A deleted form of follicle stimulating hormone (FSH) receptor, found in women with low response to FSH, impairs the function of the normal receptor when co-expressed in vitro

Abstract

OBJECTIVE: FSH mediates cyclic follicle growth and is widely used for controlled ovarian stimulation. The ovarian response to FSH is variable, and has been partly attributed to two common variants of the FSH receptor (FSHR). In addition, we have recently identified four abnormal FSHR splicing products in the cumulus cells of 37% (13 of 35) of infertile women tested [Gerasimova et al. JCEM 2010]. All alterations affected the extracellular part of the receptor. All patients with variant receptors also possessed normal FSHR. Our aim in this study was to determine the molecular mechanism of action of one of the mutant receptor forms. DESIGN: Investigations of cell lines expressing wild-type and mutant FSHR. MATERIALS AND METHODS: We established stable HEK293 cell lines expressing wild-type [FSHR(wt)] and splice-variant receptor [FSHR(del ex2)] under the control of the inducible Tet-off system. Intracellular cAMP, the first messenger produced upon FSH stimulation, was measured after the cells were stimulated with 0-1000 mIU/ml FSH for 12 h. Protein interactions were also tested by co-immunoprecipitation. Plasma membrane proteins were visualized using surface biotinylation. Subcellular localization was determined by immunofluorescence and fractionation in discontinuous sucrose gradients. RESULTS: In vitro expression of FSHR(del ex2) alone did not elicit a response to FSH, while co-expression of FSHR(del ex2) with FSHR(wt) resulted in a significant reduction of the wt activity when stimulated with FSH. Immunostaining and surface biotinylation showed that, unlike the FSHR(wt), less than half of the total amount of FSHR(del ex 2) protein is present on the cell surface, and a large amount of protein remains trapped in intracellular compartments. CONCLUSION: Our findings suggest that FSHR variants can contribute to abnormal response to stimulation in certain women undergoing IVF. The variant receptors require the presence of wt but can significantly alter the receptor's activity, resulting in less than optimal ovarian stimulation.