101 Evaluation of apoptosis in canine ovarian follicles throughout the estrous cycle

The degenerative process by which follicles are eliminated before reaching ovulation is termed atresia. Ovarian follicle atresia is the result of the highly regulated process of apoptosis. Cyclic changes of apoptosis in canine ovarian follicles are still poorly known. Thus, in the present study, we examined the apoptotic cyclic changes of canine ovarian follicular cells of different sizes throughout the oestrous cycle. The canine ovaries were collected from 26 adult female dogs (1–4 years) following routine ovariohysterectomy. The surface diameter of antral follicles was measured using a graticule in the stereomicroscope, classifying each follicle as small antral (∼0.2–0.39 mm), medium antral (∼0.4–5.9 mm), or large antral or preovulatory (∼6–10 mm). Determination of the percentage of cells with apoptosis was carried out by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) DNA nick end labelling (TUNEL) method by flow cytometry assays. According to the proportion of labelled cells, apoptosis was quantified as the percent of TUNEL-positive cells on a logarithmic scale. Percentages of TUNEL-positive cells obtained in the flow cytometric assay were compared among oestrous phases and follicular size by ANOVA. Follicles in apoptosis were observed in all canine follicles in different cycle phases and follicular development, possibly corresponding with the physiologic process of the oestrous cycle. Both oestrous phase and follicular size influenced the levels of apoptosis (P < 0.05). The percentage of apoptosis in canine follicular cells showed the greatest rate of apoptosis in large follicles at the oestrus phase (9.2%) and the lowest percentage was observed in anoestrus period small follicles (1.8%; P < 0.05). The rate of apoptosis increased (P < 0.05) according to follicular development: the largest follicles expressed the highest (P < 0.05) percentage of apoptotic cells, and small follicles expressed the lowest (P < 0.05) TUNEL-positive reactions. In conclusion, our results indicate that significant differences in the apoptosis process occur during the oestrous cycle related to follicle development in the canine ovary. In addition, flow cytometry using the TUNEL assay was a very effective method for detecting apoptosis in canine follicles.

This research was funded by FONDECYT grant 1211285.