Figure 2.

Ovarian follicles, proliferation, apoptosis and vascular state in mice after irinotecan treatment. Mature female mice were treated as described in the legend of Figure 1. Ovaries were excised from mice 1 week (1W) or 3 months (3M) after treatment, fixed, paraffin-embedded and serially sectioned for immunohistochemistry, immunofluorescence and terminal transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. (A) Representative bright field images of ovaries stained with Ki-67 (brown; Aa-f) and representative florescence images of ovaries stained anti-PCNA (green; Aa'-f'), TUNEL (green; Aa”-f”) or CD34 (red; Aa”'-f”'). Bars = 100 μm. Twenty randomly selected transverse sections of ovaries of three mice from each experimental group, each staining and each time point (1 week, white bars; 3 months, gray bars) were used for automatic analysis by Fiji software. The average amount of primordial and primary (white bars), secondary (gray bars) and antral (black bars) follicles (B) and the average amount of ovarian TUNEL positive cells/mm² as a measure of apoptosis (C) and CD34 positive blood vessels/mm² as a measure of blood vessels vascularity (D) per ovary 1 week (1W) or 3 months (3M) after treatment were measured. Bars are mean ± standard error of mean. (*), significantly different from saline value (P < 0.05). (**), CPT-11 significantly different from CTX value (P <0.05). CPT-11, irinotecan; CTX, cyclophosphamide.