Characterization of immortalized mouse granulosa cell lines.
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.
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Innogenetics N.V., Industriepark Zwijnaarde, Ghent, Belgium.
van de Voorde
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Vanderstichele
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D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D028341:Activins; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D017077:Culture Media, Conditioned; D005260:Female; D006107:Granulosa Cells / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D006133:Growth Substances / Q000494:pharmacology; D007265:Inhibins / Q000378:metabolism; D007621:Karyotyping; D051379:Mice; D011453:Prostaglandins / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis; D013256:Steroids / Q000032:analysis; D016212:Transforming Growth Factor beta / Q000378:metabolism
D017077:Culture Media, Conditioned; D006133:Growth Substances; D011453:Prostaglandins; D016271:Proto-Oncogene Proteins c-myc; D013256:Steroids; D016212:Transforming Growth Factor beta; D028341:Activins; D007265:Inhibins; D015067:17-Hydroxysteroid Dehydrogenases; D015096:3-Hydroxysteroid Dehydrogenases