonstrated the expression of REB
in Sertoli cells. REB and REBß are spliced variants of the REB gene that is implicated in cell-specific gene expression as part of dimeric bHLH complexes acting on E-box response elements. Although both the transcripts of the REB gene are widely expressed, differential expression of the REB gene transcripts REB and REBß has been shown. In the current study, a polymerase chain reaction (PCR)-based approach demonstrated that REB gene transcripts are expressed in the testis. Characterization of the REB transcripts suggested that REB is the major splice variant in Sertoli cells. PCR primers specifically designed to amplify either REB or REBß demonstrated that Sertoli cells express only REB, not REBß. REBß was present in the RNA samples obtained from whole testis, suggesting expression in other testicular cell types. A Northern blot analysis of RNA from Sertoli cells treated with or without FSH or cAMP demonstrated that REB is not hormone responsive. REB was also found to be expressed in germ cells and peritubular cells. An immunocytochemical analysis demonstrated that REB is predominantly expressed by Sertoli cells within the seminiferous tubules. The activity of REB in Sertoli cells was demonstrated with an E-box gel shift with Sertoli cell nuclear extracts. The E-box gel shift was found to contain REB and E47/E12 bHLH proteins. In summary, the Sertoli cell is one of the first cells shown to specifically express the REB isoform of the REB gene. The results are discussed in relation to the possibility that Sertoli cells may express a cell-specific bHLH protein that can preferentially dimerize with REB.
2 Correspondence. FAX: 509 335 2176; skinner{at}mail.wsu.edu
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