After removal of the cumulus cells, the denuded oocytes were placed back in their original wells containing standard maturation medium without or with 250?M stearic acid. were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apoptosis in cumulus cells. The esterified oleic/stearic acid ratio of the neutral lipid fraction in cumulus cells decreased in the presence of SCD inhibitors when COCs were exposed to saturated free fatty acids during maturation, indicating the SCD-specific conversion of saturated fatty acids under noninhibiting conditions. The observation that Rabacfosadine cumulus cells can desaturate the Rabacfosadine potentially toxic stearic acid into oleic acid via SCD activity provides a mechanistic insight into how the cumulus cells protect the oocyte against toxicity by saturated fatty acid. [22, 23], while the human and bovine genomes only contain two SCD genes: and [24, 25]. The aim of the current study is to determine how cumulus cells protect the oocyte against free fatty acids. Materials and methods Chemicals Unless stated otherwise, all chemicals used were obtained from Sigma Chemical Co (St. Louis, MO, USA) and were of the highest purity available. Solvents (acetone, acetonitrile, chloroform, methanol, and hexane) were of high-performance liquid chromatography (HPLC) grade (Labscan, Dublin, Ireland). Collection and maturation of cumulus-oocyte complexes Bovine ovaries were collected at a slaughterhouse and transported to the laboratory within 2 h of slaughter. Approval of an independent ethical committee was not needed as ovaries were a rest product of the regular slaughter process in the slaughterhouse. Antral follicles between 2 and 8 mm in diameter were aspirated by means of a suction pump under low vacuum. The follicular aspirates were pooled in a conical tube and allowed to settle for 15 min. Oocytes with a multilayered cumulus investment were selected from the follicular fluid, washed three times in HEPES-buffered M199 (Gibco BRL, Paisley, UK), and randomly allocated in groups of 35C70 COCs per well in four-well culture plates (Nunc A/S, Roskilde, Denmark). In vitro Rabacfosadine maturation (IVM) was carried out for 23 h according to our standard protocol  in maturation medium consisting of 500?l M199 per well, and no coverage of oil, supplemented with 26.2 mM NaHCO3, 0.02 IU/ml FSH (Sioux Biochemical Inc., Sioux Center IA, USA), 0.02IU/ml LH (Sioux Biochemical Inc.), 7.7?g/ml cysteamine, 10?ng/ml epidermal growth factor, and Rabbit polyclonal to ADAM18 1% (v/v) penicillin-streptomycin (Gibco BRL) at 39C in a humidified atmosphere of 5% CO2 in air. Fertilization and embryo culture After 23 h of maturation, in vitro fertilization was performed with 0.5 106/ml sperm from a bull with proven fertility. At 18C22 h after sperm addition, the cumulus cells and adhering sperm cells were removed by vortexing the presumed zygotes for 3 min in the experiment with SCD inhibition. Subsequently, the zygotes were transferred in groups of 35C70 to wells with 500?l pre-equilibrated synthetic oviductal fluid (SOF, ). Fertilization and embryo culture were performed, according to our standard procedure , in a humidified incubator at 38.5C with 5% CO2 and respectively 20% and 7%O2. At day 5 of culture, cleaved embryos were transferred to fresh SOF and cultured until day 8 on which embryonic development was assessed. In vitro maturation with free fatty acids and stearoyl-CoA desaturase 1 inhibitor The free fatty acids used in the maturation experiments were processed according to our standard protocol  and bound to 100% delipidified bovine serum albumin resulting custom-tailored lipidified BSA, and were stored in stock at a concentration of 10 mM bound to 10% (w/v) fatty acid-free BSA (fatty acid:BSA stoichiometry of 5:1). For the experiments assessing the importance of cumulus cells in protecting oocytes against free fatty acids, the cumulus cells of oocytes were removed after a maturation period of 8 Rabacfosadine h by vortexing COCs for 3 min in HEPES-buffered M199 . After removal of the cumulus cells, the denuded oocytes were placed back in their original wells containing standard maturation medium without or with 250?M stearic acid. As a control, groups of intact COCs were matured in maturation medium without or with 250?M stearic acid. For the experiment where cumulus cells were removed at 8 h, cumulus cells from the control group with maturation as intact COCs were removed before fertilization by vortexing for 3 min, which is a modification from our standard procedure. Oocytes were fertilized and cultured according.