Antisense oligodeoxynucleotide inhibits expression of recombinant porcine follicle-stimulating hormone receptor.
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.
Zhu
Changhong
C
Department of Obstetrics and Gynecology, Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0526, USA.
Wang
Yifang
Y
Nixon
Mark D
MD
La Barbera
Andrew R
AR
D000818:Animals; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D007457:Iodine Radioisotopes / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D011962:Receptors, FSH / Q000096:biosynthesis / Q000235:genetics*; D013552:Swine / Q000235:genetics / Q000378:metabolism
D007457:Iodine Radioisotopes; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D011962:Receptors, FSH; D005640:Follicle Stimulating Hormone