Different cell death types determination in juvenile mice ovarian follicles

Abstract

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.

Keywords: Apoptosis; Autophagy; Mouse; Necrosis; Ovary.

Fig. 1

The sections of the juvenile mice ovaries stained with haematoxylin and eosin. (A) The follicles are observed in different developmental stages, (B) Follicles in late stage of atresia are located at the adjacent antral follicle (yellow arrows), and (C) Antral follicle in early stage of atresia (yellow arrows) positive, but with less intensity (Fig. 5B).

Fig. 2

Apoptosis is detected in the ovaries. TUNNEL-positive (brown) granulosa cells (arrows) were detected in the antral and pre-antral follicles

Fig. 3

The sections of the juvenile mice ovaries stained by TUNEL reaction and counterstained methyl green. (A) TUNNEL-negative primordial (yellow arrow) and primary follicles (white arrow) were detected, and (B) TUNNEL-positive cells (arrows) can be also found in the ovarian surface epithelium

Fig. 4

Immunohistochemistry for beclin-1 shows that autophagy has happened in the primordial and primary follicles. The nuclei are stained with Hoechst. (A) A primary follicle (arrow) with beclin-1 expression in the oocyte and somatic cells, (B) A primordial follicle (arrow) is reacted to beclin-1 for both oocyte and squamous somatic cells

Fig. 5

Immunohistochemistry for beclin-1. (A) Lower magnification of the mouse ovary shows that beclin-1-positive cells are prominent in the theca layer of the pre-antral and antral follicles, (B) Higher magnification of the oocyte of antral follicle shows that the oopelasm and granulosa cells labeled for beclin-1 (yellow and white arrows, respectively), and (C) The theca cells with high intensity for beclin-1 (arrow) can also be detected

Fig. 6

Whole-mount staining of the ovary reveals areas of propidium iodide (PI) absorption in the cortical and medullary regions. (A) Necrosis has happened mainly in the medullary region. M: Medulla, and C: Cortex. The arrow shows follicle in cortical region, (B) Somatic cells (arrow) of primary follicles are stained with PI, and (C) Somatic cells in the primordial follicles (arrow) also show necrosis

Fig. 7

Sections of the ovary pre-exposed to propidium iodide (PI) are counterstained with Hoechst. Necrosis is shown in the theca and granulosa cells in large follicles