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

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.

Oocyte–somatic cell interactions in the human ovary—novel role of bone morphogenetic proteins and growth differentiation factors

“…In the last few years, there has been a growing interest in developing several cell lines from human GCs (Table II). These cell lines were generated through various techniques, including oncogenic transformation (Rainey et al ., 1994; Lie et al ., 1996; Hosokawa et al ., 1998; Nitta et al ., 2001; Tajima et al ., 2002; Okamura et al ., 2003), transfection with site-directed mutagenesis (Bayasula et al ., 2012) and explants of human tumors (Ishiwata et al ., 1984; van den Berg-Bakker et al ., 1993; Nishi et al ., 2001). Notably, each cell line may have different cell properties with regard to steroidogenic functions, gonadotrophin (FSH and LH) responsiveness, cAMP responsiveness, BMP responsiveness, and mitogenic and differential capabilities (Havelock et al ., 2004) (Table II).…”

“…(2001)Okamura et al . (2003)HGrC1Non-luteinized GCHPV16E6/E7, CDK4R24C, cyclin D1(+)(+)(+)(+)(+)ND(+)Bayasula et al . (2012)COV434Metastatic GC tumor(+)(+)(−)(+)(−)(+)(+)van den Berg-Bakker et al .…”

PAI‐1 in granulosa cells is suppressed directly by statin and indirectly by suppressing TGF‐β and TNF‐α in mononuclear cells by insulin‐sensitizing drugs

“…HGrC1 cells are known to respond to androstendione and LH/hCG 14 . TGF-β and TNF-α were found to significantly induce PAI-1 mRNA relative expression (Fig.…”

“…The purpose of this study was to examine the mechanisms of PAI-1 production using a newly established human non-luteinized GC line, HGrC1 14 , and seek to find new strategies to reduce PAI-1 production by GC.…”

Establishment and characterization of cell lines derived from complete hydatidiform mole

“…Construction of lentiviral vector plasmids CSII-CMV-hTERT, -CDK4R24C, -cyclin D1, -p53C234 and -TetOff, were described previously (12,14). CDK4R24C is a mutant (p16INK4a-resistant) form of CDK4, and p53C234 encodes the carboxy-terminal 234 residues of p53 and functions as a dominant-negative mutant.…”