Antioxidant Capacity of Melatonin on Preimplantation Development of Fresh and Vitrified Rabbit Embryos: Morphological and Molecular Aspects

Abstract

Embryo cryopreservation remains an important technique to enhance the reconstitution and distribution of animal populations with high genetic merit. One of the major detrimental factors to this technique is the damage caused by oxidative stress. Melatonin is widely known as an antioxidant with multi-faceted ways to counteract the oxidative stress. In this paper, we investigated the role of melatonin in protecting rabbit embryos during preimplantation development from the potential harmful effects of oxidative stress induced by in vitro culture or vitrification. Rabbit embryos at morula stages were cultured for 2 hr with 0 or 10−3 M melatonin (C or M groups). Embryos of each group were either transferred to fresh culture media (CF and MF groups) or vitrified/devitrified (CV and MV groups), then cultured in vitro for 48 hr until the blastocyst stage. The culture media were used to measure the activity of antioxidant enzymes: glutathione-s-transferase (GST) and superoxide dismutase (SOD), as well as the levels of two oxidative substrates: lipid peroxidation (LPO) and nitric oxide (NO). The blastocysts from each group were used to measure the expression of developmental-related genes (GJA1, POU5F1 and Nanog) and oxidative-stress-response-related genes (NFE2L2, SOD1 and GPX1). The data showed that melatonin promoted significantly (P<0.05) the blastocyst rate by 17% and 12% in MF and MV groups compared to their controls (CF and CV groups). The GST and SOD activity significantly increased by the treatment of melatonin in fresh or vitrified embryos, while the levels of LPO and NO decreased (P<0.05). Additionally, melatonin considerably stimulated the relative expression of GJA1, NFE2L2 and SOD1 genes in MF and MV embryos compared to CF group. Furthermore, melatonin significantly ameliorated the reduction of POU5F1 and GPX1 expression induced by vitrification. The results obtained from the current investigation provide new and clear molecular aspects regarding the mechanisms by which melatonin promotes development of both fresh and vitrified rabbit embryos.

Effect of blastocoel fluid reduction before vitrification on gene expression in mouse blastocysts

“…In contrast, Castillo‐Martín et al () reported a decrease in POU5F1 expression in in vitro‐produced porcine blastocysts following vitrification. A similar level of Nanog accompanied by a decrease in Pou5f1 expression was also observed for rabbits by Mehaisen et al when comparing vitrified morulas with fresh ones at 48 hr post‐warming (Mehaisen et al, ). This discrepancy between our data and those of Castillo‐Martin et al and Mehaisen et al could be linked to species differences as well as the in vivo versus in vitro source of embryos.…”

Antioxidants and glycine can improve the developmental competence of vitrified/warmed ovine immature oocytes

“…Oxidative damage during cryopreservation is a major concern (Tatone, Emidio, Vento, Ciriminna, & Artini, ). The overproduction of ROS in the vitrification process is detrimental for the oocyte and embryo due to the various types of cell injuries, such as membrane lipid peroxidation, impaired intracellular milieu, disturbed metabolism, amino acid and nucleic acid oxidation, mitochondrial dysfunction, the altered patterns of gene expression and apoptosis (Mehaisen et al, ; Succu et al, ). It has also been shown that vitrification by increasing the ROS activity and H 2 O 2 concentration and decreasing the glutathione content can impair the viability and developmental competence of vitrified/warmed oocytes (Gupta, Uhm, & Lee, ; Somfai et al, ).…”

Melatonin Scavenger Properties against Oxidative and Nitrosative Stress: Impact on <em>In Vitro</em> Embryo Production in Mammals

“…[87] Rabbit Morula 10 -3 M prior in vitro culture, prior vitrification Promotes the blastocyst rate, it increases SOD activity and decreases LPO and NO levels. [88] Mouse Preantral follicles 10 pM after vitrification, during culture Increases diameter of follicles and their survival. [89] Bovine Embryos produced by SCNT 10 -11 to 10 -2 M during in vitro culture It increases the total cell number, ICM and the development of bovine SCNT embryos.…”

“…Melatonin added to culture medium increases the cleavage and blastocyst rates [88,124], increases hatching rate [103], increases the total cell number (TCN) [103] and improves trophectoderm (TE) and inner cell mass (ICM) ratio in vitrified embryos [124]. Also, melatonin reduces the apoptotic index [103,124], promotes the activation of antioxidant enzymes like GST and SOD [88], decreases the level of oxidative substrates [88] and ameliorates the reduction of expression of important genes related in early embryo development, like NANOG and POU5F1 [88].…”