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.


METHODS
Experimental study.


METHODS
Academic institution.


METHODS
Cattle of slaughterhouse origin.


METHODS
Culture of granulosa cells in vitro at different cell plating density.


METHODS
Steroid secretion was measured by RIA, mRNA levels were measured by real-time polymerase chain reaction, and cell death was assessed by flow cytometry.


RESULTS
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.


CONCLUSIONS
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.
Portela
Valério M
VM
Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, St. Hyacinthe, Québec, Canada.
Zamberlam
Gustavo
G
Price
Christopher A
CA