title: FSH Regulates IGF-2 Expression in Human Granulosa Cells in an AKT-Dependent Manner.
abstract: BACKGROUND
  IGF-2 is highly expressed in the granulosa cells of human dominant ovarian follicles; however, little is known about the regulation of the IGF-2 gene or the interaction of IGF-2 and FSH during follicle development.
  
  
  OBJECTIVE
  To examine the mechanisms involved in the regulation of the IGF-2 gene by FSH and the interplay between FSH and IGF-2 during granulosa cell differentiation. Design, Setting, Patients, and Interventions: Cumulus granulosa cells were separated from cumulus-oocyte complexes isolated from the follicular aspirates of in vitro fertilization patients and cultured for in vitro studies.
  
  
  RESULTS
  Protein and mRNA levels of IGF-2 and CYP19A1 (aromatase) were quantified using Western blot and quantitative real-time PCR. IGF-2 promoter-specific activation was determined by the amplification of alternative exons by PCR. Cell proliferation was assessed after treatment with FSH and/or IGF-2.
  
  
  RESULTS
  FSH significantly enhanced IGF-2 expression after 8 hours of treatment and at low doses (1 ng/mL). Reciprocally, IGF-2 synergized with FSH to increase cell proliferation and the expression of CYP19A1. When IGF-2 activity was blocked, FSH was no longer able to stimulate CYP19A1 expression. Determination of IGF-2 promoter usage in human cumulus cells showed that the IGF-2 gene is driven by promoters P3 and P4. However, FSH exclusively increased P3 promoter-derived transcripts. Moreover, the FSH-induced stimulation of P3-driven IGF-2 transcripts was blocked by cotreatment with inhibitors of AKT or IGF-1 receptor (IGF-1R). The inhibitory effect of the IGF-1R inhibitor on FSH-induced IGF-2 mRNA accumulation was reversed by overexpression of a constitutively active AKT construct.
  
  
  CONCLUSIONS
  FSH is a potent enhancer of IGF-2 expression in human granulosa cells. In return, IGF-2 activation of the IGF-1R and AKT is required for FSH to stimulate CYP19A1 expression and proliferation of granulosa cells. These findings suggest a positive loop interaction between FSH and IGF-2 that is critical for human granulosa cell proliferation and differentiation.
authors.affiliation: Department of Physiology and Biophysics (S.C.B., S.M.C., C.S.), and Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology (A.M.Z., M.A.F., N.J.W., B.S.), University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612.
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Baumgarten
authors.affiliation: Department of Physiology and Biophysics (S.C.B., S.M.C., C.S.), and Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology (A.M.Z., M.A.F., N.J.W., B.S.), University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Convissar
authors.affiliation: Department of Physiology and Biophysics (S.C.B., S.M.C., C.S.), and Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology (A.M.Z., M.A.F., N.J.W., B.S.), University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612.
authors.forename: A Musa
authors.initials: AM
authors.lastname: Zamah
authors.affiliation: Department of Physiology and Biophysics (S.C.B., S.M.C., C.S.), and Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology (A.M.Z., M.A.F., N.J.W., B.S.), University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Fierro
authors.affiliation: Department of Physiology and Biophysics (S.C.B., S.M.C., C.S.), and Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology (A.M.Z., M.A.F., N.J.W., B.S.), University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Winston
authors.affiliation: Department of Physiology and Biophysics (S.C.B., S.M.C., C.S.), and Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology (A.M.Z., M.A.F., N.J.W., B.S.), University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612.
authors.forename: Bert
authors.initials: B
authors.lastname: Scoccia
authors.affiliation: Department of Physiology and Biophysics (S.C.B., S.M.C., C.S.), and Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology (A.M.Z., M.A.F., N.J.W., B.S.), University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612.
authors.forename: Carlos
authors.initials: C
authors.lastname: Stocco
mesh_terms: D001141:Aromatase / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D054885:Cumulus Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism; D051058:Oncogene Protein v-akt / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D007335:Insulin-Like Growth Factor II; D005640:Follicle Stimulating Hormone; D001141:Aromatase; C561410:CYP19A1 protein, human; D051058:Oncogene Protein v-akt