To evaluate changes in general structure, the ovaries were removed and immediately fixed in 4% neutral-buffered formalin for 12 h and then embedded in paraffin. Four-micrometer step sections were mounted at 50-im intervals onto microscope slides to prevent counting the same follicle twice, according to the method described by Woodruff et al.. One set of slides was stained with hematoxylin-eosin in order to count the number of different stages of follicles per ovary section, and the others were immunostained by the TUNEL technique.
Follicles were classified as preantral (PF) or early antral (EAF), according to the presence or absence of an antrum, or periovulatory follicles (POFs), which include the advanced preovulatory follicles plus the recently formed corpus luteum. Morphological characteristics of atretic follicles include the degeneration and detachment of the granulosa cell layer from the basement membrane, the presence of pyknotic nuclei in this cell type, and oocyte degeneration. The number of PFs, EAFs, POFs, and atretic follicles was determined in three ovarian sections from each ovary (three sections per ovary; six to eight ovaries per dose or time).