MOLECULAR REPRODUCTION AND DEVELOPMENT 71:389–397 (2005) Developmental Capability of Denuded Bovine Oocyte in a Co-Culture System With Intact Cumulus-Oocyte Complexes: Role of Cumulus Cells, Cyclic Adenosine 3 0 ,5 0 -Monophosphate, and Glutathione ALBERTO M. LUCIANO,* VALENTINA LODDE, MATTEO S. BERETTA, SILVIA COLLEONI, ANTONIO LAURIA, AND SILVIA MODINA Institute of Anatomy of Domestic Animals, Histology and Embryology, Faculty of Veterinary Medicine, University of Milan, Italy ABSTRACT Cumulus oophorus cells have been implicated in the regulation of female gamete development, meiotic maturation, and oocyte-sperm interaction. Nevertheless, the specific role of cumulus cells (CCs) during the final stages of oocyte maturation and fertilization processes still remains unclear. Several studies have been conducted in order to clarify the role of follicular cells using culture systems where denuded oocytes (DOs) were co-cultured with isolated CCs, or in the presence of conditioned medium. However, those attempts were ineffective and the initial oocyte competence to become a blastocyst after fertilization was only partially restored. Aim of the present study was to analyze the effect of the interactions between somatic cells and the female gamete on denuded oocyte developmental capability using a system of culture where CCs were present as dispersed CCs or as intact cumulus-oocyte complexes (COCs) in co-culture with oocytes freed of CC invest- ment immediately after isolation from the ovary. Moreover, we analyzed the specific role of cyclic adenosine 3 0 –5 0 monophosphate (cAMP) and glu- tathione (GSH) during FSH-stimulated maturation of denuded oocyte co-cultured with intact COCs. Our data confirm that denuded oocyte has a scarce develop- mental capability, and the presence of dispersed CCs during in vitro maturation (IVM) does not improve their developmental competence. On the contrary, the co- presence of intact COCs during denuded oocyte IVM partially restores their developmental capability. The absence of CCs investment causes a drop of cAMP content in DOs at the beginning of IVM and the addition of a cAMP analog in the culture medium does not restore the initial oocyte developmental competence. The relative GSH content of denuded oocyte matured in presence of intact COCs is consistent with the partial recovery of their developmental capability. However, the complete restoration of a full embryonic develop- mental potential is achieved only when DOs are co- cultured with intact COCs during both IVM and in vitro fertilization (IVF). Our results suggest that the direct interaction between oocyte and CCs is not essential during IVM and IVF of denuded oocyte. We hypothe- size that putative diffusible factor(s), produced by CCs and/or by the crosstalk between oocyte and CCs in the intact complex, could play a key role in the acquisition of developmental competence of the denuded female gamete. Mol. Reprod. Dev. 71: 389–397, 2005. ß 2005 Wiley-Liss, Inc. Key Words: denuded oocyte; cumulus-oocyte com- plex; cAMP; cysteamine; glutathione; secreted factors INTRODUCTION In the antral ovarian follicle, the cumulus oophorus is a group of closely associated granulosa cells (namely cumulus cells (CCs)), which plays a crucial role during oocyte growth and development, and ultimately during the maturation and fertilization processes in mamma- lian oocytes [reviewed in Tanghe et al. (2002)]. CCs exert important biological function: before ovula- tion they support oocyte cytoplasmic maturation and shortly after ovulation participate in the complex mechanisms controlling sperm–oocyte interaction (Tesarik, 1990). CCs communicate with each other and with the oocyte by means of an extensive network of gap junctions (Gilula et al., 1978), which permits the two-way transfer of small molecules such as nutrients and messengers between somatic and germ cells (Moor ß 2005 WILEY-LISS, INC. Grant sponsor: PRIN; Grant number: 12-2-5159205-12; Grant sponsor: FIRST; Grant number: 12-2-5159205-42. Silvia Colleoni’s present address is Laboratorio di Tecnologie della Riproduzione, CIZ srl, Via Porcellasco 7/f, 26100 Cremona, Italy. *Correspondence to: Alberto M. Luciano, Istituto di Anatomia degli Animali Domestici con Istologia ed Embriologia, Facolta ` di Medicina Veterinaria, Universita ` degli Studi di Milano, Via Celoria, 10 20133 Milano, Italy. E-mail: alberto.luciano@unimi.it Received 15 December 2004; Accepted 4 February 2005 Published online 1 April 2005 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20304