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

Fertil Steril. 2010 Apr;93(6):2050-5. doi: 10.1016/j.fertnstert.2009.01.151. Epub 2009 Mar 26.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3-Hydroxysteroid Dehydrogenases / genetics
  • 3-Hydroxysteroid Dehydrogenases / metabolism
  • Animals
  • Aromatase / genetics
  • Aromatase / metabolism
  • Cattle
  • Cell Count
  • Cell Culture Techniques
  • Cells, Cultured
  • Cholesterol Side-Chain Cleavage Enzyme / genetics
  • Cholesterol Side-Chain Cleavage Enzyme / metabolism
  • Estradiol / biosynthesis*
  • Female
  • Gene Expression Regulation, Enzymologic*
  • Granulosa Cells / cytology*
  • Granulosa Cells / enzymology
  • Granulosa Cells / metabolism*
  • Granulosa Cells / physiology
  • Luteinization / genetics
  • Luteinization / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Progesterone / biosynthesis*
  • Receptors, FSH / genetics
  • Receptors, FSH / metabolism

Substances

  • Phosphoproteins
  • Receptors, FSH
  • steroidogenic acute regulatory protein
  • Progesterone
  • Estradiol
  • 3-Hydroxysteroid Dehydrogenases
  • Aromatase
  • Cholesterol Side-Chain Cleavage Enzyme