title: Evaluation of in vivo bioactivities of recombinant hypo- (FSH21/18) and fully- (FSH24) glycosylated human FSH glycoforms in Fshb null mice.
abstract: The hormone - specific FSHβ subunit of the human FSH heterodimer consists of N-linked glycans at Asn7and Asn24residues 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 (FSH21/18, missing either an Asn24or Asn7N-glycan chain on the β - subunit, respectively) or fully glycosylated (FSH24, possessing of both Asn7and Asn24N-linked glycans on the β - subunit) FSH. The recombinant versions of human FSH glycoforms (FSH21/18and FSH24) have been purified and biochemically characterized. In vitro functional studies have indicated that FSH21/18exhibits faster FSH- receptor binding kinetics and is much more active than FSH24in 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 FSH21/18was 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 FSH21/18elicited a response similar to that by fully-glycosylated FSH24in ovaries of Fshb null mice. When injected into Fshb null males, hypo-glycosylated FSH21/18was more active than the fully-glycosylated FSH24in inducing FSH-responsive genes and Sertoli cell proliferation. Thus, our data establish that recombinant hypo-glycosylated human FSH21/18glycoform 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.
authors: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors: Huizhen
authors: H
authors: Wang
authors: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors: Jacob
authors: J
authors: May
authors: Department of Biological Sciences, Wichita State University, Wichita, KS 67260, USA.
authors: Viktor
authors: V
authors: Butnev
authors: Department of Biological Sciences, Wichita State University, Wichita, KS 67260, USA.
authors: Bin
authors: B
authors: Shuai
authors: Department of Biological Sciences, Wichita State University, Wichita, KS 67260, USA.
authors: Jeffrey V
authors: JV
authors: May
authors: Department of Biological Sciences, Wichita State University, Wichita, KS 67260, USA.
authors: George R
authors: GR
authors: Bousfield
authors: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA; Center for Reproductive Sciences, Institute for Reproductive Health and Regenerative Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA; Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA; Department of Neurosurgery, University of Kansas Medical Center, Kansas City, KS 66160, USA; Division of Reproductive Sciences, Department of Obstetrics & Gynecology, University of Colorado Denver-Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address: raj.kumar@ucdenver.edu.
authors: T Rajendra
authors: TR
authors: Kumar
mesh_terms: D000818:Animals; D015153:Blotting, Western; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D005260:Female; D043373:Follicle Stimulating Hormone, Human / Q000737:chemistry / Q000494:pharmacology*; D037201:Follicle Stimulating Hormone, beta Subunit / Q000172:deficiency* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D008297:Male; D018345:Mice, Knockout; D010053:Ovary / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013997:Time Factors
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D043373:Follicle Stimulating Hormone, Human; D037201:Follicle Stimulating Hormone, beta Subunit; D011994:Recombinant Proteins
keywords: FSH-responsive genes; N-glycosylation; Ovary; Pharmacological rescue; Testis