The induction of granulosa cell differentiation and follicle maturation is dependent upon the stimulatory actions of FSH. Our recent studies used recombinant DNA technology to fuse the carboxyl-terminal peptide (CTP) of hCG β-subunit to the carboxyl-terminus of the FSH β-subunit. The resulting FSH analog has identical in vitro receptor-binding and biological activities as wild-type FSH (WT-FSH), but an increased circulating half-life. The present studies examined further the ability of FSH with one (FSH-CTP1) or two (FSH-CTP2) appended CTPs to promote granulosa cell differentiation and follicle ovulatory potential. WT-FSH, FSH-CTP1, and FSH-CTP2 were produced from Chinese hamster ovary cells transfected with the common a-subunit and respective β-subunit. Hormone concentrations were quantitated by RIA, and relative levels confirmed by radioligand receptor assay. Both FSH-CTPl and FSH-CTP2 retained full FSH receptor-binding activity, but did not bind LH receptors. To compare in vivo bioactivity, immature estrogen-primed female rats received ip injections of FSH or the agonists at 0 and 24 h. At 48 h, substantial stimulation (up to 2.5- fold) of ovarian weight was induced by 1.0 and 3.0 IU/day FSH-CTPl or FSH-CTP2, whereas a higher dose (10 IU/day) of WT-FSH was required for an 1.8-fold stimulation. Although the in vivo potencies of FSH-CTPl and FSH-CTP2 were similar, FSH-CTPs were about 10- fold more potent than WT-FSH in inducing granulosa cell aromatase activity and LH receptors. We further reduced the frequency of hormone administration. Increasing doses (1-10 IU) of a single ip injection of FSH-CTP1 resulted in dose-dependent increases in granulosa cell aromatase activity and LH receptor content 48 h later. Although a single injection (10 IU) of WT-FSH had no effect, the same total dose of WT-FSH administered as four 2.5-IU injections 12 h apart was effective. To test the ovulatory potential of ovarian follicles, rats received a single injection of FSH-CTPl, followed 52 h later by 5 IU hCG to induce ovulation. Although hCG did not induce ovulation in females receiving a single dose (10 IU) of WT-FSH, 20 + 2 and 43 ± 5 ovulated ova/rat were found in animals primed with 3 and 10 IU FSH- CTPl, respectively. Because twice daily injections of WT-FSH (2.5 IU/injection) also increased the ovulatory potential of the ovary, the enhanced effectiveness of FSH-CTPl appears to be related to its increased circulating half-life. The present results demonstrated the stimulation of follicle maturation and ovulatory potential by long- acting human FSH agonists with extended CTPs. The availability of these recombinant FSH agonists with increased in vivo potency should provide an improved means of inducing follicle development and ovulatory potential as well as a model to elucidate the role of CTP of the CGβin hormone processing in vivo.
ASJC Scopus subject areas