236 NUCLEAR AND CYTOPLASMIC MODIFICATIONS OF GERMINAL VESICLE BOVINE OOCYTES IN RELATION TO CHROMATIN REMODELING

Abstract

We previously reported that germinal vesicle (GV) bovine oocytes can be classified on the basis of their chromatin organization and that increased chromatin condensation is accompanied by a major incidence of gap junction-mediated coupling interruption between germ and cumulus cells and by an increase in oocyte developmental competence (Lodde et al. 2005 Reprod. Fertil. Dev. 17(2), 294-295). The aim of this study was to characterize, at the ultrastructural level, both nuclear and cytoplasmic compartments of bovine oocytes classified according to their chromatin configuration because key structural modifications, such as nucleolar inactivation and remodeling of specific ooplasmic structures, take place during the later phases of oocyte growth. Cumulus-oocyte complexes collected from 0.5-2-mm early antral (EA) and 2-6-mm mid-antral (MA) follicles were freed of cumulus cells. Denuded oocytes were stained with Hoechst 33342, classified according to the degree of chromatin condensation, and processed for light microscopy of semi-thin sections (LM; n = 10 in each class) and transmission electron microscopy (TEM; n = 5 in each class). Four classes of oocytes were identified by the Hoechst staining: GV0 with filamentous chromatin diffused in the nuclear area, GV1 with few foci of condensed chromatin, GV2 with chromatin further condensed into distinct clumps, and GV3 with chromatin condensed into a single clump. Almost all oocytes collected from EA follicles were classified as GV0. Oocytes of this class were absent in MA follicles, whereas class GV1, GV2, and GV3 oocytes occurred at similar frequency. LM confirmed the chromatin condensation found by the Hoechst staining and revealed that in class GV2 and GV3 oocytes the chromatin was mainly located close to the nucleolus. Ultrastructurally, the nucleolus was fibrillo-granular in GV0 oocytes; the oocytes in the other classes displayed an electron dense fibrillar sphere with the remnant of a fibrillar center on the surface. Organelles were dispersed in the cytoplasm at GV0 while at GV1 and GV2 most organelles were homogenously distributed in the oocyte cortex. At GV3 most organelles were found in clusters in the oocyte cortex. Typical features of completion of the oocyte growth phase, like undulation of the nuclear envelope and reduction of the size of Golgi complex, were found at GV2 and GV3. Moreover, GV3 oocytes presented cortical granules that displayed varying degrees of degeneration. Our findings indicate that the process of chromatin remodeling is strictly related to structural modifications that characterize the later stages of the oocyte growth phase. Because the highest degree of chromatin condensation was combined with degenerative features of cortical granules, we hypothesize that this class of oocytes (GV3) originated from early atretic follicles, as also suggested in other species. The evaluation of oocytes on the basis of chromatin configuration may be useful for the development of new strategies for manipulating fertility in mammals.

This work was supported by a COFIN Grant.