RESUMO
Follicle-stimulating hormone (FSH) receptors (FSHR) are critically involved in mediating the responses of granulosa cells and Sertoli cells to FSH. The dynamic changes in cell surface FSH receptors (FSHR) in response to FSH remain unclear in part because of the heavy reliance on ligand-binding methodologies. This study was designed to determine the molecular and cellular properties of recombinant porcine FSHR using a novel, high-affinity purified polyclonal antibody to the ectodomain of the pFSHR. A full-length porcine FSHR cDNA was cloned and sequenced and recombinant pFSHR protein was stably expressed in a clonal cell line of Chinese hamster ovary cells (pFSHR-CHO). Recombinant receptor was stably expressed in an ovarian cell line with a density similar to that of porcine ovarian cells. A specific polyclonal antibody was generated in chickens to a 100-amino acid fragment of the pFSHR ectodomain. Immunoblotting, immunoprecipitation, indirect immunofluorescence cytochemistry and immunoelectron microscopy were performed using affinity-purified antibody to identify recombinant pFSHR in pFSHR-CHO cells. Immunoblotting of solubilized pFSHR-CHO proteins and immunoprecipitation of pFSHR-CHO protein metabolically labeled with 35S identified a single 74-kDa band in pFSHR-CHO cells; no bands were visualized in mock-transfected CHO cells. Indirect immunofluorescent labeling revealed the presence of pFSHR in pFSHR-CHO cells but not in mock-transfected CHO cells. Immunoelectron microscopy revealed the highest density of pFSHR associated with the plasma membrane and no pFSHR in mock-transfected CHO cells. The chicken anti-pFSHR antibody is a valuable tool for detecting and monitoring of FSHR using a variety of methodologies.
Assuntos
Receptores do FSH/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Imunofluorescência , Expressão Gênica , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Microscopia Imunoeletrônica , Estrutura Terciária de Proteína , Ensaio Radioligante , Receptores do FSH/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Frações Subcelulares/metabolismo , Suínos , Distribuição TecidualRESUMO
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.
