Changes in insulin-like growth factor-binding protein-2 and -3 in follicular fluid during atresia of follicles grown after ovulation in pigs

In pigs, medium-sized follicles (3–6 mm in diameter), lost during preovulatory maturation, are rapidly replenished after ovulation, increasing tenfold in number between day 3 and day 8 of the oestrous cycle. The incidence of atresia in medium-sized follicles was previously found to increase between day 5 and day 7 after the onset of oestrus (day 0). This study was conducted to determine whether changes in concentrations of insulin-like growth factor binding proteins (IGFBPs) in pig follicular fluid from medium-sized follicles were related to increased incidence of atresia. Individual follicles were isolated on days 5, 6 and 7 after oestrus from eleven pigs. Follicles were defined as atretic when they contained ≥ 10% apoptotic granulosa cells (cells containing subdiploid amounts of DNA fluorescence determined by flow cytometry). Concentrations of IGFBP-2 and -3 were determined by ligand blot analysis with125I-labelled insulin-like growth factor II and quantified by densitometry. The mean percentage of apoptotic cells per follicle increased (P ≤ 0.01) from 3.6 to 35.0 and the mean concentration of IGFBP-2 increased (P ≤ 0.001) 3.3-fold between day 5 and day 7 after oestrus. The concentration of IGFBP-3 did not differ significantly among days. Mean oestradiol concentration in follicular fluid decreased (P ≤ 0.05) from 52.3 to 0.9 ng ml−1 between day 5 and day 7. Androstenedione concentrations in follicular fluid also decreased (P ≤ 0.01) between day 5 and day 7, but progesterone did not differ significantly among days. The concentration of IGFBP-2 correlated positively with the percentage of apoptotic cells (r= 0.898, P ≤ 0.001). The concentration of IGFBP-2 did not correlate significantly with the concentration of follicular fluid steroids or of IGFBP-3. The concentration of IGFBP-2 was three times higher in atretic than in healthy follicles (P ≤ 0.001), but the concentration of IGFBP-3 did not differ significantly between atretic and healthy follicles. We conclude that increased production or accumulation of IGFBP-2 could play a role in the development of apoptosis among granulosa cells and increased follicular atresia between day 5 and day 7 after oestrus. In contrast, no physiological role for IGFBP-3 in apoptosis or atresia was indicated.