Characterization of an immortalized human granulosa cell line (COV434)

Abstract

We have investigated the biological characteristics of an immortalized granulosa cell line (COV434), which may be used to study follicular and oocyte maturation in vitro. Granulosa cell function was defined as consisting of three distinct properties: (i) production of 17beta-oestradiol in response to follicle stimulating hormone (FSH); (ii) presence of specific molecular markers of apoptosis enabling the induction of follicular atresia; and (iii) capacity to form intercellular connections with cells surrounding an oocyte. The addition of FSH to the culture medium supplemented with 10% fetal calf serum and 4-androstene-3,17-dione resulted in proliferation of the COV434 granulosa cells and in an increased synthesis of 17beta-oestradiol, indicating the presence of the FSH receptor and cytochrome P450 aromatase in these cells. The receptor for luteinizing hormone (LH) was undetectable. Similar expression of various apoptosis-associated genes was found in COV434 granulosa cells and in granulosa cells of patients stimulated with gonadotrophins for in-vitro fertilization, thus indicating that the immortalized COV434 granulosa cells were able to sustain apoptosis. Multiple intercellular connections were formed during co-culture of COV434 granulosa cells with cumulus cells containing an immature oocyte but not with cumulus cells devoid of an oocyte. Detailed morphological analysis of the intercellular connections with scanning electron microscopy and confocal light microscopy demonstrated the presence of long slender structures. It is concluded that the immortalized human granulosa cell line COV434 may be useful for experimental studies on follicular development.

Extra-ovarian expression and activity of growth differentiation factor 9

“…The mouse TM3 and TM4 (Mather 1982), LbT2 (Pernasetti et al 2001), AC (Rilianawati et al 1998, Rahman et al 2001, Farnworth et al 2006b), COV434 (Zhang et al 2000) and KGN (Nishi et al 2001) cell lines were obtained as previously described (Farnworth et al 2006b, Chand et al 2007). Rats and mice were obtained from Central Animal House, Monash University (Clayton, Victoria, Australia).…”

The FOXL2 C134W mutation is characteristic of adult granulosa cell tumors of the ovary

“…We found that the COV434 cell line, established from a metastatic granulosa cell tumor removed from a 27-year-old female, 26,27 lacked the FOXL2 mutation and did not express the FOXL2 gene. This strongly suggests that the COV434 cell line is derived from a juvenile granulosa cell tumor.…”

The bioactivity of human bone morphogenetic protein-15 is sensitive to C-terminal modification: Characterization of the purified untagged processed mature region

“…As the hGL cells have undergone a process of terminal differentiation in response to a luteinizing surge of gonadotropins, we wanted to characterize the effect of the purified rhBMP15 on granulosa cells which have not become terminally differentiated. An immortalized human granulosa cell line COV434 was chosen as it has retained the ability to produce estradiol upon FSH stimulation, thus demonstrating the presence of functional FSH receptors (Zhang et al 2000). The COV434 cells were transiently transfected with the Smad1/5/8 responsive transcriptional reporter construct, BRE-luciferase (Korchynskyi and ten Dijke 2002) and treated with the purified rhBMP15wt mature region, which activated BRE-luciferase reporter in a dose responsive manner (Fig.…”