Short Title: Gap junction’s regulation of chromatin remodeling Summary sentence: Gap junction mediated communications regulate chromatin remodeling, transcriptional activity and developmental competence acquisition during growth and differentiation of bovine oocytes isolated from small antral follicles involving cAMP mediated mechanism(s)

Oocyte development is characterized by impressive changes in chromatin structure and function in the germinal vesicle (GV), which are crucial to confer the oocyte with meiotic and developmental competences. During oogenesis, oocyte and follicular cells communicate by paracrine and junctional mechanisms. In cow, cumulus enclosed oocytes (CEOs) isolated from early antral follicles have uncondensed chromatin (GV0), functionally open gap junction (GJ) mediated communications and a limited meiotic competence. Aim of this study was to analyze the role of GJ communications on the chromatin remodeling process during the specific phase of folliculogenesis that coincides with the transcriptional silencing and the sequential acquisition of meiotic and developmental capability. CEOs were cultured in a FSH-based culture system that sustained GJ coupling, promoted oocyte growth and transition from GV0 to higher stages of condensation. When GJ functionality was experimentally interrupted, chromatin rapidly condensed and RNA synthesis suddenly ceased. These effects were prevented by the addition of cilostamide, a phosphodiesterase 3 inhibitor, indicating that the action of GJ mediated communication on chromatin structure and function is mediated by cAMP. Prolonging GJ coupling during oocyte culture prior to IVM enhanced the ability of early antral oocytes to undergo meiosis and early embryonic development. Altogether these evidences suggest that GJ-mediated communication between germinal and somatic compartments plays a fundamental role in the regulation of chromatin remodeling and transcription, which are in turn related to competence acquisition. BOR Papers in Press. Published on August 3, 2011 as DOI:10.1095/biolreprod.111.092858 Copyright 2011 by The Society for the Study of Reproduction. 2 Introduction The mammalian oocyte nucleus or germinal vesicle (GV) displays a distinctive chromatin configuration, which is subjected to dynamic modifications during oocyte growth and differentiation (reviewed in [1-3]). In both mouse [4] and cow [5], these changes have been related to the achievement of oocyte developmental competences, which are gradually acquired during the long lasting period of oogenesis [1]. Therefore, the chromatin configuration represents a morphological marker of oocyte differentiation and competence [1-3, 5]. In cow, in particular, analyzing the chromatin morphology of oocytes collected from early(0.5<2 mm) and middle(2-6 mm) antral follicles, four discrete stages of GV, from GV0 to GV3, in which chromatin becomes progressively condensed, have been identified [5, 6]. Among these, GV0 stage, which is characterized by a diffuse filamentous pattern of chromatin in the whole nuclear area, represents the high majority of oocytes in early antral follicles, while it is absent in midantral follicles [5, 6]. Notably, GV0 oocytes are transcriptionally active and unable to progress through the metaphase II of the meiotic division. The transition to GV1 stage represents the establishment of a repressed transcriptional state and the achievement of a full meiotic competence, but still a limited capacity to complete preimplantation development after in vitro fertilization [5, 6]. GV2 and GV3 show the highest developmental capability [5]. To date, the mechanisms that regulate chromatin remodeling and transcriptional silencing in the oocyte are still poorly understood. It has been suggested that companion granulosa cells contribute in modulating these events [7, 8]. Clearly, gap junction (GJ) mediated communications between the oocyte and the surrounding cumulus cells could have a pivotal role in these processes [9, 10]. Several evidences support this hypothesis. In the mouse model, for example, the presence of oocyte-associated granulosa cells is necessary for the progressive repression of transcription during the in vitro culture of oocytes isolated from pre-antral follicles [7]. On the contrary, in the absence of a patent GJ mediated communication with somatic granulosa cells, transcriptional activity remains unabated in denuded oocytes [7]. Furthermore, chromatin condensation fails to occur in knock-out mouse, where GJ mediated communications between oocyte and cumulus cells is interrupted due to targeted deletion of the connexin 37 gene, Gja4 [11]. Finally, in cow the pattern of uncondensed chromatin in GV0 oocytes associates with a fully open state of GJ mediated communications, while the percentage of oocytes with functionally open communications significantly decreases from GV1 to GV3 stage [5]. Starting from these observations, this study is aimed to better clarify the role of GJ mediated communications on the process of large-scale chromatin remodeling during the specific phase of bovine folliculogenesis that coincides with the transcriptional silencing and the sequential acquisition of meiotic and developmental capability. To this purpose, GJ functionality was modulated during the culture of cumulus oocytes complexes isolated from early antral follicles. We then assess the effect on chromatin configuration, transcriptional activity, and meiotic and developmental competences. In particular, since GJ mediated intercellular coupling is involved in the metabolic cooperation between compartments [9, 12] mainly through the regulation of cAMP [13-16], we examined the hypothesis that GJ mediated communications regulates chromatin remodeling and transcription through a cAMP mediated mechanism. This hypothesis was tested by the use of the oocyte-specific phosphodiesterase type 3 (PDE3) inhibitor, cilostamide, able to keep high the level of cAMP within the oocytes, without altering the activity of the surrounding somatic cells [17-19].