Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Abstract

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

Keywords: N-acetyl-D-glucosamine; glucuronic acid; hyaluronic acid; oocyte maturation; pig.

Figure 1

Diagram of the measurement of oocyte diameter, and the size of the perivitelline space (PVS) in porcine oocytes after in vitro maturation.

Figure 2

Evaluation of the cumulus expansion in a defined medium or complex medium. (A) Representative images of cumulus–oocyte complexes (COCs) taken at three time points (0, 22, and 44 h) during in vitro maturation. Scale bar, 200 μm. COCs were cultured in the defined medium (A–F), or 10% PFF medium (G–L); COCs were cultured without treatment (A–C, G–I) or cultured for 22 h in an IVM medium containing 0.05 mM GA and 0.05 mM AG (GAAG0-22) (D–F, J–L). (B) The area of each COCs was analyzed by drawing a line on the outer layer of the cumulus cell and measuring its inner area. The experiment was repeated three times with 25 oocytes per group. (^{a, b, c}) Values with different superscripts within the same column are significantly different (p < 0.05).

Figure 3

The mRNA expression levels of cumulus expansion- and antioxidant-related genes. (A) mRNA expression levels of genes related to cumulus cell expansion (CD44, CX43) were evaluated in cumulus cells from each treatment group, and (B) mRNA expression levels of ROS-related genes (GSR, PRDX1, TXN2, and SOD1) were evaluated in oocytes from each treatment group after in vitro maturation. (*) Values with different superscripts within the same column are significantly different (p < 0.05); four replicates.