Ganglioside GD1a promotes oocyte maturation, furthers preimplantation development, and increases blastocyst quality in pigs

Abstract

Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs.

Fig. 1.

Expression patterns of ST3GAL2 in DOs and COCs during in vitro maturation. (A) Protein levels of ST3GAL2 were examined in DOs and COCs during IVM I (22 h) and IVM II (44 h) by western blotting analysis. (B) Quantitative analysis of ST3GAL2 protein level in DOs and COCs during IVM I (22 h) and IVM II (44 h). Relative ST3GAL2 protein levels were normalized to β-actin as a control. Data in the bar graph represent the means ± SEM of three independent experiments. Differences were considered significant at * P < 0.05, ** < 0.01, and *** < 0.001.

Fig. 2.

Effect of GD1a treatment on meiotic maturation of porcine oocytes. Nuclei were classified into GV, GVBD, M I, or M II stages. (A) Diagram of oocyte maturation. (B) Summary of meiotic maturation after IVM II, with or without GD1a treatment. This experiment was replicated at three times. Data are expressed as the means ± SD. Different superscript letters denote significant differences (P < 0.05).

Fig. 3.

Effects of EGF and/or GD1a treatment on meiotic maturation of porcine oocytes in IVM I (22 h), and IVM II (44 h). Diagram of oocyte maturation at IVM I (22 h) (A), and IVM II (44 h) (B). (C) Summary of meiotic maturation after IVM I and IVM II. Nuclei were classified into GV, GVBD, M I, or M II stages. This experiment was replicated at three times. Data are expressed as the means ± SD. Different superscript letters denote significant differences (P < 0.05).

Fig. 4.

Effects of EGF and/or GD1a treatment on oocyte fertilization variables during in vitro maturation. (A) Pronuclear formation, (B) penetrated oocytes, (C) polyspermic oocytes, (D) and summary of fertilization. This experiment was replicated at three times. Data are expressed as the means ± SD. Different superscript letters denote significant differences (P < 0.05).

Fig. 5.

Effects of EGF and/or GD1a treatment on developmental competence of porcine oocytes in vitro. (A) Pattern of cleavage and blastocyst formation after IVF embryos. (B) Summary of developmental competence. This experiment was replicated at three times. Data are expressed as the means ± SD. Different superscript letters denote significant differences (P < 0.05).

Fig. 6.

Apoptosis scanning images of porcine blastocysts derived from EGF and/or GD1a treated oocytes. (A) The chromatin content was determined by DAPI staining (total DNA, blue); fragmented DNA was detected by TUNEL assay (apoptotic nuclei, green). Scale bars = 100 μm. (B) Total cell number and (C) percent of apoptotic cells in porcine blastocyst-stage embryos after EGF and/or GD1a treatment. (D) Summary of developmental competence. This experiment was replicated at three times. Data are the means ± SD. Different superscript letters denote significant differences (P < 0.05).