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.
(Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)