CONICET | Buscador de Institutos y Recursos Humanos

Background: FSH regulates Sertoli cell function during development through the FSHR, a 692-amino acid G protein-coupled receptor. In vitro studies using Sertoli cell lines are instrumental for the study of FSH action. However, no prepubertal Sertoli cell line exists that expresses the FSHR. Our group generated the murine SMAT1 cell line, as a model for in vitro study of prepubertal Sertoli cell physiology, but these cells have also lost FSHR expression. Transient FSHR transfection of SMAT1 cells is a suboptimal solution, resulting in cellular damage and heterogeneous results from one experiment to another. The objective of this work was to generate a prepubertal Sertoli cell line with stable FSHR expression.Methods: 1x106 SMAT1 cells were transfected with 0.2 µg of pCDNA3-FSHR expression plasmid (8.1 kb) using 1 µl lipofectamine in a 24-well plate, and selected by resistance to geneticin (G418). Surviving cells were subjected to limiting dilution in a 96-well plate to obtain a single cell per well. Clones that expanded under G418 were tested for FSHR expression by RT-PCR.Results: The selective condition was determined as 500 µg/ml of G418, concentration required to provoke massive death of non-transfected SMAT1 cells in 2 weeks. Upon limiting dilution, 21 wells containing 1-3 cells were identified and followed for survival and cell division for 6 weeks. Eight clones managed to expand in the presence of G418, 6 of which showed cDNA amplification when tested for FSHR expression by RT-PCR. One clone presented the expected product size for the FSHR amplified fragment (95 bp). Protein expression analysis are underway to fully characterize the obtained clone.Conclusions: By liposome transfection, we have obtained a clone of prepubertal mouse Sertoli cell line SMAT1 with stable expression of the FSHR. Following full characterization, this cell line will represent a unique tool for the study of the physiological regulation of the prepubertal Sertoli by FSH.