Hypoglycosylated hFSH Has Greater Bioactivity Than Fully Glycosylated Recombinant hFSH in Human Granulosa Cells.
BACKGROUND
Previous studies suggest that aging in women is associated with a reduction in hypoglycosylated forms of FSH.


OBJECTIVE
Experiments were performed to determine whether glycosylation of the FSHβ subunit modulates the biological activity of FSH in human granulosa cells.


METHODS
Recombinant human FSH (hFSH) derived from GH3 pituitary cells was purified into fractions containing hypoglycosylated hFSH(21/18) and fully glycosylated hFSH(24). The response to FSH glycoforms was evaluated using the well-characterized, FSH-responsive human granulosa cell line, KGN at an academic medical center.


METHODS
Granulosa cells were treated with increasing concentrations of fully- or hypoglycosylated FSH glycoforms for periods up to 48 hours.


METHODS
The main outcomes were indices of cAMP-dependent cell signaling and estrogen and progesterone synthesis.


RESULTS
We observed that hypoglycosylated FSH(21/18) was significantly more effective than fully glycosylated FSH(24) at stimulating cAMP accumulation, protein kinase A (PKA) activity, and cAMP response element binding protein (CREB) (S133) phosphorylation. FSH(21/18) was also much more effective than hFSH(24) on the stimulation CREB-response element-mediated transcription, expression of aromatase and STAR proteins, and synthesis of estrogen and progesterone. Adenoviral-mediated expression of the endogenous inhibitor of PKA, inhibited FSH(21/18)- and FSH(24)-stimulated CREB phosphorylation, and steroidogenesis.


CONCLUSIONS
Hypoglycosylated FSH(21/18) has greater bioactivity than fully glycosylated hFSH(24), suggesting that age-dependent decreases in hypoglycosylated hFSH contribute to reduced ovarian responsiveness. Hypoglycosylated FSH may be useful in follicle stimulation protocols for older patients using assisted reproduction technologies.
The Olson Center for Women's Health, Department of Obstetrics and Gynecology (C.J., X.H., C.W., J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198; Nebraska-Northwest Iowa VA Medical Center (X.H., J.S.D.), Omaha, Nebraska 68105; Department of Biological Sciences (J.V.M., V.Y.B., G.R.B.), Wichita State University, Wichita, Kansas 67260; and Department of Biochemistry and Molecular Biology (J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198.
Chao
C
Jiang
The Olson Center for Women's Health, Department of Obstetrics and Gynecology (C.J., X.H., C.W., J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198; Nebraska-Northwest Iowa VA Medical Center (X.H., J.S.D.), Omaha, Nebraska 68105; Department of Biological Sciences (J.V.M., V.Y.B., G.R.B.), Wichita State University, Wichita, Kansas 67260; and Department of Biochemistry and Molecular Biology (J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198.
Xiaoying
X
Hou
The Olson Center for Women's Health, Department of Obstetrics and Gynecology (C.J., X.H., C.W., J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198; Nebraska-Northwest Iowa VA Medical Center (X.H., J.S.D.), Omaha, Nebraska 68105; Department of Biological Sciences (J.V.M., V.Y.B., G.R.B.), Wichita State University, Wichita, Kansas 67260; and Department of Biochemistry and Molecular Biology (J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198.
Cheng
C
Wang
The Olson Center for Women's Health, Department of Obstetrics and Gynecology (C.J., X.H., C.W., J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198; Nebraska-Northwest Iowa VA Medical Center (X.H., J.S.D.), Omaha, Nebraska 68105; Department of Biological Sciences (J.V.M., V.Y.B., G.R.B.), Wichita State University, Wichita, Kansas 67260; and Department of Biochemistry and Molecular Biology (J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198.
Jeffrey V
JV
May
The Olson Center for Women's Health, Department of Obstetrics and Gynecology (C.J., X.H., C.W., J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198; Nebraska-Northwest Iowa VA Medical Center (X.H., J.S.D.), Omaha, Nebraska 68105; Department of Biological Sciences (J.V.M., V.Y.B., G.R.B.), Wichita State University, Wichita, Kansas 67260; and Department of Biochemistry and Molecular Biology (J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198.
Viktor Y
VY
Butnev
The Olson Center for Women's Health, Department of Obstetrics and Gynecology (C.J., X.H., C.W., J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198; Nebraska-Northwest Iowa VA Medical Center (X.H., J.S.D.), Omaha, Nebraska 68105; Department of Biological Sciences (J.V.M., V.Y.B., G.R.B.), Wichita State University, Wichita, Kansas 67260; and Department of Biochemistry and Molecular Biology (J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198.
George R
GR
Bousfield
The Olson Center for Women's Health, Department of Obstetrics and Gynecology (C.J., X.H., C.W., J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198; Nebraska-Northwest Iowa VA Medical Center (X.H., J.S.D.), Omaha, Nebraska 68105; Department of Biological Sciences (J.V.M., V.Y.B., G.R.B.), Wichita State University, Wichita, Kansas 67260; and Department of Biochemistry and Molecular Biology (J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198.
John S
JS
Davis
D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D043373:Follicle Stimulating Hormone, Human / Q000378:metabolism* / Q000494:pharmacology*; D006031:Glycosylation; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism* / Q000494:pharmacology*
D017362:Cyclic AMP Response Element-Binding Protein; D043373:Follicle Stimulating Hormone, Human; D020033:Protein Isoforms; D011994:Recombinant Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases