No significant differences were found in any stage of follicles 12 or 24 h after injection. Additionally, there were no differences when 0.1 ig of Trap per ovary was injected (data not shown). Therefore, the 0.5-ig and 48-h treatments were used for the following assays. In addition, histological sections showed that the Trap treatment resulted in a decrease in the density of stromal cells with an increase in the extracellular space.
As granulosa cells of the follicle die by apoptosis during the process of atresia, the TUNEL technique was performed on histological ovarian slides from ovaries obtained 48 h after the injection of Trap. Figure 2A shows representative fields of a control and a treated ovary. Specific staining was observed in granulosa cells, and Trap treatment caused a twofold increase in the number of apoptotic cells in EAFs (control: 3.17 6 0.81 apoptotic cells per field; Trap: 6.81 6 1.02 apoptotic cells per field, P < 0.05, n = 5) as shown in Figure 2B.
Agarose Gel Electrophoresis and Quantitation of DNA Fragmentation
The levels of DNA fragmentation were measured in antral follicles obtained from control and treated ovaries 48 h after surgery (Fig. 3A; lane 1, control; lane 2, Trap).
FIG. 2. A) Representative fields of ovarian sections labeled by TUNEL technique. Control ovary (a);Trap-treated ovary (b). A portion of each section shows detail observed at high magnification. EAF, Early antral follicle; AtF, atretic follicle;GC, granulosa cell;TC, theca cell. B) In situ DNA fragmentation analysis (TUNEL technique). The number of apoptotic cells was determined by counting labeled cells in X400 randomly selected fields of antral follicles. Data were expressed as the apoptotic cell mean/ field (control: 3.17 6 0.82;Trap: 6.81 6 1.02 apoptotic cells per field). The number of follicles analyzed is shown between parentheses. Three sections per ovary were analyzed (five ovaries/group). *P < 0.05 Paired Student t-test;n = 5.
FIG. 3. Effect of Trap injection on DNA fragmentation in cultured preovulatory follicles. A) Agarose gel showing DNA fragmentation. B) Quantitative estimation of DNA cleavage. Animals were injected under the bursa of one ovary with 0.5 ig of Trap. The other ovary was injected with vehicle and used as a control. Forty-eight hours after surgery, antral follicles were isolated and cultured for 24 h in serum-free medium. Four micrograms of follicular DNA extracted from each culture was analyzed by ethidium bromide staining. Low-molecular-weight DNA (