A polysaccharide gel made of gellan gum improves oocyte maturation and embryonic development in pigs

Abstract

Gellan gum (GG) is a soft, tractable, and natural polysaccharide substrate used for cell incubation. In this study, we examined the effects of GG on porcine oocyte maturation. Cumulus cells and oocyte complexes (COCs) were collected from slaughterhouse-derived porcine ovaries and cultured on plastic plates containing 0.05% or 0.1% GG gels. The 0.1% GG gel improved the maturation rate and quality of blastocysts, as determined by the total cell number and the rate of abnormally condensed nuclei. GG gels have antioxidant abilities and oocytes cultured on GG gels (0.05% and 0.1%) have reduced reactive oxygen species (ROS) content. Furthermore, GG gels (0.05% and 0.1%) increased F-actin formation, whereas treatment of oocytes with H₂O₂ reduced F-actin levels. GG gels increased the ATP content in oocytes but did not affect the mitochondrial DNA copy number or mitochondrial membrane potential. In addition, the medium cultured on 0.05% GG increased the glucose consumption of COCs. In conclusion, GG gel reduced ROS content, increased energy content, and improved subsequent embryonic development in pigs.

Keywords: Antioxidant; F-actin; Gellan gum; Oocyte maturation.

Fig. 1.

The effect of a gellan gum gel (Gel) on the antioxidant ability and reactive oxygen species content of oocytes. The antioxidant abilities of the phosphate-buffered saline (PBS) and gellan gum (Gel) was measured. Y-axis: µM (A). Data were analyzed using Student’s t-test. Reactive oxygen species (ROS) levels in mature oocytes (B). Values for the control group (Con.) was defined as 1.0. Data were analyzed using the Steel–Dwass test. Representative images of ROS content in oocytes (C). Data are presented as the mean ± standard error of the mean (SEM), * P < 0.05. The number of samples is indicated in each figure.

Fig. 2.

The effect of the GG gel on actin polymerization in oocytes. F-actin (A) and actin cap (B) expression levels in mature oocytes. Data were analyzed using the Tukey–Kramer test. Values for the control group (Con.) was defined as 1.0. Representative images of F-actin (C) and actin cap (D) in oocytes. Arrow, actin cap. Data are presented as the mean ± SEM, * P < 0.05. The number of samples is indicated in each figure.

Fig. 3.

The effect of H₂O₂ on actin polymerization in oocytes. Denuded oocytes were cultured without (0 µM) or with H₂O₂ (100 µM, 200 µM, and 300 µM) for 5 h. F-actin expression in oocytes (A). Data were analyzed using the Tukey–Kramer test. The value for the 0 µM group was defined as 1.0. Representative images of F-actin expression in oocytes (B). Data are presented as the mean ± SEM, * P < 0.05. The number of samples is indicated in each figure.

Fig. 4.

The effect of the GG gel on mitochondrial functions in oocytes. Adenosine triphosphate (ATP) content of oocytes (A). The proportion of orange (active mitochondria)/green (whole mitochondria)-stained cells (B). Representative images of oocytes stained with MitoTracker Orange and Green (C). Mt-cn Oocytes (D). Mitochondrial membrane potential (MMP) and Mt-cn data were analyzed using the Steel–Dwass test. ATP data were analyzed using the Tukey–Kramer test. Data are presented as the mean ± SEM, * P < 0.05. The number of samples is indicated in each figure.

Fig. 5.

Effect of the gellan gum (GG) gel on glucose consumption. Glucose consumption (ratio of the initial concentration to the concentration of the spent culture medium) of COCs in the first (0–24 h: A) and later steps (24–48 h: B). Data were analyzed using the Tukey–Kramer test. Data are presented as the mean ± SEM, * P < 0.05. The number of samples is indicated in each figure. Y-axis: mg/dl.