Delineation of regions in the extracellular domain of follicle-stimulating hormone receptor involved in hormone binding and signal transduction

PROBLEM: To use antipeptide antibodies to potential surface-oriented regions of the extracellular domain (ECD) of the human follicle-stimulating hormone receptor (hFSHR) to delineate regions involved in FSH binding and FSH-induced signal transduction. METHOD OF STUDY: We developed and characterized antipeptide antibodies to different, potentially surface-oriented regions of the ECD of hFSHR. The ability of these antibodies to recognize the receptor was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting. The ability to modulate FSH binding and cAMP generation was studied by the radioreceptor assay and in vitro FSH bioassay respectively. RESULTS: Antipeptide antibodies to regions 15-31, 216-235, 285-300 and 327-341 hFSHR inhibited both FSH binding and cAMP production. Regions 15-31 and 216-235 were accessible to their cognate antipeptide antibodies both before and after FSH binding,. while regions 285-300 and 327-341 hFSHR were accessible only prior to FSH binding. CONCLUSIONS: Based on the observations made with respect to accessibility to antipeptide antibodies, ability of antibodies to inhibit FSH binding and the subsequent cAMP generation and kinetics of antibody binding, regions 285-300 and 327-341 hFSHR appear to be the chief FSH-binding sites, while regions 15-31 and 216-235 hFSHR serve as ancillary FSH-binding sites

<span style="font-size:11.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language: HI" lang="EN-GB">Polymeric nanoparticle formulation of Octapeptide (NP-OP): <i style="mso-bidi-font-style:normal">In vitro</i> release and <i style="mso-bidi-font-style:normal">in vivo</i> effect in common marmosets, <i style="mso-bidi-font-style:normal">Callithrix jacchus </i>Linn.</span>

1055-1062Octapeptide (OP)/FSH-Receptor Binding Inhibitor-8 (FRBI-8), is a synthetic peptide corresponding to N-terminal sequence of purified fraction of Follicle Stimulating Hormone Binding-Inhibitor (FSHBI), isolated earlier from human ovarian follicular-fluid. In order to avoid the repeated drug-administration, OP-loaded, polymeric polylactide (PLA) nanoparticle formulation (NP-OP), was developed using multiple-emulsion technique. This yielded an average particle size of 120 nm with 70% encapsulation-efficiency. In vitro release profile of NP-OP showed sustained release of OP for 21 days. In vivo anti-fertility studies were conducted in marmosets. Results indicated that control animals conceived in the same cycle while two of three treated animals failed to conceive in treatment cycle. The <i style="mso-bidi-font-style: normal">in vivo studies thus corroborate with in vitro release of OP, demonstrating its anti-fertility activity in 66% of animals. </span