Abstract
Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation.
Highlights
Cryopreservation is a long-term storage technique for preserving cells, embryos, or tissues without damage induced by chemical reactivity or time [1, 2]
Motility and VCL were significantly decreased after cryoprotective agent (CPA) addition (Figs 1 and 2, p < 0.05); STR and beat-cross frequency (BCF) were significantly increased (Fig 2, p < 0.05)
The densities of superoxide dismutase 2 (SOD2) and NDUFV2 were highly increased after CPA addition (Fig 5, p < 0.05), and these results show a similar pattern to the 2-DE results
Summary
Cryopreservation is a long-term storage technique for preserving cells, embryos, or tissues without damage induced by chemical reactivity or time [1, 2]. It is a valuable technique for storing cells with a limited life span and reducing the risk of microbial contamination, cross contamination with other cell lines, genetic drift, and morphological changes [3, 4]. In several cases such as valuable deceased males, unexpected death, and catastrophic injury, cryopreservation of epididymal sperm play an important role to reserve genetic information [7, 8].
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