Evaluation of In vivo Bioactivities of Recombinant Hypo- (FSH) and Fully- (FSH) Glycosylated Human FSH Glycoforms in Fshb Null Mice

The hormone - specific FSHβ subunit of the human FSH heterodimer consists of N-linked glycans at Asn and Asn 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, missing either an Asn or Asn N-glycan chain on the β - subunit, respectively) or fully glycosylated (FSH, possessing of both Asn and Asn N-linked glycans on the β - subunit) FSH. The recombinant versions of human FSH glycoforms (FSH and FSH) have been purified and biochemically characterized. In vitro functional studies have indicated that FSH exhibits faster FSH- receptor binding kinetics and is much more active than FSH in every assay tested to date. However, the in vivo bioactivity of the hypo-glycosylated 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 48 h and induced ovarian genes in a dose- and time-dependent manner. Quantification by real time qPCR assays indicated that hypo-glycosylated FSH was bioactive in vivo and induced FSH-responsive ovarian genes similar to fully-glycosylated FSH. Western blot analyses followed by densitometry of key signaling components downstream of the FSH-receptor confirmed that the hypo-glycosylated FSH elicited a response similar to that by fully-glycosylated FSH in ovaries of Fshb null mice. When injected into Fshb null males, hypo-glycosylated FSH was more active than the fully-glycosylated FSH in inducing FSH-responsive genes and Sertoli cell proliferation. Thus, our data establish that recombinant hypo-glycosylated human FSH 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.