Proteomic identification of cryostress in epididymal spermatozoa

Abstract

Background: Cryopreservation of epididymal spermatozoa is important in cases in which it is not possible to collect semen using normal methods, as the sudden death of an animal or a catastrophic injury. However, the freezing and thawing processes cause stress to spermatozoa, including cold shock, osmotic damage, and ice crystal formation, thereby reducing sperm quality. We assessed the motility (%), motion kinematics, capacitation status, and viability of spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, we identified proteins associated with cryostress using a proteomic approach and performed western blotting to validate two-dimensional electrophoresis (2-DE) results using two commercial antibodies. Results: Cryopreservation reduced viability (%), motility (%), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and capacitated spermatozoa, whereas straightness (STR) and the acrosome reaction increased after cryopreservation (P < 0.05). Nine proteins were differentially expressed (two proteins decreased and seven increased) (>3 fold, P < 0.05) before and after cryopreservation. The proteins differentially expressed following cryopreservation are putatively related to several signaling pathways, including the ephrinR-actin pathway, the ROS metabolism pathway, actin cytoskeleton assembly, actin cytoskeleton regulation, and the guanylate cyclase pathway.

Comparative sperm protein profiling in bulls differing in fertility and identification of phosphatidylethanolamine‐binding protein 4, a potential fertility marker

“…An ejaculate having less percentage of total motile spermatozoa may fail to reach the site of fertilization due to lack of motility regulating factors secreted in epididymis (Fu-Jun & Xiao-Fang, 2012;Yoon et al, 2016) and proteins such as CatSper, IGF1, and ODF1 (Tamburrino et al, 2014;Cabrillana et al, 2017;Selvaraju et al, 2016b) BSP1 (Somashekar et al, 2015), and TIP39 (Selvaraju et al, 2016a). An ejaculate having less percentage of total motile spermatozoa may fail to reach the site of fertilization due to lack of motility regulating factors secreted in epididymis (Fu-Jun & Xiao-Fang, 2012;Yoon et al, 2016) and proteins such as CatSper, IGF1, and ODF1 (Tamburrino et al, 2014;Cabrillana et al, 2017;Selvaraju et al, 2016b) BSP1 (Somashekar et al, 2015), and TIP39 (Selvaraju et al, 2016a).…”

“…In the present study, total sperm motility, percentage of type-A sperm, sperm acrosome reaction, sperm fructose uptake, and sperm intracellular calcium signaling were significantly higher in fertile group than the low fertile and infertile groups. An ejaculate having less percentage of total motile spermatozoa may fail to reach the site of fertilization due to lack of motility regulating factors secreted in epididymis (Fu-Jun & Xiao-Fang, 2012;Yoon et al, 2016) and proteins such as CatSper, IGF1, and ODF1 (Tamburrino et al, 2014;Cabrillana et al, 2017;Selvaraju et al, 2016b) BSP1 (Somashekar et al, 2015), and TIP39 (Selvaraju et al, 2016a). The low percentages of acrosomal reacted spermatozoa of low and infertile groups might suggest the inability of spermatozoa to undergo acrosome exocytosis and lack of calcium-mediated signaling cascades further affects penetration of zona pellucida (Tranter et al, 2000).…”

“…Although the presence of spermatozoa-specific proteins is reported, the majority of the spermatozoal proteins involved in successful fertilization and embryonic development (McReynolds et al, 2014) are acquired either from accessory sex glands or from epididymis during the final stages of spermatozoa maturation and ejaculation (Amaral et al, 2014;Jodar et al, 2016;Yoon et al, 2016). The presence or absence (Soggiu et al, 2013) and abundance (Somashekar et al, 2015) of particular proteins might affect spermatozoa fertilizing ability, but the role of a single protein in regulating fertilization process needs to be firmly established.…”

Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality

“…These findings, together with the reduced antioxidant activity of SOD and reduced glutathione (GSH) observed in bull and ram sperm after cryopreservation, could explain in part the increased susceptibility of frozen-thawed sperm to suffer lipid peroxidation and oxidative damage [45,46]. Conversely, other studies showed that antioxidant enzymes such as SOD2 and PRDX5 increased after cryopreservation in bull sperm [44,47,48]. Whether this increment is the result of a protein reorganization like in ram sperm, or, on the contrary, if it really reflects a higher activity of these enzymes to protect sperm against oxidative attack, needs to be further verified.…”

“…In ruminants, two main metabolic pathways, oxidative phosphorylation and glycolysis, produce the energy required to maintain sperm motility in the form of ATP [55]. Comparative proteomics studies between fresh and cryopreserved sperm revealed that freezing-thawing procedures alter the abundance of several enzymes implicated in oxidative phosphorylation and glycolysis in ram, bull and gazelle sperm [38,43,44,47]. Among them, different ATP synthases, COX5B, AK1, NDUFV2, ODPB2, ACO2 and NDPK7 were some of those proteins related to oxidative phosphorylation, while different hexokinases, GPI, ALDOA, GAPDH5, PGK2, PGAM2, PKM2 and TPI were some of those proteins related to glycolysis.…”