Effect of 7,8-Dihydroxyflavone as an Antioxidant on <i>In Vitro</i> Maturation of Oocytes and Development of Parthenogenetic Embryos in Pigs

Abstract

One of the factors that impairs in vitro produced porcine embryos is the oxidative stress that is mainly caused by the imbalance between reactive oxygen species (ROS) generation and antioxidants activity, especially that of glutathione (GSH). Here, we examined the effect of 7,8-dihydroxyflavone (7,8-DHF), a kind of flavonoid antioxidant, on porcine oocyte maturation and its developmental competence. Porcine oocytes were cultured in media supplemented with 0, 1, 5 and 10 μM 7,8-DHF during both in vitro maturation (IVM) and in vitro culture (IVC) after parthenogenetic activation. Maturation of oocytes was evaluated based on first polar body (PB) extrusion and intracellular GSH level, and developmental competence was assessed through observing cleavage and blastocyst formation. In each step, the levels of intracellular GSH and ROS were assessed by fluorescence intensity, and the apoptosis-related gene expression was examined using semiquantitative RT-PCR. The group treated with 1 μM 7,8-DHF during IVM and IVC showed increased cytoplasmic maturation and reached the blastocysts stage (36.1%) at a higher rate than the other groups (24.7, 16.0 and 10.3% for 0, 5 and 10 μM, P<0.05). In that group, the intracellular GSH level was significantly increased while ROS generation was significantly decreased after IVM and IVC (P<0.05). Moreover, it showed high expression of an anti-apoptotic gene (BCL2L1) and low expression of a pro-apoptotic gene (BAK1) (P<0.05). In conclusion, treatment with 1 μM 7,8-DHF during IVM and IVC showed an anti-apoptotic effect by increasing intracellular GSH synthesis and scavenging ROS and therefore improved the developmental competence of porcine embryos.

Effects of supplementation of medium with different antioxidants during in vitro maturation of bovine oocytes on subsequent embryo production

“…Yet, regardless of the use of one or two associated antioxidants, these substances may have other benefits. The flavonoids (quercetin and resveratrol), cysteamine (via GSH), vitamin C and carnitine interact with gene expression, altering the expression of certain genes (Bax, Blc‐2, TP53, Oct‐4, Cx‐43, IGF‐1 and Glut‐1), which may cause a likely reduction in cell damage (Choi et al., ; Elamaran et al., ; Kawashima et al., ; Mukherjee et al., ; You, Lee, Hyun, & Lee, ). The antioxidants quercetin, resveratrol, vitamin C and carnitine can also present a protective effect on mitochondrial functions (Carrasco‐Pozo, Mizgier, Speisky, & Gotteland, ; Lee., Li, Chen, & Chang, ; Somfai et al., ; Sugiyama et al., ) to new mitochondrial synthesis in oocytes from antral follicles cultured in vitro with resveratrol.…”

“…For evaluation, the oocytes (10–15 oocytes) were incubated in 10 μM 2′,7′‐dichlorodihydrofluorescein diacetate (H 2 DCFDA, diluted in DMSO) in PBS/PVA, for 20 min in a cell culture incubator (38.5°C; 5% CO 2 in air) to determine ROS levels (Mukherjee et al., ; Wang et al., ). For GSH levels evaluation followed the same procedure, but the oocytes were incubated in 10 μM 4‐chloromethyl‐6,8‐difluoro‐7‐hydroxycoumarin—Cell Tracker Blue—diluted in DMSO (Thermo Fisher Scientific Inc. USA) (Choi et al., ; Kwak et al., ; Mukherjee et al., ; Wang et al., ). As technical control, oocytes stained for ROS or GSH were also evaluated in groups cultured with 0.5 mg/ml catalase (diluted in H 2 O) or 0.003% hydrogen peroxide (H 2 O 2 , diluted in H 2 O), respectively (Nasr‐Esfahani, Aitken, & Johnson, ).…”

Methods of RNA preparation affect mRNA abundance quantification of reference genes in pig maturing oocytes

“…(), while the other four ( YWHAG/RPL4/HRPT1/PPIA ) are different. The reason we selected these seven genes for RT‐qPCR normalization is that they were reported within recent 5 years (year 2012 to 2015) (Choi et al., ; Gu et al., ; Kuchipudi et al., ; Nagyova et al., ; Park et al., ; Santiquet et al., ; Spate et al., ), comparing to the report published in year 2007 (Kuijk et al., ). In addition, YWHAG, one candidate gene included in the present study but not in Kuijk et al.…”

“…All seven candidate genes ( GAPDH, 18S, YWHAG, BACT, RPL4, HRPT1 and PPIA ) were chosen primarily based on their relatively stable expression in different types of pig tissues, cells and oocytes, as reported previously (Choi et al., ; Gu et al., ; Kuchipudi et al., ; Nagyova, Nemcova, Mlynarcikova, Scsukova, & Kalous, ; Park et al., ; Santiquet, Robert, & Richard, ; Spate et al., ). Detailed information on these genes was summarized as in Table .…”

Thymoquinone reduces intracytoplasmic oxidative stress and improves epigenetic modification in polycystic ovary syndrome mice oocytes, during in‐vitro maturation

“…Thus in PCOS patients, in-vitro maturation (IVM), not only rescues oocytes from ovarian damage, but also compensates the deficiency of antioxidants activity, preserves immature oocytes from unfavorable in-vivo maturation, and avoids ovarian hyperstimulation (Nikmard et al, 2017;Savic-Radojevic et al, 2015;Zhao et al, 2009). However, the activation of the oxidative stress pathway in the IVM medium is unavoidable (Choi et al, 2013). Recent studies showed that the environmental factors, such as light and culture medium composition, can induce metabolic alterations in the cytoplasm, resulting in mitochondrial dysfunction, augmentation of lipid peroxidation, DNA fragmentation, apoptosis, and embryo growth arrest in cultured oocytes (Agarwal, Said, Bedaiwy, Banerjee, & Alvarez, 2006).…”