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

Objective: To determine if initial cell plating density alters steroidogenesis and the E-2:P ratio in granulosa cells in long-term serum-free culture. Design: Experimental study. Setting: Academic 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 E-2 secretion and mRNA encoding estrogenic enzymes, whereas higher density inhibited E-2 secretion and enhanced P secretion and levels of mRNA encoding progestagenic enzymes. Increasing plating density decreased the E-2: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. (Fenil Steril (R) 2010;93:2050 5. (C)2010 by American Society for Reproductive Medicine.)