Leptin improves the in vitro development of preimplantation rabbit embryos under oxidative stress of cryopreservation.

Tarek A. Alshaheen,Mohamed H. H. Awaad,Gamal M. K. Mehaisen,Wilfried A. Kues

doi:10.1371/journal.pone.0246307

Tarek A. Alshaheen, Mohamed H. H. Awaad + Show 2 more

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https://doi.org/10.1371/journal.pone.0246307

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Journal: PloS one	Publication Date: Feb 2, 2021
Citations: 4	License type: CC BY 4.0

Affiliation: King Faisal University, Cairo University

Abstract

Vitrification is an economically effective method for embryo cryopreservation in human and livestock animals; however, it carries the risk of damage by the exposure to severe oxidative stress. The present study was conducted to evaluate the effect of leptin at different levels on the in vitro development of fresh and vitrified preimplantation embryos in a rabbit model. Normal embryos at morulae stage were randomly cultured for 2 h with 0, 10, 20 or 100 ng/mL of leptin, then were cultured for further 48 h as freshly or after vitrification. Thereafter, developed blastocysts form the best leptin level in fresh and vitrified embryos along with their controls were allocated to analyze the pro-oxidant (malondialdehyde, MDA; nitric oxide, NO), antioxidant (total antioxidant capacity, TAC; superoxide dismutase, SOD; glutathione peroxidase, GPx), apoptotic (Bcl-2 associated X protein, BAX; heat shock 60kD protein member 1, HSP60; tumor necrosis factor alpha, TNFα) and developmental (sex determining region Y box protein 2, SOX2; Nanog homeobox protein, NANOG; Octamer-binding protein 4, OCT4) biomarkers. Results indicate that expanding and hatching rates of embryos were significantly higher at 20 ng/mL leptin than the other levels, while vitrification had an independent suppression effect on the in vitro development rates. The MDA and NO were significantly higher, while TAC, SOD and GPx were significantly lower in the vitrified than fresh embryos. In contrast, leptin treatment significantly decreased the pro-oxidant biomarkers and increased the antioxidant biomarkers in both fresh and vitrified embryos. Vitrification significantly increased the antiapoptotic biomarkers, and decreased the developmental biomarkers in embryos. In contrast, leptin decreased the BAX and TNFα, increased the HSP60, and moreover, ameliorated the reduction of developmental biomarkers in the vitrified embryos. These results conclude that leptin could be used as antiapoptotic and antioxidant promotor to support the in vitro embryonic development, particularly under oxidative stress emerged from cryopreservation programs.

Highlights

Embryo cryopreservation is widely used as an assisted reproductive technology for the preservation, reconstitution and distribution of valuable and/or endangered livestock populations [1,2]
Recent studies reported that vitrification and oxidative stress negatively affected the embryo expression of several proteins encoded by developmentally-related-genes such as gap junction protein alpha 1 (GJA1), nanog homeobox protein (NANOG) and octamer-binding protein 4 (OCT4) [7,15,16]
We recently found that culture of rabbit embryos with antioxidant materials such as melatonin [39] and retinol [15] can protect embryos from the oxidative stress and support the in vitro development of embryos

Summary

Introduction

Embryo cryopreservation is widely used as an assisted reproductive technology for the preservation, reconstitution and distribution of valuable and/or endangered livestock populations [1,2]. Embryo vitrification techniques have been studied and developed using various cryoprotectant solutions, carriers and devices, and resulting in various in vitro development and in vivo survival rates [5,6]. Previous works has demonstrated that in vitro produced and vitrified embryos could be injured by the exposure to oxidative stress through induction of membrane lipid peroxidation, protein oxidation, cellular anti-ROS-defense suppression, metabolism disruption, mitochondrial dysfunction, cell death and apoptosis [7,10,11,12,13,14]. Recent studies reported that vitrification and oxidative stress negatively affected the embryo expression of several proteins encoded by developmentally-related-genes such as gap junction protein alpha 1 (GJA1), nanog homeobox protein (NANOG) and octamer-binding protein 4 (OCT4) [7,15,16]. Other reports correlated the low developmental competence of embryos to the presence of some pro-oxidant proteins such as malondialdehyde (MDA) and nitric oxide (NO) [7] and some apoptotic proteins like Bcl-2 associated X protein (BAX), heat shock 60kD protein member 1 (HSP60) and tumor necrosis factor alpha (TNFα) [17,18]

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