Dihydromyricetin supplementation during in vitro culture improves porcine oocyte developmental competence by regulating oxidative stress

Abstract

Dihydromyricetin (DHM), a dihydroflavonoid compound, exhibits a variety of biological activities, including antitumor activity. However, the effects of DHM on mammalian reproductive processes, especially during early embryonic development, remain unclear. In this study, we added DHM to porcine zygotic medium to explore the influence and underlying mechanisms of DHM on the developmental competence of parthenogenetically activated porcine embryos. Supplementation with 5 μM DHM during in vitro culture (IVC) significantly improved blastocyst formation rate and increased the total number of cells in porcine embryos. Further, DHM supplementation also improved glutathione levels and mitochondrial membrane potential; reduced natural reactive oxygen species levels in blastomeres and apoptosis rate; upregulated Nanog, Oct4, SOD1, SOD2, Sirt1, and Bcl2 expression; and downregulated Beclin1, ATG12, and Bax expression. Collectively, DHM supplementation regulated oxidative stress during IVC and could act as a potential antioxidant during in vitro porcine oocytes maturation.

Keywords: Dihydromyricetin; In vitro culture; Oxidative stress; Porcine oocyte.

Fig. 1.

Effects of various DHM concentrations (0 μM, 2.5 μM, 5 μM, and 10 μM) on porcine embryo development. (A) Embryo development on different days. Scale bar = 200 μm. (B) Blastocyst formation rates on different days. Numbers of oocytes (n) used in this experiment were 189, 173, 191, and 175 with 0 μM, 2.5 μM, 5 μM, and 10 μM DHM, respectively. R = 5. (C) On the seventh day, the blastocysts were stained with Hoechst 33342. Scale bar = 100 μm. (D) Total cell number of blastocysts on day 7 in the Con (n = 72) and DHM-treated (n = 80) groups. R = 4. Data are presented as the mean ± SD. Significant differences are represented by *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Fig. 2.

Effects of DHM on proliferation and apoptosis of blastocyst cells. (A) TUNEL staining of blastocysts on day 7. Scale bar = 100 μm. (B) The proportion of apoptotic cells in the Con (n = 31) and DHM-treated (n = 30) groups. R = 3. (C) Representative EdU stained images of blastocysts. (D) Proportion of proliferating cells in embryos treated with DHM (n = 39) and not treated with DHM (n = 45) on day 7. R = 4. Data are presented as the mean ± SD. Significant differences are represented by *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Fig. 3.

Effects of DHM on ROS and GSH levels in 4-cell embryos. (A) Representative ROS staining images of 4-cell-stage embryos. Scale bar = 200 μm. (B) Relative changes in ROS fluorescence intensity levels of 4-cell-stage embryos in the Con (n = 83) and DHM-treated (n = 89) groups. ROS fluorescence intensity in DHM-treated 4-cell stage embryos was significantly decreased to 0.16 ± 0.07-fold compared to that in the Con group. Blue and red dots represent the measurement distributions in the control and DHM groups, respectively. R = 3. (C) Representative GSH-stained images of 4-cell-stage embryos. Scale bar = 200 μm. (D) Relative GSH fluorescence intensity levels of 4-cell-stage embryos in the Con (n = 76) and DHM-treated (n = 85) groups. DHM treatment of 4-cell stage embryos led to a 0.11 ± 0.01-fold increase in GSH fluorescence intensity levels. R = 3. Data are presented as mean ± SD. Significant differences are represented by *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Fig. 4.

Effects of DHM on mitochondrial function in 4-cell stage embryos. (A) Representative JC-1 staining images of 4-cell stage embryos in the con and DHM-treated groups. Scale bar = 100 μm. (B) Relative fluorescence levels of JC-1 red/green in 4-cell stage embryos with (n = 95) and without DHM treatment (n = 98). R = 3. Four-cell stage embryos with DHM treatment led to a 0.42 ± 0.07-fold increase in JC-1 fluorescence intensity levels. Data are presented as the mean ± SD. Significant differences are represented by *(P < 0.05), **(P < 0.01), and ***(P < 0.001).

Fig. 5.

Differential gene expression in blastocysts. Gene expression levels were analyzed in porcine blastocysts with or without DHM treatment on day 7. Significant differences are represented by *(P < 0.05) and **(P < 0.01).