Enhanced stimulation of follicle maturation and ovulatory potential by long acting follicle-stimulating hormone agonists with extended carboxyl-terminal peptides.
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 beta-subunit to the carboxyl-terminus of the FSH beta-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 alpha-subunit and respective beta-subunit. Hormone concentrations were quantitated by RIA, and relative levels confirmed by radioligand receptor assay. Both FSH-CTP1 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-CTP1 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-CTP1 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-CTP1, 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-CTP1, 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-CTP1 appears to be related to its increased circulating half-life.(ABSTRACT TRUNCATED AT 400 WORDS)