Evaluation of in vivo bioactivities of recombinant hypo- (FSH21/18) and fully- (FSH24) glycosylated human FSH glycoforms in Fshb null mice

摘要

The hormone - specific FSHβ subunit of the human FSH heterodimer consists of N-linked glycans at Asn⁷ and Asn²⁴ residues that are co-translationally attached early during subunit biosynthesis. Differences in the number of N-glycans (none, one or two) on the human FSHβ subunit contribute to macroheterogeneity in the FSH heterodimer. The resulting FSH glycoforms are termed hypo-glycosylated (FSH^21/18, missing either an Asn²⁴ or Asn⁷ N-glycan chain on the β - subunit, respectively) or fully glycosylated (FSH²⁴, possessing of both Asn⁷ and Asn²⁴ N-linked glycans on the β - subunit) FSH. The recombinant versions of human FSH glycoforms (FSH^21/18 and FSH²⁴) have been purified and biochemically characterized. In vitro functional studies have indicated that FSH^21/18 exhibits faster FSH- receptor binding kinetics and is much more active than FSH²⁴ in every assay tested to date. However, the in vivo bioactivity of the hypoglycosylated FSH glycoform has never been tested. Here, we evaluated the in vivo bioactivities of FSH glycoforms in Fshb null mice using a pharmacological rescue approach. In Fshb null female mice, both hypo- and fully-glycosylated FSH elicited an ovarian weight gain response by 48h and induced ovarian genes in a dose- and time-dependent manner. Quantification by real time qPCR assays indicated that hypo-glycosylated FSH^21/18 was bioactive in vivo and induced FSH-responsive ovarian genes similar to fully-glycosylated FSH²⁴. Western blot analyses followed by densitometry of key signaling components downstream of the FSH-receptor confirmed that the hypo-glycosylated FSH^21/18 elicited a response similar to that by fully-glycosylated FSH²⁴ in ovaries of Fshb null mice. When injected into Fshb null males, hypo-glycosylated FSH^21/18 was more active than the fully-glycosylated FSH²⁴ in inducing FSH-responsive genes and Sertoli cell proliferation. Thus, our data establish that recombinant hypo-glycosylated human FSH^21/18 glycoform elicits bioactivity in vivo similar to the fully-glycosylated FSH. Our studies may have clinical implications particularly in formulating FSH-based ovarian follicle induction protocols using a combination of different human FSH glycoforms.