FLOW CYTOMETRIC DETERMINATION OF DEGRADED DEOXYRIBONUCLEIC-ACID IN GRANULOSA-CELLS TO IDENTIFY ATRETIC FOLLICLES DURING PREOVULATORY MATURATION IN THE PIG

Granulosa cells of individual follicles were analyzed by DNA fluorescence now cytometry to determine how the percentage of cells with degraded DNA and the distribution of cells in the phases of the cell cycle (G(0)/G(1), S-1, G(2)/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 (A(0) cells), containing less fluorescence than the G(0)/G(1) peak, was found in the DNA histogram of every follicle. The percentage of A(0) cells ranged from 0.02 to 83.6% per follicle. The percentage of A(0) cells was inversely related to the percentage of G(0)/G(1) cells (r = -0.9611, p = 0.0001). The percentage of A(0) cells (mean +/- SEM) was greater (p = 0.0001) in MA (45.9 +/- 6.3%) than in MH follicles (5.3 +/- 1.6%). Follicular estradiol-17 beta 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 A(0) granulosa cells in the DNA histogram; 27 follicles were biochemically atretic (BA) with greater than or equal to 10% A,cells (mean 44.5 +/- 5.0%), while 86 follicles were biochemically healthy (BH) with < 10% A(0) 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 G(0)/G(1) and S+G(2)/M cells and less estradiol-17 beta and androstenedione (P less than or equal to 0.05). The increased incidence of granulosa cells with low DNA fluorescence, measured by how cytometry, is a new, biochemical marker for atretic follicles in the pig.