Establishment of a Human Nonluteinized Granulosa Cell Line That Transitions From the Gonadotropin-Independent to the Gonadotropin-Dependent Status

Endocrinology. 2012 Jun;153(6):2851-60. doi: 10.1210/en.2011-1810. Epub 2012 Mar 30.

Abstract

The ovary is a complex endocrine organ responsible for steroidogenesis and folliculogenesis. Follicles consist of oocytes and two primary steroidogenic cell types, the granulosa cells, and the theca cells. Immortalized human granulosa cells are essential for researching the mechanism of steroidogenesis and folliculogenesis. We obtained granulosa cells from a 35-yr-old female and immortalized them by lentivirus-mediated transfer of several genes so as to establish a human nonluteinized granulosa cell line (HGrC1). We subsequently characterized HGrC1 and investigated its steroidogenic performance. HGrC1 expressed enzymes related to steroidogenesis, such as steroidogenic acute regulatory protein, CYP11A, aromatase, and gonadotropin receptors. Stimulation with FSH increased the mRNA levels of aromatase, which consequently induced the aromatization of androstenedione to estradiol. Activin A increased the mRNA levels of the FSH receptor, which were synergistically up-regulated with FSH stimulation. HGrC1 also expressed a series of ligands and receptors belonging to the TGF-β superfamily. A Western blot analysis showed that bone morphogenetic protein (BMP)-4, BMP-6, and BMP-7 phosphorylated small mother against decapentaplegic (Smad)1/5/8, whereas growth differentiation factor-9 phosphorylated Smad2/3. BMP-15 and anti-Müllerian hormone phosphorylated Smad1/5/8 while also weakly phosphorylating Smad2/3. These results indicate that HGrC1 may possess the characteristics of granulosa cells belonging to follicles in the early stage. HGrC1 might also be capable of displaying the growth transition from a gonadotropin-independent status to gonadotropin-dependent one.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activins / pharmacology
  • Adult
  • Aromatase / genetics
  • Aromatase / metabolism
  • Blotting, Western
  • Bone Morphogenetic Proteins / genetics
  • Bone Morphogenetic Proteins / metabolism
  • Cell Differentiation*
  • Cell Line
  • Cell Proliferation*
  • Cholesterol Side-Chain Cleavage Enzyme / genetics
  • Cholesterol Side-Chain Cleavage Enzyme / metabolism
  • Female
  • Follicle Stimulating Hormone / pharmacology
  • Gene Expression / drug effects
  • Gonadotropins / metabolism*
  • Granulosa Cells / cytology*
  • Granulosa Cells / metabolism*
  • Humans
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phosphorylation / drug effects
  • Receptors, Gonadotropin / genetics
  • Receptors, Gonadotropin / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Smad Proteins / metabolism
  • Transforming Growth Factor beta / genetics
  • Transforming Growth Factor beta / metabolism

Substances

  • Bone Morphogenetic Proteins
  • Gonadotropins
  • Phosphoproteins
  • Receptors, Gonadotropin
  • Smad Proteins
  • Transforming Growth Factor beta
  • activin A
  • steroidogenic acute regulatory protein
  • Activins
  • Follicle Stimulating Hormone
  • Aromatase
  • Cholesterol Side-Chain Cleavage Enzyme