Establishment of a mouse sertoli cell line producing rat ABP
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文摘
ABP/SHBG gene products interact with spermatogenic cells of different species by receptor-mediated mechanism depending on the maturation step (Gérard et al., 1990-95) and are supposed to play a role of paracrine factor in the testis. To elucidate the physiological significance of ABP action on germ cells and because germ cells did not survive without Sertoli cells, we needed to create an experimental model of co-culture with Sertoli cells producing ABP or not producing ABP. TM4 cells were chosen as Sertoli cell line (Mather et al., 1980). They are able to grow on plastic without hormones. However, the secretion of ABP was lost except in the presence of peritubular cells (Mather et al., 1982). We have first checked the absence of ABP secretion by immunodetection using polyclonal antibodies against mouse ABP. We used a rat ABP construct consisting in a 1600-base-pair rat ABP ADNc cloned into HindIII-Xbal site of the eukaryotic expression vector pRc/CMV (Bocchinfuso et al., 1991) for transfection by either a liposome methodology (Dotap, Boehringer) or a polyethyleamine (PEI, Exgen 500, Euromedex) into TM4 cells to generate stable clones producing recombinant rat ABP. Neomycin-resistant clones were selected by adding G418 in the culture medium and grown for 3 weeks. Controls were made in TM4 and CHO cells transfected with the parent vector only. Analyses of over 25 clones revealed the presence of recombinant rat ABP when probed with a rabbit polyclonal antibody raised against the testicular rat ABP (kindly provided by J. Closset) in cell homogenates and culture medium, indicating the traduction and the secretion of a protein similar to the testicular rat ABP. In addition, rat ABP was detectable within the cytoplasm of transfected cells by immunocytochemistry. Clones analyzed by Southern blotting using a 1600-base-pair rat ABP ADNc probe assessed that we have generated an immortalized Sertoli cell line growing on plastic without hormone supplementation and continuously secreting rat ABP. We conclude that we have now a functional model to study co-cultures with spermatogenic cells and TM4 producing no ABP. TM4 and extratesticular cells (CHO) producing ABP.