MORPHOLOGICAL EVIDENCE OF APOPTOSIS AND THE PREVALENCE OF APOPTOTIC VERSUS MITOTIC CELLS IN THE MEMBRANA GRANULOSA OF OVARIAN FOLLICLES DURING SPONTANEOUS AND INDUCED ATRESIA IN EWES

Apoptosis is a process by which granulosa cells are thought to be dele ted during ovarian follicular atresia. The aims of the present studies , using sheep as the experimental model, were to determine 1)whether m orphological changes in cells composing the membrana granulosa during the process of atresia conformed with the general criteria of apoptoti c cell death as assessed using tissue sections stained with hematoxyli n and eosin; 2) whether cells classified as apoptotic on the basis of their morphology contained fragmented DNA using an in situ 3' end-labe ling technique; and 3) the degree of apoptosis and mitosis within the granulosa cell populations of large antral follicles (greater than or equal to 3 mm in diameter) during both spontaneous and experimentally induced atresia using stereological methods. The results showed that m ost degenerate granulosa cells in follicles undergoing atresia display the morphological characteristics of apoptosis, suggesting that this is the most common pathway of cell deletion. Typical features were cel ls containing nuclei with marginated chromatin; cells with a single sm all densely staining nucleus (pyknotic appearance); cells with multipl e smaller, densely staining nuclear fragments; and densely staining me mbrane-bound bodies (apoptotic bodies) either singly or in clusters. C ells with morphological features more typical of oncosis or necrosis w ere sometimes observed, but mainly during the later stages of atresia. All cells classified as apoptotic on the basis of morphological crite ria contained fragmented DNA as measured by 3' end-labeling. Apoptotic bodies and/or cells were found in all follicles examined, including t hose classified as healthy. The overall prevalence of apoptotic cells plus apoptotic bodies expressed as a percentage of the total granulosa cell number per follicle varied from 0.02% to 0.20% in healthy follic les, varied from 0.21% to 2.00% in follicles in early (primary) atresi a, and was > 2.0% in follicles in later (secondary) atresia. Percentag es of mitotic cells in healthy follicles were > 0.5% in all but one of those examined and were < 1.0% in all follicles classified as atretic . Both morphological and 3' end-labeling results indicated that apopto tic cells were widely disseminated throughout the membrana granulosa, including the cell layer adjacent to the basement membrane. Collective ly, these observations indicate that during early atresia, apoptosis o ccurs randomly and is not limited to specific areas within follicles. Our finding that apoptotic cell death and mitosis occur simultaneously within the same follicle is consistent with the notion that atresia i s determined by a dynamic equilibrium between cell division, different iation, and death.