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Prakash Thiruppathi1 , James Dias2 *, Judith Luborsky1 *
Dept Obstet & Gynecol, Rush Medical College, Chicago, IL 1
Wadsworth Center, NYSDOH, Albany, NY 2
Expression of gonadotropin receptors is essential for normal gonadal responsiveness to gonadotropins. The objective of this study was to assess FSH receptors (hFSHR) on human granulosa cells (GC) by flow cytometry. Labeling of hFSHR with rabbit antiserum to a peptide domain (R265-S296) of the human receptor (Liu et. al., Endocrinol, 1994, 135:682) and antirabbit-FITC was optimized with CHO cells transfected with hFSHR (Ares Advanced Technologies). Controls included normal rabbit serum and antiserum adsorbed with antigen. Binding and antiserum dilution (1:50-1:500) were linearly related. Binding saturation was achieved at 100,000 cells/0.2 ml antiserum (1:100). The fluorescent signal was converted to number of bound molecules with beads containing a known amount of antimouse immunoglobulin (FCS Corp). The estimated receptors/CHO-hFSHR cell was 15,423 or about 10-fold less than estimates by radiolabeled hormone binding. A similar procedure was used to assess FSHR on GCs obtained at the time of oocyte retrieval (36 hours post-hCG stimulation) during IVF; in the 10 subjects studied (mean age 31±6.5), peak estradiol response to hMG averaged 1337.5±743 pg/ml (range 604-2340 pg/ml). Lymphocytes were identified and gated out with antiCD45-PE. The number of FSHR/GC ranged from 1806-6113 (3612±1874). Bound FSH and hCG on GC were also detected using rabbit antibody to FSH or hCG, correlating with the presence of both FSHR and LHR. Thus, hFSHR could be detected and receptor number estimated by flow cytometry on transfected CHO cells and GC. The results show FSHR expression on human GC at the transition into the early luteal phase. It also provides a basis for determining if gonadotropin resistance in some women with compromised fertility can be explained by altered receptor expression rather than by diminished ovarian reserve. Supported by HD18407.
This abstract is being presented on Tuesday, August 3 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.