ATRESIA IN FOLLICLES GROWN AFTER OVULATION IN THE PIG - MEASUREMENT OF INCREASED APOPTOSIS IN GRANULOSA-CELLS AND REDUCED FOLLICULAR-FLUID ESTRADIOL-17-BETA

The incidence of atresia in the first group of follicles grown after o vulation was investigated in the pig. At slaughter, 113 follicles 3-6 mm in diameter were dissected from the ovaries of four pregnant pigs p er day on Days 5, 6, and 7 after the onset of estrus. Granulosa cells were isolated from each follicle. The percentage of granulosa cells co ntaining sub-diploid amounts of DNA (%A(0) cells), a measure of apopto sis, was determined for each follicle by DNA fluorescence flow cytomet ry of propidium iodide (PI)-stained nuclei of ethanol-fixed cells. Gra nulosa cell DNA condition was used to classify follicles. Follicles wi th greater than or equal to 10% A(0) cells (n = 33) were designated bi ochemically atretic (BA), and follicles with < 10% A(0) cells (n = 80) were designated biochemically healthy (BH). Internucleosomal cleavage , also indicative of apoptosis, was determined by autoradiographic ana lysis of [P-32]-3'-end-labeled DNA from granulosa cells. Densitometric analysis showed that optical density of [P-32]-3'-end-labeled DNA fra gments in the 0.18-20-kb size range was correlated with the %A(0) cell s (R greater than or equal to 0.90, N = 22, p < 0.001). The incidence of pigs with BA follicles was 2 of 4, 3 of 4, and 4 of 4 on Days 5, 6, and 7, respectively. The %BA follicles per pig (mean +/- SEM) increas ed (p less than or equal to 0.01) between Days 5 and 7; values were 6. 2 +/- 3.6, 28.1 +/- 13.5, and 50.0 +/- 7.1, respectively, on Days 5, 6 , and 7. The %A(0) cells in BA follicles increased (p < 0.01) between Days 5 and 7; values were 15.9 +/- 3.9, 30.8 +/- 5.7, and 43.8 +/- 5.3 , respectively, on Days 5, 6, and 7. Estradiol-17 beta concentration w as greater (p < 0.001) in BH than in BA follicles on Days 5 and 6, but decreased to a mean < 1 ng/ml in both BH and BA follicles on Day 7. F ollicular androstenedione concentration decreased (p < 0.01) in both B H and BA follicles between Days 5 and 7. Progesterone concentration in creased (p < 0.01) in BA follicles between Days 5 and 7, and was great er (p < 0.01) in BA than in BH follicles on Day 7. In conclusion, we s uggest that granulosa cells with sub-diploid levels of DNA were underg oing internucleosomal DNA cleavage indicative of apoptotic death. Furt her we suggest that an increased percentage of granulosa cells undergo ing DNA degradation and a loss of estradiol-17 beta production in vivo between Days 5 and 7 post-estrus indicate a high incidence of atresia among the 3-6-mm follicles that began to grow after ovulation, signal ing the end of their growth period.