Elsevier

Cryobiology

Volume 100, June 2021, Pages 158-163
Cryobiology

Peroxiredoxin 6 translocates to the plasma membrane of human sperm under oxidative stress during cryopreservation

https://doi.org/10.1016/j.cryobiol.2021.02.002Get rights and content

Abstract

Peroxiredoxin 6 (PRDX6) is one antioxidant enzyme which could control the levels of reactive oxygen species and to avoid oxidative damage of sperm. In this study, we aimed to investigate the position change of PRDX6 in human sperm under oxidative stress during cryopreservation. Semen samples were obtained from 98 healthy donors and 27 asthenozoospermic donors. The plasma membrane protein and cytoplasmic protein of sperm samples were extracted and analyzed after cryopreservation. Western blot and immunofluorescence were used to measure the expressions of PRDX6. Liquid chromatography mass spectrometric (LC–MS/MS) analysis was performed to confirm the component of sperm membrane complex. Western blot showed that the detection rate of PRDX6 in plasma membranes with low sperm motility (≤20%) was significantly higher than that with high sperm motility (≥40%). Western blot and Immunofluorescence revealed that cryopreservation and thawing induced the position change of the PRDX6 from cytoplasm to sperm membrane. LC–MS/MS analysis showed that PRDX6, ADP/ATP translocase 4 (ANT4) and glyceraldehyde-3-phosphte dehydrogenase (GAPDHS) were present in the components of membrane complex after cryopreservation. The present study indicated that the presence of PRDX6 in sperm plasma membrane was related to sperm motility. GAPDHS and ANT4 may be involved the position change of the PRDX6 from cytoplasm to sperm membrane under oxidative stress during cryopreservation.

Introduction

The cryopreservation of human semen is widely used both in the laboratory and in clinical practice. It is an important routine procedure used for different purposes, including donor insemination and fertility preservation from patients undergoing chemotherapy [3,23,30]. In addition to the deleterious effect of cryopreservation on the motility, fertilization capacity and viability of sperm, it may also cause sperm DNA fragmentation [23,32]. Oxidative stress is the result of imbalance between antioxidants and reactive oxygen species (ROS) in the body, which is considered as a potential mechanism of sperm DNA damage [4]. Therefore, it is necessary to have an antioxidant enzyme system in the sperm to balance the level of ROS to ensure its normal performance.

The peroxiredoxin (PRDX) protein family consists of six anti-oxidant enzymes (PRDX1–6), which are abundant in human semen and can scavenge a wide variety of ROS [5,28]. PRDX6 is a sole 1-Cystein enzyme with phospholipase A2 (PLA2) and glutathione (GSH) peroxidase activities [7]. Previous studies have showed that PRDX6 could protect cells against ROS-induced damage or play a role in signaling by controlling the levels of hydrogen peroxide (H2O2) [26,33]. Moreover, PRDX have been found to be linked to the membrane under oxidative stress conditions and can be used as a candidate biomarker to test oxidative stress levels [2,27,29]. However, the position change of the PRDX6 in human sperm under oxidative stress remains unclear. Thus, the aim of present study was to investigate the position change of in human sperm under oxidative stress during cryopreservation. In present study, we utilized fresh and cryopreserved human sperm to investigate the position change of PRDX6 in human sperm under oxidative stress during cryopreservation.

Section snippets

Subjects and semen sample preparation

This study was conducted in accordance with Declaration of Helsinki and human semen studies [15], and was approved by the ethics committee of National Research Institute for Family Planning (2020006–1). Written informed consent was obtained from each donor and the experimental protocol was established according to the associated national guidelines from the Ministry of Science and Technology of China.

The study included 98 normozoospermic men selected from community volunteers and 27

Effect of sperm motility on the presence of PRDX6 in sperm plasma membrane

In order to verify the relationship between sperm motility and PRDX6, a preliminary analysis was performed. In this study, 98 normozoospermic donors and 27 asthenozoospermic donors were included. Sperm samples were divided into three groups [Group A (sperm motility≥40%, n = 70); Group B (30%≤sperm motility<40%, n = 10); Group C (sperm motility≤20%, n = 27)] according sperm motility. The plasma membrane protein and cytoplasmic protein of all sperm samples were extracted and immunoblotted with

Discussion

It is well known that oxidative stress is an important factor leading to male fertility problems [17,18,22]. Previous reports have suggested that sperm viability was associated with ROS levels in human sperm, and the increase of ROS levels is related to the decrease of sperm motility [1,25]. Oxidative damage could induce the change of sperm membranes, proteins and DNA. Evidences have showed that PRDX6 could protect cells against ROS-induced damage [2,26]. Maria et al. found that PRDX6 is the

Author contributions

X.L: Conception and design, Data collection, Data Formal analysis and interpretation, Drafting article, Administrative support. Y.G: Data collection, Data Formal analysis and interpretation, Providing materials and samples. H.Y: Administrative support. All the authors have read and approved the final manuscript

Declaration of conflicting interests

The authors declare that they have no conflict of interest.

Funding

This work was supported by the Non-profit Central Research Institute Fund of National Research Institute For Family Planning [number 2020GJM03] and CASM Innovation Fund for Medical Sciences (CIFMS) [number 2018-12M-1-004].

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