Improving Sperm Cryopreservation With Type III Antifreeze Protein: Proteomic Profiling of Cynomolgus Macaque (Macaca fascicularis) Sperm.

Bingbing Chen,Briauna Marie Inglis,Yanchao Duan,Angbaji Suo,Hao Ding,Wendell Q Sun,Shuai Qiu,Wei Si,Xi Li,Shanshan Li,Shengnan Wang

doi:10.3389/fphys.2021.719346

Abstract

Antifreeze protein III (AFP III) is used for the cryopreservation of germ cells in various animal species. However, the exact mechanism of its cryoprotection is largely unknown at the molecular level. In this study, we investigated the motility, acrosomal integrity, and mitochondrial membrane potential (MMP), as well as proteomic change, of cynomolgus macaque sperm after cryopreservation. Sperm motility, acrosomal integrity, and MMP were lower after cryopreservation (p < 0.001), but significant differences in sperm motility and MMP were observed between the AFP-treated sperm sample (Cryo+AFP) and the non-treated sample (Cryo–AFP) (p < 0.01). A total of 141 and 32 differentially expressed proteins were, respectively, identified in cynomolgus macaque sperm cryopreserved without and with 0.1 μg/ml AFP III compared with fresh sperm. These proteins were mainly involved in the mitochondrial production of reactive oxygen species (ROS), glutathione (GSH) synthesis, and cell apoptosis. The addition of AFP III in the sperm freezing medium resulted in significant stabilization of cellular molecular functions and/or biological processes in sperm, as illustrated by the extent of proteomic changes after freezing and thawing. According to the proteomic change of differentially expressed proteins, we hypothesized a novel molecular mechanism for cryoprotection that AFP III may reduce the release of cytochrome c and thereby reduce sperm apoptosis by modulating the production of ROS in mitochondria. The molecular mechanism that AFP III acts with sperm proteins for cellular protection against cryoinjuries needs further study.

Highlights

Similarities between non-human primates and humans in physiology, genetics, and behavior make primates one of the widely used animal models in biomedical research
Sperm samples that were frozen with 0.1 μg/ml Antifreeze protein III (AFP III) (Cryo+Antifreeze proteins (AFPs)) showed significantly higher post-thaw motility and membrane potential (MMP) than those samples frozen without AFP III (Cryo–AFP) (p < 0.01)
This study demonstrates that sperm cryopreservation changes the levels of some proteins in both the Cryo–AFP and Cryo+AFP groups, which results in a decrease in the number of differential proteins between the two groups

Summary

Introduction

Similarities between non-human primates and humans in physiology, genetics, and behavior make primates one of the widely used animal models in biomedical research. Primate models play essential roles in human disease research, drug development, and therapeutic strategy validation. The generation of primate models has been greatly accelerated as a result of the newly developed nuclease-based genome editing tools, such as CRISPR-Cas technology, and improvements of Improving Sperm Cryopreservation With AFP III assisted reproduction technologies in primates (David, 2016). The cost, space, and labor required to maintain these models as living animals have created a huge burden to the biomedical community. In combination with the established assisted reproductive technologies, such as artificial insemination, in vitro fertilization, and embryo transfer, in primate, cryopreservation of sperm and embryo provides efficient and cost-effective methods to safeguard primate models

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