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Antisense oligodeoxynucleotide inhibits expression of recombinant porcine follicle‐stimulating hormone receptor
Author(s) -
Zhu Changhong,
Wang Yifang,
Nixon Mark D.,
La Barbera Andrew R.
Publication year - 2003
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.10310
Subject(s) - follicle stimulating hormone receptor , biology , chinese hamster ovary cell , microbiology and biotechnology , messenger rna , receptor , recombinant dna , follicle stimulating hormone , endocrinology , gene , biochemistry , hormone , luteinizing hormone
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 μM 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 μM 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 μM) 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 μM) 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. Mol. Reprod. Dev. 65: 188–193, 2003. © 2003 Wiley‐Liss, Inc.