Antisense oligodeoxynucleotide inhibits expression of recombinant porcine follicle-stimulating hormone receptor.

Abstract

Follicle-stimulating hormone (FSH) regulates folliculogenesis in the ovary and spermatogenesis in the testis via specific, high affinity membrane-bound receptors (FSHR). To assess the role of FSHR gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN) on FSHR mRNA levels and (125)I-FSH binding were determined in Chinese hamster ovary cells stably expressing recombinant porcine FSHR (pFSHR-CHO cells). An 18-mer phosphorothioated antisense ODN corresponding to the region surrounding the translation initiation codon of the porcine FSHR cDNA was synthesized. An 18-mer phosphorothioated nonsense sequence of identical nucleotide composition was synthesized for use as a control. pFSHR-CHO cells were cultured in the absence or presence of 1-20 microM antisense or nonsense ODN for 24 hr and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for (125)I-FSH binding activity. Treatment with 10 microM antisense ODN caused a paradoxical increase in porcine FSHR mRNA. Nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 microM ) inhibited membrane binding of (125)I-FSH by 13.6 +/- 0.8% (mean +/- SEM, n = 3, P < 0.05) in 24 hr. Treatment of cells with antisense ODN (10 microM) for 48 hr resulted in a 76 +/- 1.5% (P < 0.05) inhibition of (125)I-FSH binding. These results indicate that an FSHR antisense ODN effectively inhibits porcine FSHR synthesis and inhibition of receptor synthesis causes a decrease in functional membrane-bound FSHR.