Zygote 13 (May), pp. 177–185. C  2005 Cambridge University Press doi:10.1017/S0967199405003126 Printed in the United Kingdom Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development A. Nader Fatehi 1 , Bernard A.J. Roelen 1 , Ben Colenbrander 1 , Eric J. Schoevers 1 , Bart M. Gadella 1,2 , Mart M. Bevers † and Rob van den Hurk 1 Utrecht University, Utrecht, The Netherlands Date submitted: 15.12.04. Date accepted: 25.01.05 Summary The present study was conducted to evaluate the function of cumulus cells during bovine IVF. Oocytes within cumulus–oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization. Keywords: Blastocyst, Bovine, IVF, Oxidative stress Introduction The importance of cumulus cells for maturation and acquisition of developmental competence in mam- malian oocytes has been well established (Mochizuki et al., 1991; Younis & Brackett 1991; Younis et al., 1988; Zhang et al., 1995). The oocyte and its surrounding cumulus cells are metabolically coupled through gap junctions, which provide an important means for entry of various metabolites into the ooplasm (Colonna & All correspondence to: B.A.J. Roelen, Department of Farm Animal Health, Yalelaan 7, 3584 CL, Utrecht, The Netherlands. Tel: +31 30 2533352. Fax: +31 30 2534811. e-mail: b.a.j.roelen@vet.uu.nl 1 Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. 2 Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. † Deceased. Mangia, 1983; Downs, 1995; Haghighat & Van Winkle, 1990). Cumulus cells support the nuclear maturation of oocytes to the metaphase II stage and greatly enhance cytoplasmic maturation, resulting in oocytes that are capable of undergoing normal fertilization and sub- sequent embryo development (Suzuki et al., 2000). Re- moval of cumulus cells from the oocytes prior to in vitro maturation adversely affects maturation, fertilization and embryo development in rat (Vanderhyden & Armstrong, 1989), sheep (Staigmiller & Moor, 1984) and cattle (Leibfried-Rutledge et al., 1989; Zhang et al., 1995). However, information on the role of the cumulus oophorus during fertilization and subsequent embryo development is limited. Presence of cumulus cells dur- ing in vitro fertilization (IVF) increases the fertilization rate in mouse (Fraser, 1985; Itagaki & Toyoda, 1991; Siddiquey & Cohen, 1982), hamster (Bavister, 1982), pig (Kikuchi et al., 1993) and buffalo (Nandi et al., 1998) and we previously reported that removing cumulus cells prior to fertilization of in vitro matured