Synergistic Effects of Glutathione and .BETA.-Mercaptoethanol Treatment During In Vitro Maturation of Porcine Oocytes on Early Embryonic Development in a Culture System Supplemented with L-cysteine

Abstract

Abstract. Various methods have been used to remove reactive oxygen species (ROS) generated from in vitro culture (IVC) conditions that can cause cell injury or death, including the application of low oxygen (O2) tension and the addition of antioxidants. The beneficial effects of antioxidants and O2 tension on IVC of porcine embryos, however, are controversial among researchers. In this study, we sought to determine the effects and optimal concentrations of antioxidants for the development of porcine embryos in an IVC system. Specifically, we examined the synergistic effects of antioxidants on development to the blastocyst stage in a culture system supplemented with L-cysteine during IVM. Of the antioxidants tested (melatonin, glutathione (GSH), β-mercaptoethanol (β-ME), N-acetylcysteine (NAC) and dithiothreitol (DTT)), addition of GSH (1 mM) or β-ME (25 μM) significantly increased development to the blastocyst stage compared with the controls without antioxidant treatment (22.2 ± 4.2% for 1 mM GSH, 25.9 ± 2.2% for 25 μM β-ME and 12-13% for the control, P<0.05). In addition, the mean cell number per blastocyst was increased by approximately 1.7-fold in the presence of GSH or β -ME. These GSH-and β-ME-induced increases in development to the blastocyst stage and total cell number, however, were not mimicked by melatonin, NAC or DTT, all of which are ROS scavengers. The combination of GSH or β-ME with L-cysteine significantly reduced high O2 tension-induced ROS production (P<0.05). These results suggest that a combination of 1 mM GSH or 25 μM β-ME with 1 mM L-cysteine could be used for production of high quality porcine blastocysts in IVC systems. Key words: Antioxidant system, Embryo, Oxidative stress, Pig (J. Reprod. Dev. 56: [575][576][577][578][579][580][581][582] 2010) he academic and industrial value of porcine embryonic technology is very high, as this technology allows for production of transgenic porcine embryos with disease-resistance genes, for xenogenic organ transplantation and for preservation of superior genotypes that are at risk of extinction. Obtaining many high-quality embryos, however, is a prerequisite for improvement of embryo technology. A large number of immature oocytes can be collected from the ovaries of slaughtered pigs, and these oocytes can then be matured, fertilized and cultured in vitro. At present, the quality of in vitro produced (IVP) porcine embryos is considerably lower than that of in vivo-derived embryos, although many investigators have attempted to improve in vitro culture systems to help produce higher quality porcine embryos.Earlier studies have shown that in vitro cultured (IVC) embryos cannot develop normally, as the oxygen (O2) tension in vitro is higher than that in the oviduct. This high O2 tension induces excessive production of reactive oxygen species (ROS), which leads to oxidative stress and impedes oocyte maturation and embryonic development [1,2]. To remove the ROS generated from IVC conditions, various methods have been used such as the application of low O2...

Melatonin and tannic acid supplementation in vitro improve fertilization and embryonic development in pigs

“…Our results agree with this study, as we found supplementation of melatonin to decrease the ROS produced during maturation. The level of melatonin appears to impact the success of oocyte maturation, as a previous study reported minimal improvements, however the levels were lower than those used in the current study (Choe et al, 2010). Shi et al (2009) reported that the concentrations of melatonin change as the follicle changes size, suggesting an effect of melatonin in oocyte maturation: as the follicle size increased, the levels of melatonin decreased.…”

Effects of systemic progesterone during the early luteal phase on the availabilities of amino acids and glucose in the bovine uterine lumen

“…Orn is an intermediary in the synthesis of polyamines, derived from the conversion of Arg to Orn by arginase followed by conversion of Orn to polyamines by ornithine decarboxlyase (ODC1), which is essential for embryogenesis and placental development (Fozard et al 1980;Bazer et al 2012). Cys, a member of the functional amino acids (defined as regulators of key metabolic pathways benefiting survival, growth, development, reproduction and health; Wu et al 2009), has been shown to improve the development of porcine blastocysts in vitro (Choe et al 2010).…”

Male pronucleus formation after ICSI: effect of oocyte cysteine or sperm Triton X-100 treatments

“…In this regard, several IVM approaches have been applied, such as oocyte co-culture with follicular cells (Zheng and Sirard 1992;Ding and Foxcroft 1994;Agung et al 2010), or supplement addition to the IVM defined media with: porcine follicular fluid (Naito et al 1988;Yoshida et al 1992;Ka et al 1997), follicular fluid meiosis-activating sterol (Faerge et al 2006), bovine serum albumin (Zheng and Sirard 1992), fetal bovine serum (Kishida et al 2004), fetal calf serum (Naito et al 1988), epidermal growth factor (EGF) (Ding and Foxcroft 1994;Uhm et al 2010), β-mercaptoethanol (Abeydeera et al 1998), cysteine (CYS) (Yamauchi and Nagai 1999;Yoshioka et al 2003;Kobayashi et al 2007;Choe et al 2010), ascorbic acid (Tatemoto et al 2001), leptin (Craig et al 2005;Jin et al 2009) or lycopene (Watanabe et al 2010), among others.…”