title: A human FSHB transgene encoding the double N-glycosylation mutant (Asn(7Δ) Asn(24Δ)) FSHβ subunit fails to rescue Fshb null mice.
abstract: 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 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 FSHβ WT subunit or an HFSHB(dgc) transgene that encodes a human FSHβ(Asn7Δ 24Δ) 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 FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, unlike human FSHβ WT subunit, inefficiently combines with the mouse α-subunit in pituitaries of Fshb null mice. FSH dimer containing this mutant FSHβ 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 FSHβ 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 FSHβ subunit are essential for its efficient assembly with the α-subunit to form FSH heterodimer in pituitary. Our studies also reveal that N-glycans on FSHβ subunit are essential for FSH secretion and FSH in vivo bioactivity to regulate gonadal growth and physiology.
authors.affiliation: Department of Molecular and Integrative Physiology.
authors.forename: Huizhen
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Biological Sciences, Wichita State University, Wichita, KS 67260, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Butnev
authors.affiliation: Department of Biological Sciences, Wichita State University, Wichita, KS 67260, USA.
authors.forename: George R
authors.initials: GR
authors.lastname: Bousfield
authors.affiliation: Department of Molecular and Integrative Physiology; Center for Reproductive Sciences, Institute for Reproductive Health and Regenerative Medicine; Department of Pathology and Laboratory Medicine; Department of Neurosurgery, University of Kansas Medical Center, Kansas City, KS 66160, USA. Electronic address: tkumar@kumc.edu.
authors.forename: T Rajendra
authors.initials: TR
authors.lastname: Kumar
mesh_terms: D000818:Animals; D005260:Female; D037201:Follicle Stimulating Hormone, beta Subunit / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D008297:Male; D018345:Mice, Knockout; D010053:Ovary / Q000002:abnormalities / Q000473:pathology; D011499:Protein Processing, Post-Translational; D013737:Testis / Q000002:abnormalities / Q000473:pathology; D019076:Transgenes
chemical_list: D037201:Follicle Stimulating Hormone, beta Subunit
keywords: Genetic rescue; Gonadotropin; N-glycosylation; Ovary; Pituitary; Testis