Flow cytometric determination of degraded deoxyribonucleic acid in granulosa cells to identify atretic follicles during preovulatory maturation in the pig

Abstract

Granulosa cells of individual follicles were analyzed by DNA fluorescence flow cytometry to determine how the percentage of cells with degraded DNA and the distribution of cells in the phases of the cell cycle (G0/G1, S1, G2/M) related to the incidence of morphological atresia and to changes in follicular steroid concentrations. Follicles were dissected from ovaries recovered at slaughter on Days 1, 3, 5, or 7 of altrenogest-synchronized preovulatory maturation. Twenty-one follicles with debris among their isolated granulosa cells were classified as morphologically atretic (MA); 92 follicles with debris-free granulosa cells were classified as morphologically healthy (MH). Granulosa cells were prepared for flow cytometry by fixation in 80% ethanol and staining with propidium iodide (PI) containing RNase. DNA fluorescence intensity was determined by use of the 488-nm line of an argon laser. A subpopulation of granulosa cells with degraded DNA (A0 cells), containing less fluorescence than the G0/G1 peak, was found in the DNA histogram of every follicle. The percentage of A0 cells ranged from 0.02 to 83.6% per follicle. The percentage of A0 cells was inversely related to the percentage of G0/G1 cells (r = -0.9611, p = 0.0001). The percentage of A0 cells (mean ± SEM) was greater (p = 0.0001) in MA (45.9 ± 6.3%) than in MH follicles (5.3 ± 1.6%). Follicular estradiol-17β was less in MA than in MH follicles, but androstenedione or progesterone did not differ significantly. A follicle biochemical health classification was derived from the percentage of A0 granulosa cells in the DNA histogram; 27 follicles were biochemically atretic (BA) with ≥ 10% A0 cells (mean 44.5 ± 5.0%), while 86 follicles were biochemically healthy (BH) with < 10% A0 cells (mean 2.2 ± 0.4%). The percentage of BA follicles per pig was greater (p = 0.0001) on Day 5 (47%) than on any other day (10-22%). Compared to BH follicles, BA follicles contained a reduced percentage of G0/G1 and S+G2/M cells and less estradiol-17β and androstenedione (p ≤ 0.05). The increased incidence of granulosa cells with low DNA fluorescence, measured by flow cytometry, is a new, biochemical marker for atretic follicles in the pig.