Glycine and Melatonin Improve Preimplantation Development of Porcine Oocytes Vitrified at the Germinal Vesicle Stage

Yu Tang,Yan Wang,Baozeng Xu,Yifeng Yang,Ying Zhang,Lixiang Liu

doi:10.3389/fcell.2022.856486

Abstract

Lipid-rich porcine oocytes are extremely sensitive to cryopreservation compared to other low-lipid oocytes. Vitrification has outperformed slowing freezing in oocyte cryopreservation and is expected to improve further by minimizing cellular osmotic and/or oxidative stresses. In this study, we compared the effects of loading porcine cumulus-oocyte complexes with glycine (an organic osmolyte) or glycine plus melatonin (an endogenous antioxidant) during vitrification, thawing and subsequent maturation to mitigate osmotic injuries or osmotic and oxidative damages on the developmental potential of porcine oocytes. Our data demonstrated that glycine treatment significantly increased the vitrification efficiency of porcine oocytes to levels comparable to those observed with glycine plus melatonin treatment. It was manifested as the thawed oocyte viability, oocyte nuclear maturation, contents of reactive oxygen species, translocation of cortical granules and apoptotic occurrence in mature oocytes, levels of ATP and transcripts of glycolytic genes in cumulus cells (markers of oocyte quality), oocyte fertilization and blastocyst development. However, the latter was more likely than the former to increase ATP contents and normal mitochondrial distribution in mature oocytes. Taken together, our results suggest that mitigating osmotic and oxidative stresses induced by vitrification and thawing can further enhance the developmental competency of vitrified porcine oocytes at the germinal vesicle stage.

Highlights

Cryopreservation is widely used to preserve structurally intact living oocytes of mammals for the widespread and long-term storage of animal genetic resources in very low temperatures, typically in liquid nitrogen at −196°C
Oocyte viability and cumulus expansion index assay after maturation culture demonstrated that 6 mM glycine and 10−9 M melatonin were the best concentration with the least side effects under our experimental conditions and were used in the following experiments
Our current results demonstrate that glycine or glycine plus melatonin supplementation during vitrification, thawing, and oocyte maturation improves the quality of oocyte meiotic maturation and subsequent embryonic development after vitrification of porcine cumulus-oocyte complexes (COCs)

Summary

Introduction

Cryopreservation is widely used to preserve structurally intact living oocytes of mammals for the widespread and long-term storage of animal genetic resources in very low temperatures, typically in liquid nitrogen at −196°C. In comparison with oocytes of mouse, bovine, or sheep, porcine oocytes have the highest lipid content and are most sensitive to chilling and cryopreservation (Loewenstein and Cohen, 1964; Isachenko et al, 1998; McEvoy et al, 2000). Cryopreserved oocytes still yield very low blastocysts compared to Vitrification of Porcine GV Oocytes fresh oocytes (Casillas et al, 2018). This implies that cryopreservation may cause a certain degree of sublethal damage to porcine or other lipid-rich oocytes, further optimization is needed to enhance the cryopreservation efficiency of lipid-rich oocytes

Methods

Results

Conclusion