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

Abstract

To assess the role of follicle-stimulating hormone receptor (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 125I-FSH binding were determined in Chinese hamster ovary cells expression recombinant porcine FSHR (pFSHR-CHO cells). An 18-mer phosphorothioate-endcapped antisense ODN that corresponded to the region surrounding the translation initiation codon of the porcine FSHR cD-NA was synthesized. An 18-mer nonsense sequence of identical nucleotide composition, which had little homology to known DNA sequences, was synthesized for use as a control. pFSHR-CHO cells were cultured in 24-well plates (10⁵ cells/ well) in the absence or presence of 1–20 μmol/L antisense or nonsense ODN for 24 h and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for¹²⁵I-FSH binding activity. Treatment with 10 μmol/L antisense ODN caused a paradoxical increase in porcine FSHR mRNA from 0.89±0.06 to 1.64±0.08 ng/mg total RNA (P< 0.05). Transfection with lipofectamine and 0.33 μmol/L antisense ODN caused an increase in porcine mRNA from 0.95±0.08 to 1.53±0.07 ng/mg total RAN. This was probably due to upregulation of mRNA synthesis resulting from inhibition of porcine FSHR protein translation. The nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 μmol/L) inhibited membrane binding of¹²⁵I-FSH by 13.6±0.8%(P<0.05) in 24 h. Treatment of cells with antisense ODN (10 μmol/L) for 48 h resulted in a 76±1.5% (P 0.05) inhibition of¹²⁵I-FSH binding. In contrast, transfection with lipofectamine and 0.33 μmol/L antisense ODN at 0 h caused a 76.1± 1.3% (P<0.05) reduction in binding within 24 h. Binding had returned to 52.3 ± 2.3% (P< 0.05) of normal by 48 h. These results indicate that an antisense ODN corresponding to the region of the translation start site of the porcine FSHR cDNA is an effective specific inhibitor of porcine FSHR synthesis and that inhibition of receptor synthesis causes a decrease in functional membrane-bound FSHR.

Keywords

follicle-stimulating hormone / antisense oligodeoxynucleotide / folliculogenesis / mRNA / spermatogenesis

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