Carboxyethylgermanium sesquioxide (Ge-132) treatment during in vitro culture protects fertilized porcine embryos against oxidative stress induced apoptosis

Abstract

Compared with the in vivo environment, porcine in vitro embryo-culture systems are suboptimal, as they induce oxidative stress via the accumulation of reactive oxygen species (ROS). High ROS levels during early embryonic development cause negative effects, such as apoptosis. In this study, we examined the effects of the antioxidant carboxyethylgermanium sesquioxide (Ge-132) during in vitro culture (IVC) on embryonic development in porcine in vitro fertilization (IVF) embryos. Zygotes were treated with different concentrations of Ge-132 (0, 100, 200 and 400 μg/ml). All of the Ge-132 treatment groups displayed greater total cell numbers after IVC (98.1, 98.5 and 103.4, respectively) compared with the control group (73.9). The 200 μg/ml Ge-132 treatment group exhibited significantly increased intracellular GSH levels compared with the control group, whereas the ROS generation levels decreased in Ge-132 dose-dependent manner (P < 0.05). The mRNA expression levels of the KEAP1 gene and proapoptotic genes BAX and CASPASE3 were lower in the Ge-132 treated blastocysts compared with the control group (P < 0.05). The percentages of apoptotic and necrotic cells in the Ge-132 treated embryos on day 2 (48 h) were significantly lower than the untreated embryos (9.1 vs. 17.1% and 0 vs. 2.7%, respectively). In the day 7 blastocysts, the percentages of apoptotic cells in 200 µg/ml Ge-132 treated group were lower compared to controls (1.6 vs. 2.5%). More KEAP1 protein was found to be localized in cytoplasm of the 200 μg/ml Ge-132 treated blastocysts, whereas KEAP1 protein was predominantly nuclei in the control blastocysts. These results indicate that the developmental competence of embryos cultured under Ge-132 treatment may be associated with KEAP1 signaling cascades involved in oxidative stress and apoptosis during porcine preimplantation embryo development.

Keywords: Apoptosis; Ge-132; In vitro culture (IVC) porcine embryos; Oxidative stress.

Fig. 1.

Effect of various concentrations of Ge-132 treatment during in vitro culture (IVC) on the cleavage pattern of in vitro fertilization (IVF) embryos at day 2 (A) and the percentage of IVF embryos that developed to the blastocyst stage at day 7 (B). For each end point, bars with different letters (a–c) are significantly (P < 0.05) different for different concentrations of Ge-132. EarBL, early blastocyst; ExpBL, expanded blastocyst; HatBL, hatched blastocyst. The experiment was replicated five times.

Fig. 2.

Epifluorescentphotomicrographic images of in vitro cultured porcine embryos. (A) Embryos were stained with CellTracker Blue (a–d) and 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (e–h) to detect the intracellular levels of glutathione (GSH) and reactive oxygen species (ROS), respectively. Porcine embryos on day 2 at the 4- to 5-cell stages derived from the in vitro culture (IVC) medium supplemented with 0 µg/ml Ge-132 (a and e), 100 µg/ml Ge-132 (b and f), 200 µg/ml Ge-132 (c and g) and 400 µg/ml Ge-132 (d and h). (B) The relative levels of intracellular GSH and ROS in in vitro cultured porcine embryos among the four groups (0 µg/ml, 100 µg/ml, 200 µg/ml and 400 µg/ml Ge-132). Within each group end point (GSH and ROS), the bars with different letters (a–c) are significantly (P < 0.05) different. The experiment was replicated three times.

Fig. 3.

mRNA expression levels (Mean ± SEM) of POU5F1, CDX2, NRF2, KEAP1, BAX, and CASPASE3 in blastocysts treated with Ge-132 during in vitro culture (IVC). Within the same target mRNA, values with different superscript letters (a and b) are significantly (P < 0.05) different. The experiment was replicated at least three times.

Fig. 4.

Effect of Ge-132 on the protein expression of KEAP1 and BCL2 in blastocysts treated with Ge-132 during in vitro culture (IVC). (A) Green fluorescence signals reveal KEAP1 protein localization, while red fluorescence signals showed BCL2 protein expression. The cells were counterstained with Hoechst (blue fluorescence) to visualize nuclear morphology. The white arrow indicates the absent of KEAP1 in nuclei (localization in cytosol). Each image shown is representative of at least 14 random fields observed. Indicated scale bars signify 100 µm distance. (B) The intensity of KEAP1 and BCL2 was quantitated by densitometric analysis and is shown in the histogram. The data is represented as the mean ± SD from three independent experiments. Within each group end point (KEAP1 and BCL2), the bars with different letters (a, b) are significantly (P < 0.05) different.