Different cell death types determination in juvenile mice ovarian follicles

Follicular atresia is a phenomenon that leads to evacuation of the ovary from the oocytes and the occurrence of menopause. The contribution of various types of cell death in atresia at different follicular developmental stages requires extensive investigation. In this study, we evaluated 3 types of programmed cell death (PCD), apoptosis, autophagy, and necrosis, in juvenile mouse ovary when we can observe all follicular stages as well as atresia. Ovaries from juvenile mice on the 21st post-natal (PN) day were prepared histologically for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to evaluate apoptosis and immunohistochemistry for beclin-1 to evaluate the autophagy marker. Necrotic cell death was also assessed by penetration of propidium iodide (PI). The count and percentage of the labeled follicles at different stages in the ovaries were evaluated and compared using the Kruskal-Wallis and Mann-Whitney tests. We detected TUNEL-positive granulosa cells in pre-antral and antral follicles but not in the primordial and primary follicles. Somatic cells and oocytes of primordial, primary, pre-antral and antral follicles reacted to beclin-1. The percentage of the PI-labeled primordial and primary follicles were significantly higher than the beclin-1 positive (P=0.01 and P=0.01). In conclusion, we showed that apoptosis, autophagy, and necrosis play a role in follicular atresia and the contributions of each one depends on the follicular stages. It was also demonstrated that necrosis happens particularly in the small follicles while in the large one, all three cell death types occurred with an equal ratio.