Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells

ObjectiveTo determine if initial cell plating density alters steroidogenesis and the E2:P ratio in granulosa cells in long-term serum-free culture.DesignExperimental study.SettingAcademic institution.Animal(s)Cattle of slaughterhouse origin.Intervention(s)Culture of granulosa cells in vitro at different cell plating density.Main Outcome Measure(s)Steroid secretion was measured by RIA, mRNA levels were measured by real-time polymerase chain reaction, and cell death was assessed by flow cytometry.Result(s)Low plating density favored E2 secretion and mRNA encoding estrogenic enzymes, whereas higher density inhibited E2 secretion and enhanced P secretion and levels of mRNA encoding progestagenic enzymes. Increasing plating density decreased the E2:P ratio and cell health.Conclusion(s)Lower cell density favors an estrogenic granulosa cell phenotype, whereas higher density favors luteinization. Serum-free culture systems should be optimized with this in mind.