Wedelolactone facilitates the early development of parthenogenetically activated porcine embryos by reducing oxidative stress and inhibiting autophagy

Abstract

Wedelolactone (WDL) is a coumaryl ether compound extracted from the traditional Chinese medicinal plant, Eclipta prostrata L. It is a natural polyphenol that exhibits a variety of pharmacological activities, such as anti-inflammatory, anti-free radical, and antioxidant activities in the bone, brain, and ovary. However, its effect on embryonic development remains unknown. The present study explored the influence of WDL supplementation of porcine oocytes culture in vitro on embryonic development and the underlying mechanisms and its effect on the levels of Kelch-like ECH-associated protein 1/nuclear factor-erythroid 2-related factor 2/antioxidant response element (Keap1/Nrf2/ARE). The results showed that WDL (2.5 nM) significantly increased the blastocyst formation rate, mitochondrial activity, and proliferation ability while reducing the reactive oxygen species accumulation, apoptosis, and autophagy. These findings suggested that WDL can enhance the growth and development of early porcine embryos to alleviate oxidative stress and autophagy through regulating NRF2 and microtubule-associated protein 1 light chain 3 (MAP1LC3) gene expression levels.

Keywords: Autophagy; Nrf2/ARE; Oxidative stress; Porcine embryo; Wedelolactone.

Figure 1. Effects of different concentrations (0, 0.25, 2.5, or 25 nM) of wedelolactone (WDL) on porcine blastocyst formation rate on day 6 and day 7.

(A) Embryonic development rate from day 6 to day 7 in the control and WDL-treated groups. Scale bars represent 100 μm. (B) Blastocyst formation rates on day 6 and day 7. Significant differences are represented by two asterisks (**) indicating P < 0.01. (C) Total number of cells in blastocysts on day 7 with (n = 90) or without WDL treatment (n = 86). Data are presented as the mean ± standard deviation (SD). Significant differences are represented by two asterisks (**) indicating P < 0.01.

Figure 2. Effects of wedelolactone (WDL) on blastocyst cell proliferation and apoptosis.

(A) Representative 5-ethynyl-2′-deoxyuridine (EdU) staining images of blastocysts. Scale bars represent 200 μm. (B) Representative terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate (dUTP) nick-end labeling (TUNEL) staining images of blastocysts. (C) Relative levels of proliferating cells in embryos treated with (n = 52) or without WDL (n = 41) on day 7. Significant differences are represented by three asterisks (***) indicating P < 0.001. (D) Proportions of apoptotic cells in embryos treated with (n = 102) or without (n = 96) WDL on day 7. Significant differences are represented by three asterisks (***) indicating P < 0.001. (E) Effect of WDL on expression levels of blastocyst multipotency and apoptosis-related genes.

Figure 3. Effects of wedelolactone (WDL) on the levels of reactive oxygen species (ROS) and glutathione (GSH) in porcine embryos.

(A) Representative 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining images of 4-cell stage embryos. Scale bars represent 100 μm. (B) Relative H2DCFDA fluorescence intensity level changes in 4-cell stage embryos in the control (n = 83) and WDL-treated (n = 81) groups. Significant differences are represented by three asterisks (***) indicating P < 0.001. (C) Representative 4-chloromethyl-6, 8-difluoro-7-hydroxycoumarin (CMF2HC) staining images of 4-cell stage embryos. Scale bars represent 100 μm. (D) Relative CMF2HC fluorescence intensity changes of 4-cell stage embryos in control (n = 89) and WDL-treated (n =92) groups. Significant differences are represented by three asterisks (***) indicating P < 0.001. (E) Effect of WDL on expression levels of blastocyst oxidative stress-related genes (three asterisks (***) indicating P < 0.001).

Figure 4. Effect of wedelolactone (WDL) on autophagy expression in porcine blastocysts.

(A) Representative fluorescence images show the presence of the light chain 3 (LC3) protein in porcine blastocysts treated with (n = 46) or without WDL (n = 34). Scale bars represent 200 μm. (B) Fluorescence dots denote the number of LC3 in blastocysts. Significant differences are represented by two asterisks (**) indicating P < 0.01. (C) Effect of WDL on expression levels of blastocyst autophagy-related genes.

Figure 5. Effect of wedelolactone (WDL) on mitochondrial function in 4-cell stage embryos.

(A) Representative fluorescent images of JC-1-stained 4-cell stage embryos in control and WDL-treated groups. Scale bars represent 100 μm. (B) Relative fluorescence levels of JC-1 red/green in 4-cell stage embryos treated with (n = 67) or without (n = 60) WDL. Significant differences are represented by three asterisks (***) indicating P < 0.001.

Figure 6. Effect of wedelolactone (WDL) on the expression levels of the nuclear factor-erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway-related genes in porcine blastocysts.

(A) Representative porcine blastocysts were stained with Nrf2 and counterstained with Hoechst 33342 in control (n = 60) and WDL-treated groups (n = 79). Scale bars represent 200 μm. (B) The experiment was replicated at least three times. Fluorescence intensity of Nrf2 in blastocysts. Significant differences are represented by two asterisks (**) indicating P < 0.01. (C) Nrf2/ARE signaling pathway-related genes in blastocysts.