Hypoglycosylated hFSH Has Greater Bioactivity Than Fully Glycosylated Recombinant hFSH in Human Granulosa Cells

Jiang, Chao; Hou, Xiaoying; Wang, Cheng; May, Jeffrey V.; Butnev, Viktor Y.; Bousfield, George R.; Davis, John S.

doi:10.1210/jc.2015-1317

Cited by 60 publications

(73 citation statements)

References 43 publications

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“…Western blot analysis of FSH preparations was performed with the FSHβ subunit-specific primary antibody RFSH20 (1:5000) that detected two bands corresponding to 18 KDa and 21 KDa in the FSH 21/18 preparation (Fig.1B, Lane 1), and a single band at 24KDa in the case of FSH 24 and hFSH AFP 7298 reference preparation (Fig. 1B, Lanes 2 and 3) as previously reported [6, 17, 18]. An independent blot similarly loaded as above with FSH preparations, was probed with an α-subunit antiserum, which detected a band in all the lanes (Fig.…”

Section: Methodsmentioning

confidence: 79%

“…The existence of macro-heterogeneity in FSH preparations contributed by FSHβ subunits differing in N-glycosylation was recently confirmed [4-6, 14, 17-19]. Four FSH glycoforms were identified by direct purification from pituitary extracts [4-7, 14, 17-19] and following recombinant expression of genes encoding specific N-glycosylation mutant FSHβ subunits [4-7, 14, 17-19].…”

Section: Discussionmentioning

confidence: 99%

“…1A). The purification and characterization of the hormones from GH 3 -conditioned media were described in detail elsewhere [6, 17, 18]. Western blot analysis of FSH preparations was performed with the FSHβ subunit-specific primary antibody RFSH20 (1:5000) that detected two bands corresponding to 18 KDa and 21 KDa in the FSH 21/18 preparation (Fig.1B, Lane 1), and a single band at 24KDa in the case of FSH 24 and hFSH AFP 7298 reference preparation (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…It was predicted that such age-related FSH glycoforms act differently in different target cells [4, 14, 17]. Direct purification of FSH glycoforms from human pituitaries and/or urine collected at different ages and their characterization indeed identified hypo-glycosylated FSH is a mixture of FSH 18 and FSH 21 glycoforms present as the dominant form in young age and fully-glycosylated FSH, designated as FSH 24 is the dominant form in old age [4, 6, 14, 17-20]. Recombinant expression in a heterologous GH 3 cell culture system followed by purification and biochemical characterization revealed that these FSH glycoforms differ in the number or location of N-linked glycan chains on FSHβ subunit [4-7, 14].…”

Section: Introductionmentioning

confidence: 99%

“…More recently, these FSH glycoforms have also been tested in an FSH-responsive KGN granulosa cell line [17]. However, the in vivo bioactivities of GH 3 -derived recombinant human FSH glycoforms have not yet been directly tested.…”

Section: Introductionmentioning

confidence: 99%

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

Wang

May

Butnev

et al. 2016

Molecular and Cellular Endocrinology

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The hormone - specific FSHβ 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 FSHβ 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 β - subunit, respectively) or fully glycosylated (FSH24, possessing of both Asn7 and Asn24 N-linked glycans on the β - 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 hypoglycosylated 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 48h 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.

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Section: Methodsmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Wang

May

Butnev

et al. 2016

Molecular and Cellular Endocrinology

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Gonadotropins

Ulloa‐Aguirre¹,

Dias²,

Bousfield³

2017

Endocrinology

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Gonadotrophin Receptors

Casarini¹,

Huhtaniemi²,

Simoni³

et al. 2016

Endocrinology

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The two gonadotrophin receptors (GnRs), luteinizing hormone receptor (LHCGR) and follicle-stimulating receptor (FSHR), belong to the glycoprotein hormone receptor subgroup of type A G protein-coupled receptors (GPCRs). LHCGR binds specifically the two structurally similar gonadotrophins, luteinizing hormone (LH) and human chorionic gonadotrophin (hCG), and FSHR binds follicle-stimulating hormone (FSH). The receptors reside on plasma membrane and transmit the gonadotrophin signal to target cells using the classical Gs/adenylyl cyclase/cyclic AMP/protein kinase A signaling cascade. Other signaling pathways (e.g., inositol phosphate, calcium) are activated at pharmacological hormone concentrations or at high receptor density. LHCGR is expressed in testicular Leydig cells and in ovarian theca, luteinizing granulosa and luteal cells. FSHR is expressed in testicular Sertoli cells and ovarian granulosa cells. LHCGR activation stimulated Leydig cell steroidogenesis, in particular testosterone production, while FSHR maintains Sertoli cell metabolism, thereby indirectly stimulating spermatogenesis. Recent basic research, using GnR, expressing cells in vitro and genetically modified mice in vivo, has elucidated novel aspects of the molecular mechanisms of gonadotrophin receptor function. The crystal structure of GnRs has also been partly resolved. Numerous inactivating and activating GnR mutations that have been discovered in patients have unraveled the molecular basis of hypogonadism and other aberrations of reproductive endocrine functions. The purpose of this chapter is to review the recent trends of GnR research and how it has elucidated the molecular mechanisms of GnR function and the role of GnR in human reproductive physiology and pathophysiology

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Hypoglycosylated hFSH Has Greater Bioactivity Than Fully Glycosylated Recombinant hFSH in Human Granulosa Cells

Cited by 60 publications

References 43 publications

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

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

Gonadotropins

Gonadotrophin Receptors

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