Comparison of Medium Supplementation on Proliferation and Hormone Production of Bovine Granulosa Cells in a Defined Culture System

Abstract

Aims: The objective of this study was to develop a serum-free bovine granulosa cell (GC) culture system in which estradiol (E2) production could be maintained in a defined media, with polyvinyl alcohol (PVA), insulin and insulin-like growth factor-1 (IGF-1) and without FSH and compared the effects of two different macromolecules (PVA vs, BSA) on steroids output and cell proliferation during in vitro culture. Study Design: Bovine granulosa cell culture. Place and Duration of Study: Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, Brazil, 2011. Methodology: Bovine ovaries were collected from adult cows at a local abattoir and were transported in warm saline solution. Small follicles were dissected according to their vascularization and follicular fluid conditions. GC were cultured in α-MEM containing IGF-I, insulin, androstenedione, 0.1% PVA or 0.1% BSA, without FSH. After 48, 96 and 144 h of culture were analyzed GC morphology and secretion of E2 and P4. The relationship among cell shape, cell proliferation, and GC time course of steroidogenesis in vitro was further explored. Results: In the presence of PVA and BSA, E2 production reached its highest production at 144 h. There was a significant increase on P4 production on the medium containing BSA at 48 and 96 h. The changes in E2 production/P4 production ratio in PVA-cultures indicate that there was a larger increase in E2 production by these cells at 48 and 96 hrs than by cells from medium with BSA. The results of the cellular proliferation demonstrated significant tritiated thymidine incorporation in both PVA and BSA cells cultured at 144 hours. Conclusion: These results demonstrate the development of a relevant culture system for bovine GC under defined conditions with PVA. This chemically defined culture system will enable us to study the factors that regulate the physiological control of GC proliferation, differentiation and steroidogenic characteristics.