Recent studies have shown that Chinese hamster ovary (CHO) cells transfected with the FSH subunit genes secrete bioactive FSH. Here, we determined the in vitro and in vivo bioactivity of recombinant FSH produced by CHO mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase-I (NAGT-), resulting in glycoproteins with asparagine-linked (GlcNAc)2(Mannose)5 oligosaccharides, or mutant cells defective in sialic acid transport into the Golgi (ST-). In the latter, glycoproteins are secreted lacking terminal sialic acids. Determination of in vitro bioactivity, using the granulosa cell aromatase bioassay, indicated that both FSH variants are as active as FSH secreted by the wild type (WT) cells and purified pituitary FSH. Also, these normal and variant forms of FSH are equipotent in a radioligand receptor assay using rat testis membranes. However, the variant FSH molecules are more basic than the WT FSH as determined using a chromatofocusing column (pI: wild type 3.6-5.0, NAGT- greater than 7.0, ST- approximately 6.0 and greater than 7.0). Injection of immature estrogen-treated rats with WT FSH induced high aromatase activity in their granulosa cells whereas treatment with either one of the FSH variants was ineffective; the lack of in vivo activity of the FSH variants was correlated with rapid clearance of these molecules in serum. Thus, recombinant human FSH produced by cells deficient in NAGT-I or defective in sialic acid transport retains normal receptor binding and in vitro bioactivity, but exhibits minimal in vivo activity and a shortened half-life when compared to WT FSH, indicating the important role of terminal sugars for FSH action in vivo.

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Ovulation in mammals is preceded by surges of the two pituitary gonadotropins, LH and FSH. Although previous studies have shown that purified FSH induces ovulation when administered to hypophysectomized rats, proof that FSH has inherent ovulatory potential is lacking because all FSH preparations have varying degrees of residual LH. To determine if FSH alone can induce ovulation, we generated LH-free recombinant FSH (RCFSH) by culturing eukaryotic cells transfected with the human common alpha- and FSH beta-subunit genes. Immature hypophysectomized rats were implanted with estrogen and then primed with PMSG (15 IU, sc). Fifty-two hours later, either RCFSH or hCG was injected (sc) to induce ovulation. A dose-dependent increase in the ovulation rate was stimulated by RCFSH, reaching 100% ovulation at 18 IU/rat, comparable to that achieved with 12 IU hCG. The maximum number of oocytes ovulated per ovary was similar for both groups. Ovulation induced by either RCFSH or hCG was time dependent and associated with a periovulatory increase in the ovarian activity and message levels of tissue-type plasminogen activator, a protease important in the preovulatory degradation of the follicle wall. Because PMSG has inherent LH-like activity in rats, we also implanted hypophysectomized rats with a minipump (sc) that released RCFSH (4 IU/day) to induce follicle growth. Fifty-two hours later, a single sc injection of a surge dose (20 IU) of RCFSH also induced ovulation, further indicating the ability of FSH alone to induce both follicle growth and ovulation. To test whether FSH can also induce ovulation in adult animals, rats were hypophysectomized on proestrous morning and treated with increasing doses of RCFSH (ip) to induce ovulation. At 7.8 IU RCFSH, all rats ovulated, with about 10 oocytes/rat. These results demonstrate that RCFSH is capable of inducing ovulation in hypophysectomized immature and adult rats, with associated increases in ovarian tissue-type plasminogen activator gene expression. Thus, FSH may be involved in follicular rupture in addition to its role in follicle recruitment and maturation. The preovulatory surges of both LH and FSH may represent a protective mechanism to ensure an optimal ovulatory stimulus. The present finding also serves as the basis to formulate new ovulation induction protocols.