Characterization of an immortalized human granulosa cell line (COV434)

We have investigated the biological characteristics of an immortalized gran ulosa cell line (COV434), which may be used to study follicular and oocyte maturation in vitro. Granulosa cell function was defined as consisting of t hree distinct properties: (i) production of 17 beta-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 oocy te. The addition of FSH to the culture medium supplemented with 10% fetal c alf serum and 4-androstene-3,17-dione resulted in proliferation of the COV4 34 granulosa cells and in an increased synthesis of 17 beta-oestradiol, ind icating the presence of the FSH receptor and cytochrome P450 aromatase in t hese cells. The receptor for luteinizing hormone (LH) was undetectable. Sim ilar expression of various apoptosis-associated genes was found in COV434 g ranulosa cells and in granulosa cells of patients stimulated with gonadotro phins for in-vitro fertilization, thus indicating that the immortalized COV 434 granulosa cells were able to sustain apoptosis. Multiple intercellular connections were formed during co-culture of COV434 granulosa cells with cu mulus cells containing an immature oocyte but not with cumulus cells devoid of an oocyte. Detailed morphological analysis of the intercellular connect ions with scanning electron microscopy and confocal light microscopy demons trated the presence of long slender structures. It is concluded that the im mortalized human granulosa cell line COV434 may be useful for experimental studies on follicular development.