A human FSHB transgene encoding the double N-glycosylation mutant (Asn7Δ Asn24Δ) 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, macro-heterogeneous 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 HFSHBWT transgene that encodes human FSHβ WT subunit or an HFSHBdgc transgene that encodes a human FSHβAsn7Δ 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 FSHβAsn7Δ 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 HFSHBdgc 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, HFSHBdgc unlike HFSHBWT 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.

Evaluation of in vivo bioactivities of recombinant hypo- (FSH 21/18 ) and fully- (FSH 24 ) glycosylated human FSH glycoforms in Fshb null mice

“…This strategy permitted us to monitor the in vivo bioactivity of the injected FSH glycoforms on an identical Fshb null genetic background and in the absence of endogenous FSH. We took advantage of our previously made observation that Fshb null mice retain full FSH-responsiveness to exogenous FSH [22] and these mice could be rescued both genetically [21, 23, 24] and pharmacologically [22, 41, 42]. Our data in this manuscript are consistent with these previously published reports and confirm that the GH 3 -cell-derived recombinant FSH glycosylation variants are biologically active in vivo .…”

“…Fshb null mice were generated and genotyped by genomic PCR assays performed on tail DNA samples as described before [23, 24]. For the in vivo bioactivity testing experiments, immature Fshb null female mice at 21d-22d of age or Fshb null male pups at postnatal day 3 or 5 were used.…”

“…Aliquots of 10-15 μg of protein were denatured in SDS-PAGE sample buffer (final concentration = 32 mM Tris-HCl, pH 6.8, 12.5% glycerol (v/v), 1% SDS, and 31 μM β-mercaptoethanol) at 100C for 5 min., separated on 12 % polyacrylamide gels, and transferred onto PVDF membranes as described [23-25]. The membranes were blocked in 5 % non-fat dry milk and incubated with primary antibodies (Cell Signaling Systems, 1:2, 000) at room temperature for 4-5 h, washed in 0.1 % Tween-20 containing buffer, incubated in goat anti-rabbit HRP conjugate (1:4,000) as described [23-25]. The antigen-antibody complexes were visualized by an enhanced chemiluminescence, ECL III detection kit (GE Health Care).…”

In Vivo and In Vitro Impact of Carbohydrate Variation on Human Follicle-Stimulating Hormone Function

“…Real-time qPCR assays, immuno co-localization, and Western blot analyses under denaturating conditions confirmed that the transgene encoded mRNA and the corresponding subunits were abundantly expressed in pituitaries ( 21 ). While WT human FSHβ subunit-containing, inter-species hybrid FSH was readily detectable by Western blot analysis under non-denaturing conditions of HFSHB WT mouse pituitaries, FSH dimer containing double N-glycosylation-mutant human FSHβ subunit was barely detectable in pituitaries of HFSHB WT mice on an Fshb null genetic background ( 21 ). Consistent with these expression data, mutant FSHβ subunit-containing FSH dimer was not detectable in either short-term pituitary organ culture media or serum samples by specific RIAs ( 21 ).…”

“…Pituitary extracts also possess a non-glycosylated, 15-kDa FSHβ variant ( 20 ). However, the corresponding FSH 15 does not appear to be physiologically relevant, because subunit association is extremely inefficient when both FSHβ glycans are missing, and little, if any, FSH heterodimer is secreted ( 21 ). FSH 24 and FSH 21 are detected in FSH derived from human pituitary extracts, as well as from urinary protein preparations (Table 1 ).…”