Evaluation of in vivo bioactivities of recombinant hypo- (FSH21/18) and fully- (FSH24) glycosylated human FSH glycoforms in Fshb null mice. (5th December 2016)

• Record Type: Journal Article
• Authors: Wang, Huizhen; May, Jacob; Butnev, Viktor; Shuai, Bin; May, Jeffrey V.; Bousfield, George R.; Kumar, T. Rajendra
• Is Part Of: Molecular and cellular endocrinology. Volume 437(2016)
• Page Start: 224
• Page End: 236
• Publication Date: 2016-12-05
• Subjects: FSH-responsive genes -- N-glycosylation -- Ovary -- Testis -- Pharmacological rescue; AMH anti-Müllerian hormone -- cAMP Cyclic adenosine 5′-monophosphate -- CHO Chinese hamster ovary -- CREB Cyclic AMP-responsive element binding protein -- FSH Follicle-stimulating hormone -- LH Luteinizing hormone -- PKA Protein kinase-A -- PVDF Polyvinylidene difluoride -- RIA Radioimmuno assay -- RRA Radioreceptor assay -- r-h Recombinant human; Endocrinology -- Periodicals; Molecular biology -- Periodicals; Cytology -- Periodicals; Endocrinology -- Periodicals; Hormones -- Periodicals; Endocrinologie -- Périodiques; Cytology; Endocrinology; Molecular biology; Periodicals; 573.4
• DOI: 10.1016/j.mce.2016.08.031
• Languages: English
• ISSNs: 0303-7207

Abstract

Abstract: The hormone - specific FSHβ subunit of the human FSH heterodimer consists of N-linked glycans at Asn 7 and Asn 24 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 Asn 24 or Asn 7 N-glycan chain on the β - subunit, respectively) or fully glycosylated (FSH 24, possessing of both Asn 7 and Asn 24 N-linked glycans on the β - subunit) FSH. The recombinant versions of human FSH glycoforms (FSH 21/18 and FSH 24 ) 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 FSH 24 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 21/18 was bioactive in vivo and induced FSH-responsive ovarian genes similar to fully-glycosylated FSH 24 . 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 FSH 24 in ovaries of Fshb null mice. When injected into Fshb null males, hypo-glycosylated FSH 21/18 was more active than the fully-glycosylated FSH 24 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. Highlights: In vivo bioactivity of recombinant hypo (FSH 21/18 ) - and fully (FSH 24 ) - glycosylated FSH was tested in Fshb −/- mice. Both the FSH glycoforms elicited an ovarian weight gain response and induced antrum formation in Fshb −/- female mice. In ovarian gene induction assays, FSH glycoforms were found active in the absence of endogenous FSH. Both the FSH glycoforms elicited a testis weight gain response in Fshb −/- male mice. In Sertoli cell proliferation and testis gene expression assays, FSH 21/18 glycoform was found more active than FSH 24 .