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 fluoresce nce now cytometry to determine how the percentage of cells with degrad ed 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 atre sia and to changes in follicular steroid concentrations. Follicles wer e dissected from ovaries recovered at slaughter on Days 1, 3, 5, or 7 of altrenogest-synchronized preovulatory maturation. Twenty-one follic les with debris among their isolated granulosa cells were classified a s morphologically atretic (MA); 92 follicles with debris-free granulos a cells were classified as morphologically healthy (MH). Granulosa cel ls were prepared for flow cytometry by fixation in 80% ethanol and sta ining with propidium iodide (PI) containing RNase. DNA fluorescence in tensity was determined by use of the 488-nm line of an argon laser. A subpopulation of granulosa cells with degraded DNA (A(0) cells), conta ining 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%). Follicula r estradiol-17 beta was less in MA than in MH follicles, but androsten edione or progesterone did not differ significantly. A follicle bioche mical health classification was derived from the percentage of A(0) gr anulosa cells in the DNA histogram; 27 follicles were biochemically at retic (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 wa s 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 androst enedione (P less than or equal to 0.05). The increased incidence of gr anulosa cells with low DNA fluorescence, measured by how cytometry, is a new, biochemical marker for atretic follicles in the pig.