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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:Although the mechanisms by which mutant huntingtin (mHtt) results in Huntington's disease (HD) remain unclear, mHtt-induced mitochondrial defects were implicated in HD pathogenesis. The effect of mHtt could be mediated by transcriptional alterations, by direct interaction with mitochondria, or by both. In the present study, we tested a hypothesis that mHtt directly damages mitochondria. To test this hypothesis, we applied brain cytosolic fraction from YAC128 mice, containing mHtt, to brain non-synaptic and synaptic mitochondria from wild-type mice and assessed mitochondrial respiration with a Clark-type oxygen electrode, membrane potential and Ca2+ uptake capacity with tetraphenylphosphonium (TPP+ )- and Ca2+ -sensitive electrodes, respectively, and, reactive oxygen species production with Amplex Red assay. The amount of mHtt bound to mitochondria following incubation with mHtt-containing cytosolic fraction was greater than the amount of mHtt bound to brain mitochondria isolated from YAC128 mice. Despite mHtt binding to wild-type mitochondria, no abnormalities in mitochondrial functions were detected. This is consistent with our previous results demonstrating the lack of defects in brain mitochondria isolated from R6/2 and YAC128 mice. This, however, could be because of partial loss of mitochondrially bound mHtt during the isolation procedure. Consequently, we increased the amount of mitochondrially bound mHtt by incubating brain non-synaptic and synaptic mitochondria isolated from YAC128 mice with mHtt-containing cytosolic fraction. Despite the enrichment of YAC128 brain mitochondria with mHtt, mitochondrial functions (respiration, membrane potential, reactive oxygen species production, Ca2+ uptake capacity) remained unchanged. Overall, our results suggest that mHtt does not directly impair mitochondrial functions, arguing against the involvement of this mechanism in HD pathogenesis. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

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.

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