Expression of the basic helix-loop-helix protein REBalpha in rat testicular Sertoli cells

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 REBalpha in Sertoli cells. REBalpha and REBbeta 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 REBalpha and REBbeta 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 REBalpha is the major splice variant in Sertoli cells. PCR primers specifically designed to amplify either REBalpha or REBbeta demonstrated that Sertoli cells express only REBalpha, not REBbeta. REBbeta 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 REBalpha is not hormone responsive. REBalpha was also found to be expressed in germ cells and peritubular cells. An immunocytochemical analysis demonstrated that REBalpha is predominantly expressed by Sertoli cells within the seminiferous tubules. The activity of REBalpha 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 REBalpha and E47/E12 bHLH proteins. In summary, the Sertoli cell is one of the first cells shown to specifically express the REBalpha 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 REBalpha.