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Alvaro G. Hernandez1 *, Hung-Chang Yao1 *, Janice M. Bahr1 *
University of Illinois at Urbana-Champaign, Urbana, IL 1
In the chicken ovary, one 6-8mm diameter follicle 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 6-8mm diameter follicles undergo apoptosis within 6 h in suspension culture. We are reporting a culture method in which granulosa cells from 6mm diameter follicles remain viable in vitro for 24 h. Granulosa layers were isolated from 6mm diameter follicles (Gr-6) and from the largest follicle (Gr-F1) and dispersed in 0.15% collagenase for 15 min. Cells (1x106/0.5ml for Gr-6 and 2x105/0.5ml for Gr-F1) were cultured in 24 well plates in Dulbecco’s modified medium with 25mM Hepes, 0.2% BSA, transferrin (5µg/ml), insulin (5µg/ml) and sodium selenite (5ng/ml) for 0, 6, 12 and 24 h in an incubator with 5% CO2/ 95% O2 at 39oC. Theca cell explants (8mm diameter) were cultured in 1 ml of the same medium for 6 and 12 h. Cell proliferation (n= 6) and DNA integrity (n= 4) were evaluated at each time by a colorimetric assay and DNA gel electrophoresis, respectively. Proliferation, expressed as fold increase with 1 h = 1.0, increased in Gr-6 and Gr-F1 at 6, 12 and 24 h (1.5, 2.15 and 3.8 for Gr-6 and 1.5, 1.8 and 2.0 for Gr-F1). DNA fragmentation was observed in theca cells at 6 and 12 h but not in granulosa cells. In conclusion, we have identified culture conditions that will allow culture of granulosa cells obtained from 6-8mm diameter follicles, the stage prior to selection. (Supported in part by NSF-IBN-96-30957 and USDA-CSREES 35-324).
This abstract is being presented on Monday, August 2 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.