Expression of the basic helix-loop-helix protein REBα 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 fell bHLH proteins demonstrated the expression of REB alpha in Sertoli cells. REB alpha and REB beta 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 alpha and REB beta has been shown. In the current study, a polymerase chain reaction (PCR)-ased approach demonstrated that REB gene transcripts are expressed in the testis. Characterization of the REB transcripts suggested that REB alpha is the major splice variant in Sertoli cells. PCR primers specifically designed to amplify either REB alpha or REB beta demonstrated that Sertoli cells express only REB alpha, not REB beta. REB beta 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 alpha is not hormone responsive. REB alpha was also found to be expressed in germ cells and peritubular cells. An immunocytochemical analysis demonstrated that REB alpha is predominantly expressed by Sertoli cells within the seminiferous tubules, The activity of REB alpha 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 alpha and E47/E12 bHLH proteins. In summary, the Sertoli cell is one of the first cells shown to specifically express the REB alpha 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 alpha.