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A human FSHB transgene encoding the double N-glycosylation mutant (Asn(7?) Asn(24?)) FSH? subunit fails to rescue Fshb null mice.

ABSTRACT: Follicle-stimulating hormone (FSH) is a gonadotrope-derived heterodimeric glycoprotein. Both the common ?- and hormone-specific ? subunits contain Asn-linked N-glycan chains. Recently, macroheterogeneous FSH glycoforms consisting of ?-subunits that differ in N-glycan number were identified in pituitaries of several species and subsequently the recombinant human FSH glycoforms biochemically characterized. Although chemical modification and in vitro site-directed mutagenesis studies defined the roles of N-glycans on gonadotropin subunits, in vivo functional analyses in a whole-animal setting are lacking. Here, we have generated transgenic mice with gonadotrope-specific expression of either an HFSHB(WT) transgene that encodes human FSH? WT subunit or an HFSHB(dgc) transgene that encodes a human FSH?(Asn7? 24?) double N-glycosylation site mutant subunit, and separately introduced these transgenes onto Fshb null background using a genetic rescue strategy. We demonstrate that the human FSH?(Asn7? 24?) double N-glycosylation site mutant subunit, unlike human FSH? WT subunit, inefficiently combines with the mouse ?-subunit in pituitaries of Fshb null mice. FSH dimer containing this mutant FSH? subunit is inefficiently secreted with very low levels detectable in serum. Fshb null male mice expressing HFSHB(dgc) transgene are fertile and exhibit testis tubule size and sperm number similar to those of Fshb null mice. Fshb null female mice expressing the mutant, but not WT human FSH? subunit-containing FSH dimer are infertile, demonstrate no evidence of estrus cycles, and many of the FSH-responsive genes remain suppressed in their ovaries. Thus, HFSHB(dgc) unlike HFSHB(WT) transgene does not rescue Fshb null mice. Our genetic approach provides direct in vivo evidence that N-linked glycans on FSH? subunit are essential for its efficient assembly with the ?-subunit to form FSH heterodimer in pituitary. Our studies also reveal that N-glycans on FSH? subunit are essential for FSH secretion and FSH in vivo bioactivity to regulate gonadal growth and physiology.

SUBMITTER: Wang H

PROVIDER: S-EPMC5130991 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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A human FSHB transgene encoding the double N-glycosylation mutant (Asn(7Δ) Asn(24Δ)) FSHβ subunit fails to rescue Fshb null mice.

Wang Huizhen H Butnev Vladimir V Bousfield George R GR Kumar T Rajendra TR

Molecular and cellular endocrinology 20160219

Follicle-stimulating hormone (FSH) is a gonadotrope-derived heterodimeric glycoprotein. Both the common α- and hormone-specific β subunits contain Asn-linked N-glycan chains. Recently, macroheterogeneous FSH glycoforms consisting of β-subunits that differ in N-glycan number were identified in pituitaries of several species and subsequently the recombinant human FSH glycoforms biochemically characterized. Although chemical modification and in vitro site-directed mutagenesis studies defined the ro ...[more]

PMID: 26911932

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Project description:The hormone - specific FSH? 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 FSH? subunit contribute to macroheterogeneity in the FSH heterodimer. The resulting FSH glycoforms are termed hypo-glycosylated (FSH21/18, missing either an Asn24 or Asn7 N-glycan chain on the ? - subunit, respectively) or fully glycosylated (FSH24, possessing of both Asn7 and Asn24 N-linked glycans on the ? - subunit) FSH. The recombinant versions of human FSH glycoforms (FSH21/18 and FSH24) have been purified and biochemically characterized. In vitro functional studies have indicated that FSH21/18 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 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 FSH21/18 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 FSH21/18 elicited a response similar to that by fully-glycosylated FSH24 in ovaries of Fshb null mice. When injected into Fshb null males, hypo-glycosylated FSH21/18 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 FSH21/18 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.

Dataset Information

A human FSHB transgene encoding the double N-glycosylation mutant (Asn(7?) Asn(24?)) FSH? subunit fails to rescue Fshb null mice.

Publications

A human FSHB transgene encoding the double N-glycosylation mutant (Asn(7Δ) Asn(24Δ)) FSHβ subunit fails to rescue Fshb null mice.

Similar Datasets

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