Assessment of recombinant porcine follicle-stimulating hormone receptor using a novel polyclonal ectodomain antibody.
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.
Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Jianqiang
J
Wu
Yifang
Y
Wang
Weihong
W
Xiao
Kevin B
KB
Meyer
Kara M
KM
Schmidt
Randal E
RE
Morris
Sandra J F
SJ
Degen
Andrew R
AR
La Barbera
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D016253:Microscopy, Immunoelectron; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D011962:Receptors, FSH / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine; D014018:Tissue Distribution
D011962:Receptors, FSH; D011994:Recombinant Proteins