Characterization of immortalized mouse granulosa cell lines

Abstract

Cell cultures of primary mouse granulosa cells were transfected with a v-myc-containing plasmid, and the resulting stable cell lines were tested for their steroidogenic properties and physiologic status. Granulosa cells were obtained from 22-day-old NMRI mice injected with 8 IU pregnant mare serum gonadotropin i.p. 2 days earlier. In Passage 1 the cells were transfected with pSVv-myc using calcium phosphate precipitation or lipofectin. The 3 beta- and 17 beta-hydroxy steroid dehydrogenase activity was visualized in control cultures. The three cell lines obtained have been in culture for over 1 yr and have been subcultured for more than 90 passages. The cell line GRM01, with a doubling time of 37 +/- 3 h and a diploid modal chromosome number, produced progesterone, estradiol, as well as inhibinlike and activinlike material under basal conditions. A combination of follicle-stimulating hormone and luteinizing hormone was able to increase the secretion of progesterone. GRM01L, a fast growing clone of the GRM01 line with a doubling time of 10 +/- 1 h, retained only the capacity to produce activinlike material and transforming growth factor-beta, and it was the only one with a tumorigenic capacity. Epidermal growth factor, insulin, and interleukin-6 were able to induce the [3H]thymidine incorporation into DNA in these two cell lines. GRM02, with a doubling time of 36 +/- 2 h and a hypertriploid modal chromosome number, produced progesterone and activinlike and inhibinlike material. Follicle-stimulating hormone and luteinizing hormone were able to enhance the secretion of progesterone. For this cell line, only insulin was shown to induce [3H]thymidine incorporation into DNA.

Secretion of Steroids, Growth Factors, and Cytokines by Immortalized Mouse Granulosa Cell Lines1

“…Preliminary data on basal and gonadotropin-stimulated steroidogenic activity, together with evidence for secretion of activins and TGFP have been presented [38]. This paper extends these observations and gives more details on the expression and/or secretion of gonadotropin receptors, steroid hormones, growth factors, and cytokines by these immortalized mouse granulosa cell lines.…”

“…The immortalized mouse granulosa cells, which are morphologically similar to primary mouse granulosa cells in culture [38], conserved the main features of ovarian granulosa cells, including the capacity to synthesize steroids, transferrin, and inhibins, together with responsiveness to LH and/or FSH. Normally, granulosa cells cease to divide in primary cultures or luteinize spontaneously in vitro.…”

“…Indeed, highly differentiated granulosa cells in preovulatory follicles and luteinized cells, subsequent to ovulation, produce only extensive amounts of progesterone and no estrogens. This celltype classification was further substantiated by the secretion of low amounts of prostaglandins (PGE 2 or PGF 2 ,,) [38], known to be secreted just before ovulation, and by an EGF-induced increase of progesterone secretion. Normal granulosa cells acquire EGF receptors after two days of treatment with FSH in vitro and in association with a transformation to granulosa-lutein cells [59].…”

“…The lack of steroidogenic activity could be due to the fact that the cell line was obtained after specific subculturing methods selecting for a fast-growing cell type with neoplastic properties [38], or could result from a lack of available substrate, since granulosa cells and luteal cells have a relatively low capacity for de novo cholesterol synthesis [60].…”

“…The technique of immortalization, together with the morphological characteristics of the newly established cell lines are extensively described by Briers et al [38]. Briefly, granulosa cells were collected by trituration from the ovaries of 20-day-old NMRI mice that had been treated with eCG.…”