288 RELATIONSHIP BETWEEN CHROMATIN ORGANIZATION AND OOCYTE-CUMULUS CELL COMMUNICATION IN GERMINAL VESICLE STAGE BOVINE OOCYTES

Abstract

Chromatin configuration in the germinal vesicle (GV) undergoes dynamic changes during oocyte growth, and the progressive chromatin condensation has been related to the acquisition of embryonic developmental potential. However, little is known about the mechanisms that regulate chromatin remodeling. In immature mouse oocytes, chromatin condensation and redistribution around the nucleolus are associated with transcriptional repression in both in vivo-derived and in vitro-cultured oocytes in the presence of an intact cumulus oophorus (de la Fuente et al. 2001 Dev. Biol. 229, 224). It is widely accepted that oocyte communication with the somatic cell compartment is essential for both oocyte growth and acquisition of meiotic competence (Eppig et al. 1997 Hum. Reprod. 12, 127). In particular, cumulus cells play an active role in modulating the levels of transcription in the nucleoplasm and in perinuclear domains as well as in chromatin configuration of GV stage oocytes. In cattle, a heterogeneous population of cumulus-oocyte complexes (COCs) has been found after isolation from the follicle, and this is characterized by a different functional degree of gap junction-mediated communication (Luciano et al. 2004 Biol. Reprod. 70, 465). This study was aimed at investigating the possible correlation between the chromatin configuration of immature bovine oocytes and the status of communication between the oocyte and cumulus cells, and oocyte developmental competence. In the first experiment, 138 COCs, isolated from follicles 2–6 mm in diameter, were injected with a 3% solution of Lucifer Yellow to assess the communication status between oocytes and cumulus cells. Successively, COCs were freed of cells, and denuded oocytes (DOs) were stained with Hoechst 33342 to determine the chromatin configuration. In a second experiment, 330 COCs were denuded and stained with Hoechst 33342 in order to assess chromatin configuration and then matured in vitro according to their GV stage. After IVM, DOs were fertilized, and presumptive zygotes were cultured for 7 days at which time blastocyst rate was assessed. Data were analyzed by ANOVA and Fisher's PLSD test. Three stages of GV oocytes were identified: GVI, with filamentous chromatin distributed in the nucleoplasm; GVII, with chromatin condensed into thick clumps; and GVIII, with chromatin condensed into a single clump. The GVIII stage showed a lower proportion of functional open communication than the GVI and GVII groups (8.5 vs. 45.7 and 46.1, respectively, P < 0.05). However, when compared with each other, the GVI stage oocytes showed lower embryonic developmental competence (12.9 in GVI vs. 22.1 and 24.2 in GVII and GVIII, respectively, P < 0.05). Our findings indicate that the status of communication between oocytes and cumulus cells could be related to the chromatin organization in immature bovine oocytes. A direct correlation between the communications grade, the modulation of oocyte transcriptional activity, and the acquisition of oocyte developmental competence remain to be confirmed.

This work was supported by a 2003 UniMi Grant.