Atresia in Follicles Grown after Ovulation in the Pig: Measurement of Increased Apoptosis in Granulosa Cells and Reduced Follicular Fluid Estradiol-17β1

The incidence of atresia in the first group of follicles grown after ovulation was investigated in the pig. At slaughter, 113 follicles 3–6 mm in diameter were dissected from the ovaries of four pregnant pigs per day on Days 5, 6, and 7 after the onset of estrus. Granulosa cells were isolated from each follicle. The percentage of granulosa cells containing sub-diploid amounts of DNA (%Ao cells), a measure of apoptosis, was determined for each follicle by DNA fluorescence flow cytometry of propidium iodide (PI) stained nuclei of ethanol-fixed cells. Granulosa cell DNA condition was used to classify follicles. Follicles with ≥ 10% Ao cells (n = 33) were designated biochemically atretic (BA), and follicles with < 10% Ao cells (n = 80) were designated biochemically healthy (BH). Internucleosomal cleavage, also indicative of apoptosis, was determined by autoradiographic analysis of [32P]-3′-end-labeled DNA from granulosa cells. Densitometric analysis showed that optical density of [32P]-3′-end-labeled DNA fragments in the 0.18–20-kb size range was correlated with the %Ao cells (R ≥ 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) increased (p ≤ 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. 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-17β concentration was 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. Follicular androstenedione concentration decreased (p < 0.01) in both BH and BA follicles between Days 5 and 7. Progesterone concentration increased (p < 0.01) in BA follicles between Days 5 and 7, and was greater (p < 0.01) in BA than in BH follicles on Day 7. In conclusion, we suggest that granulosa cells with sub-diploid levels of DNA were undergoing internucleosomal DNA cleavage indicative of apoptotic death. Further we suggest that an increased percentage of granulosa cells undergoing DNA degradation and a loss of estradiol-17β 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, signaling the end of their growth period.