COMPARATIVE INVITRO AND INVIVO STUDIES ON THE BIOLOGICAL CHARACTERISTICS OF RECOMBINANT HUMAN FOLLICLE-STIMULATING-HORMONE

The in vitro and in vivo activities of recombinant human FSH (recFSH) produced by a Chinese hamster ovary cell line were studied and compared with those of natural FSH preparations. The specific FSH activities of recFSH established by immunoassay and in vivo bioassay were greater than 10,000 IU/mg protein and considerably higher than the activities of tested urinary FSH references, while the in vivo bio/immuno ratios of these preparations were not significantly different. Compared to a highly purified pituitary standard (IS 83/575), recFSH had a comparable high specific in vivo bioactivity, but the specific immunoreactivity of IS 83/575 was about 2 times lower. In receptor displacement and in vitro bioassay studies recFSH provided dose-response curves parallel to those of pituitary and urinary FSH references. When equal amounts of immunoreactive FSH were tested, recFSH and urinary and pituitary FSH displayed comparable activities in both assays. The in vitro bioactivity of recFSH could be neutralized effectively by each of three monoclonal antibodies raised against recFSH (alpha-specific), urinary FSH (beta-specific), and pituitary FSH (alpha-beta-specific), respectively. Moreover, 50% inhibition of comparable responses induced by recFSH, urinary "pure" FHS, or pituitary FSH was established by the same amount of monoclonal antibody. These results support the structural and functional similarity of recFSH and natural FSH. To test whether recFSH is capable of inducing LH-specific biological responses, the in vitro induction of testosterone production in mouse Leydig cells was assessed. At least 16 IU recFSH/ml incubate were needed to increase testosterone production, indicating that the intrinsic LH bioactivity of recFSH is negligible (< 0.025 mIU LH/IU FSH). The in vivo efficacy of recFSH was examined by treating immature female hypophysectomized rats during 4 days with recFSH only or with recFSH supplemented with hCG. RecFSH only treatment increased ovarian weight and aromatase activity in a dose-dependent manner. When recFSH dosages providing submaximal responses were supplemented with 1 IU hCG, both ovarian weight and aromatase activity were largely augmented. Neither recFSH nor urinary pure FSH, administered in a high dose was able to increase plasma estradiol levels, while ovarian weight and aromatase activity were increased to the same extent. However, when recFSH was supplemented with only 0.1 IU hCG, a 3-fold increase in median plasma estradiol level was obtained. These findings support the two-cell two-gonadotropin theory, holding that both FSH and LH are required for estrogen biosynthesis, but also reveal that only very small amounts of LH activity are sufficient to increase estrogen secretion up to measurable plasma levels. The increases in ovarian weight, aromatase activity, and plasma estradiol induced by recFSH supplemented with 1 and 10 IU hCG were comparable to those induced by two human menopausal gonadotropin references. In conclusion, the present study demonstrates that the in vitro and in vivo biological characteristics of recFSH are indistinguishable from those of FSH isolated from natural sources.