Culture of Chicken Granulosa Cells from Small Yellow Follicles: A Suitable Culture System

Abstract

In the chicken ovary, one small yellow follicle (SYF) is selected daily to enter the hierarchy and ovulates a few days later. Cellular changes that occur in these follicles may provide an understanding of the mechanism of follicular selection. It was reported that granulosa cells isolated from SYF undergo apoptosis within 6 h in suspension culture. In this study, a culture method in which granulosa cells from SYF remain viable in vitro for 24 h is reported. Granulosa layers were isolated from SYF (Gr-SYF) and from the largest preovulatory follicle (Gr-F1) and dispersed in collagenase (1.5 mg/ml) for 15 min. Cells (1×10⁶/500μl for Gr-SYF and 2×10⁵/500μl for Gr-F1) were cultured either in 24 well plates in an incubator with 5% CO₂/95% O₂ at 39°C or in suspension in falcon tubes and incubated in water bath at 37C, for 0, 6, 12 and 24 h. Theca cell explants (8 mm diameter) were cultured in 1 ml of the same medium for 6 and 12 h. Cell proliferation (n=6) at 6, 12 and 24 h, DNA integrity and membrane permeability (n=4) were determined by a colorimetric assay, gel electrophoresis and flow cytometry, respectively. Proliferation increased in Gr-SYF cultured in a plate at 12 and 24 h whereas it did not change in Gr-F1 cells at any time of culture. DNA fragmentation was observed in Gr-SYF cultured in suspension at 12 h and theca cells at 6 and 12 h but not in Gr-SYF cultured in the plate. Cell permeability and DNA stainability (indicative of apoptosis) were only increased in Gr-SYF cultured in suspension. In conclusion, we have identified culture conditions that will allow the culture of granulosa cells obtained from SYF, the stage prior to selection.