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

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.

The association of resveratrol and AFPI did not enhance the cryoresistance of ram sperm

“…Moreover, the addition of seminal plasma proteins associated with membrane protectors, such as vitamin E, increases sperm viability after heat shock ( Pérez-Pé et al, 2001 ). In Macaca fascicularis , the addition of 0.1 µg/mL of AFP III in semen extender significantly reduced the number of differential proteins in cryopreserved compared to fresh spermatozoa ( Chen et al, 2021 ). Thus, the greater amount of seminal plasma might have potentiated the cryoprotective effect of AFP I.…”

A brief review of the methodology and cryoprotectants in selected fish and mammalian species

“…It seems that AFPs bind to the lipid bilayer, increasing the phase transition temperature of the membranes and stabilizing them. Based on the data in Table 3, increasing the survival of frozen-thawed semen and embryos using AFPs has been reported in some mammals, including mice (Jo et al, 2011), rabbits (Nishijima et al, 2014), monkeys (Chen et al, 2021), rams (Correia et al, 2021) and goats (Monteiro et al, 2023).…”

Antifreeze Proteins: Novel Applications and Navigation towards Their Clinical Application in Cryobanking

“…AFPs including AFP I, AFP III, DAFP ( Dendroides canadensis antifreeze protein) and AFGPs have been applied in sperm banking research, e.g. sea bream sperm [ 85 , 86 ], buffalo bull ( Bubalus bubalis ) sperm [ 87 , 88 , 89 , 90 ], ram sperm [ 91 ], rabbit sperm [ 71 ], Persian sturgeon ( Acipenser persicus ) sperm [ 92 ], rooster sperm [ 93 ], common carp ( Cyprinus carpio ) sperm [ 94 ], human sperm [ 95 ] and crab-eating macaque ( Macaca fascicularis ) sperm [ 96 ], where later research has provided more insight into the possible mechanism of action of AFP III [ 97 ]. AFP III improved survival by preserving protein expression and modulating the release of cytochrome C and free radicals at the molecular level.…”