Anti-Human FSH Receptor Monoclonal Antibodies:  Immunochemical and Immunocytochemical Characterization of the Receptor

Abstract

The extracellular domain of the human FSH receptor was expressed in Escherichia coli as a fusion protein with ubiquitin. It was tagged with a poly-His tract which was used for its purification. Immunization of mice allowed the preparation of high affinity antireceptor monoclonal antibodies. The latter fell into two categories: some of them were inhibited hormone binding and adenylate cyclase activation whereas others were devoid of these properties. None of the antibodies had agonistic activity (i.e., stimulated adenylate cyclase). Immunoaffinity chromatography allowed us to purify the native receptor in a single step either from a permanently transfected L cell line (75% recovery) or from human ovaries (33% recovery). Immunoblotting of the receptor in human ovaries showed the presence of a major band of 87 kDa and of a minor band of 81 kDa. Endoglycosidase digestion and pulse-chase experiments showed the former to be the mature receptor and the latter the precursor containing mannose-rich carbohydrates. Thus, as in the case for the LH receptor, there was an accumulation (albeit to a lower degree) of the precursor in target cells. We did not detect variant forms of the protein corresponding to the alternative mRNA transcripts previously described. Additive binding to the receptor of several antibodies, but not of the same antibody, allowed us to establish a sandwich-type ELISA for the receptor (sensitivity approximately 1 fmol) and to obtain evidence against the existence of previously described oligomeric forms of the protein. All monoclonal antibodies were able to label the receptor immunocytochemically in transfected cells, and two of them were also able to detect it at the markedly lower physiological concentrations, i.e., in human Sertoli and granulosa cells.

Basolateral Localization and Transcytosis of Gonadotropin and Thyrotropin Receptors Expressed in Madin-Darby Canine Kidney Cells

“…Establishment of MDCK Cell Lines Permanently Expressing FSH, LH, and TSH Receptors-MDCK cell lines were obtained and maintained as described (28,29) after cotransfection with expression vectors encoding either hFSHR (pSG5-hFSHR) (21), hTSHR (pSG5-hTSHR) (30), or pLHR (pCMV-LHRA) (8) and with the plasmid pSV-Neo which confers resistance to the antibiotic G418 (31). These clones were screened for receptor expression by immunocytochemistry using monoclonal anti-receptor antibodies.…”

“…Purification and Western Blot Analysis of the Gonadotropin and TSH Receptors Expressed in MDCK Cells-These procedures were performed as described previously (20,21,26,30).…”

“…LH receptor-driven hormone transcytosis was observed through endothelial cells of testicular blood vessels (19). Recently immunocytochemistry using specific monoclonal antibodies demonstrated the basolateral distribution of the TSH receptor in thyroid cells (20) and of the FSH receptor in Sertoli cells (21), whereas the LH receptor was present all over the cell surface of thecal, granulosa, and luteal cells in the ovary and Leydig cells in the testes (22,23).…”

Follicle stimulating hormone‐induced growth promotion and gene expression profiles on ovarian surface epithelial cells

“…Snap-frozen samples were homogenized in homogenizing buffer containing 10 g/mL of leupeptin, 10 g/mL of pepstatin, 10 g/mL of aprotinin, 10 g/mL of benzamidine and 10 g/mL of phenylmethylsulfonyl fluoride. 10 After the lysates were sonicated for 15 sec and centrifuged at 4,000g for 10 min, the supernatant was removed and recentrifuged at 100,000g for 15 min. The pellet fractions were resuspended in ice-cold homogenizing buffer.…”

“…In Western blot analysis, 2 bands at 87 and 81 KDa were detected by FSHR antibody as previously reported. 10 …”