Stable transfection of the rat follicle-stimulating hormone receptor complementary DNA into an immortalized murine Sertoli cell line

Abstract

A plasmid expressing the rat FSH receptor (R) cDNA under the Simian virus (SV) 40 promoter/enhancer was stably transfected into a mouse Sertoli cell (SC) line (MSC-1) established from transgenic mice carrying a fusion gene of the human anti-Müllerian hormone (AMH) promoter sequences linked to the SV40 T-antigen gene ( Peschon et al., 1992). The original cell line has numerous SC characteristics, but it was reported not to express the inhibin- α and follicle-stimulating hormone (FSH)R genes. The new FSHR expressing cell line possessed ≈2000 per cell with equilibrium association constant ( K a) of 1.5×10 9 l/mol. In Northern blots, an FSHR mRNA species of 2.6 kb was found. The cells responded to recombinant human FSH (recFSH) and pertussis toxin (PT) with stimulated cAMP production. Moreover, PT enhanced the FSH-stimulated cAMP production in these cells, indicating the presence of a functional Gi protein. 12- O-Tetradecanoylphorbol-13-acetate (TPA) suppressed the FSH-stimulated cAMP production of the cells, which effect was similar to that observed previously upon protein kinase C (PKC) activation in rat seminiferous tubules in vitro. Hence, the FSHR signalling, and its modulatory pathways, were intact in the FSHR expressing MSC-1 cell line. RT-PCR with inhibin- α specific oligonucleotide primers, followed by Southern hybridization, indicated that, unlike previously shown, the original and the FSHR expressing MSC-1 cells do express the inhibin α gene. FSH stimulation of the cells decreased their proliferation and, unexpectedly, the inhibin- α mRNA levels. The cells have functional features both from neonatal and mature SC. A feature of the former cells is the lack of FSH-stimulated up-regulation of inhibin- α expression; in fact FSH decreased this message. The antiproliferative, and apparently differentiating, effect of FSH on these cells resembled mature SC functions. Since adult SC do not proliferate in vitro, the new FSHR expressing and proliferating cell line provides a useful in vitro model for studying some facets of SC functions, though keeping in mind that these transformed cells do not behave identically with adult SC in vivo. The constitutive expression of FSHR in these cells allows the study of posttranscriptional events in the FSHR regulation.