Sublethal sperm freezing damage: Manifestations and solutions

doi:10.1016/j.theriogenology.2018.06.006

Theriogenology

Volume 118, 15 September 2018, Pages 172-181

https://doi.org/10.1016/j.theriogenology.2018.06.006 Get rights and content

Highlights

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Cryopreservation of spermatozoa results in lethal and sublethal damage.
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Sublethal damage includes alterations to sperm proteins, lipids and sugars.
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These changes may underlie significant issues with in vivo fertility of cryopreserved semen.
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Seminal plasma and isolated proteins may be useful in preventing freezing damage.

Abstract

Semen cryopreservation is an important tool for artificial breeding, species conservation and human reproductive medicine. However, sublethal freezing damage remains an important limitation of the cryopreservation process, inevitably leading to reduced fertility in vivo. This review explores several facets of sublethal freezing damage, touching on cryocapacitation, the generation of reactive oxygen species and alterations to sperm proteins, lipids and sugars. The effects of sublethal freezing damage on sperm performance in vivo are also discussed, examining fertile lifespan and interaction with female reproductive tract immune cells, mucus and oviductal cells. Finally, the cryoprotective potential of whole seminal plasma and individual proteins are explored.

Introduction

The ability to cryopreserve the male gamete has major benefits for artificial reproduction, making international semen transport, optimally timed inseminations and indefinite storage of genetic material entirely achievable. Yet these benefits come at a cost; the process of cryopreservation is a challenging treatment for spermatozoa, with risk of injury due to high solute concentrations and osmotic damage if freezing and thawing curves are not optimal [1,2]. Even with improvements in the cryopreservation process over several decades, reports of ultrastructural damage and decreased viability and motility following freezing are ubiquitous [3]. This loss of fundamental sperm viability due to lethal damage is certainly a setback, however it is the sublethal freezing damage which spermatozoa experience that underlies the significant issues with fertility compared to fresh semen [4,5]. For the purposes of this review, sublethal freezing damage is defined as any non-fatal biochemical or physiological alteration induced during any stage of cryopreservation. While the physical mechanisms of freezing damage have been thoroughly researched [1,6], its many manifestations are still coming to light. Cryopreservation causes changes to membrane phospholipids, proteins and sugars, possibly due to a process termed ‘cryocapacitation’. Many of these changes mirror hallmarks of the normal capacitation process, but the pathways and signalling cascades involved may reflect cellular damage rather than normal physiological progression, likely tied to increases in intracellular ROS [7].

While there has been a collective push to improve cryopreservation protocols [8], solutions to curb freezing damage remain elusive. Sublethal freezing damage has gained a considerable amount of attention in the previous few decades and as such, not all aspects of this phenomenon can be covered here in depth. Instead, this review will touch on recent findings of select biochemical and physiological changes induced by cryopreservation, particularly focusing on proteomic alterations. In addition, the consequences of these changes in vivo will be explored in the context of emerging theories. Finally, the use of seminal plasma and isolated proteins as a potential mechanism for improving outcomes of sperm cryopreservation is discussed.

Section snippets

Proteomic alterations to cryopreserved spermatozoa

The sperm membrane is clearly disturbed by freezing, and as a result, proteins which are either intrinsic to or associated with the membrane undergo significant changes in their distribution and abundance, and in some cases, their post-translational modifications. Changes to protein distribution following freezing have been characterised using immunofluorescence, typically profiling the movement of a single protein [9,10]. This redistribution appears unique to each protein, as SP22 staining was

Mechanisms of fertility failure in cryopreserved spermatozoa

The reasons why frozen thawed spermatozoa often perform poorly when inseminated may appear obvious on the surface; cryopreservation almost universally results in significant losses of motility and viability. Yet these obvious changes are not enough to explain fertility losses. While post thaw motility has certainly been correlated to in vivo fertility [95,96], it does not completely account for it. Further, frozen thawed spermatozoa require not only more total, but also more motile spermatozoa

Using proteins to prevent and reverse cryopreservation damage

A large body of evidence has accumulated regarding the effects of seminal plasma supplementation on post thaw function in a range of species, most particularly in sheep (Table 2). The majority of studies report significant benefits of seminal plasma addition on post thaw motility, viability and acrosome integrity [[142], [143], [144], [145], [146], [147], [148], [149], [150], [151], [152], [153]] [ [154,155] (cold shock)]. In addition, seminal plasma has been shown to prevent and revert

Conclusion

While some spermatozoa in an inseminate of frozen thawed semen are clearly capable of achieving fertilisation, attempts to improve the in vivo fertility of cryopreserved spermatozoa are well warranted. From the angle of cryopreservation, this task largely centres around improving post thaw outcomes by minimising the lethal and sublethal damage discussed. In addition, any factors which could improve the motility, mucus penetration or OEC binding, or limit freezing induced capacitation, have real

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This study aimed to evaluate the effect of phospholipase C (PLC) on the capacitation of cryopreserved ovine semen. Sixteen semen samples were cryopreserved with diluent added by 0, 10, or 20 μM of U73122, a PLC inhibitor. The sperm kinetics of the thawed samples were evaluated using the “Computer-assisted Sperm Analysis” system, and the integrity of the plasma and mitochondrial membranes was evaluated using fluorescent probes. Additionally, sperm capacitation and the acrosome reaction with chlortetracycline hydrochloride were evaluated before and after capacitation induction. The results were analysed using analysis of variance and Tukey's test with a 95% probability. Concentrations of 10 or 20 μM of U73122 did not affect the kinetics or number of sperm with intact plasma and mitochondrial membranes. However, after thawing, 10 and 20 μM of the inhibitor reduced the percentage of capacitated and acrosome-reacted sperm. After induction of capacitation, there was a reduction in the number of non-capacitated sperm in all treatment groups, suggesting a reversible effect of U73122. In conclusion, U73122 at concentrations of 10 or 20 μM prevents premature capacitation and acrosome reaction induced by the freezing procedure, without affecting the kinetics and integrity of the sperm membranes.
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Cryopreservation may reduce sperm fertility due to cryodamage including physical-chemical and oxidative stress damages. As a powerful antioxidant, melatonin has been reported to improve cryoprotective effect of sperm. However, the molecular mechanism of melatonin on cryopreserved ram sperm hasn't been fully understand. Give this, this study aimed to investigate the postthaw motility parameters, antioxidative enzyme activities and lipid peroxidation, as well as proteomic, metabolomic changes of Huang-huai ram spermatozoa with freezing medium supplemented with melatonin. Melatonin was firstly replenished to the medium to yield five different final concentrations: 0.1, 0.5, 1.0, 1.5, and 2.0 mM. A control (NC) group without melatonin replenishment was included. Protective effects of melatonin as evidenced by postthaw motility, activities of T-AOC, T-SOD, GSH-Px, CAT, contents of MDA, 4-HNE, as well as acrosome integrity, plasma membrane integrity, with 0.5 mM being the most effective concentration (MC group). Furthermore, 29 differentially abundant proteins involving in sperm functions were screened among Fresh, NC and MC groups of samples (n = 5) based on the 4D-LFQ, with 7 of them upregulated in Fresh and MC groups. 26 differentially abundant metabolites were obtained involving in sperm metabolism among the three groups of samples (n = 8) based on the UHPLC-QE-MS, with 18 of them upregulated in Fresh and MC groups. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the abundance changes of vital proteins and metabolites related to sperm function. Particularly, several proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine may be potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa. In conclusion, a dose-dependent replenishment of melatonin to freezing medium protected ram spermatozoa during cryopreservation, which can improve motility, antioxidant enzyme activities, reduce levels of lipid peroxidation products, modify the proteomic and metabolomic profiling of cryopreserved ram spermatozoa through reduction of oxidative stress, maintenance of OXPHOS and microtubule structure.
Melatonin, a powerful antioxidant protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner, with 0.5 mM being the most effective concentration. Furthermore, sequencing results based on the 4D-LFQ combined with the UHPLC-QE-MS indicated that melatonin modifies proteomic and metabolomic profiling of ram sperm during cryopreservation. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the expression changes of vital proteins and metabolites related to sperm metabolism and function. Particularly, several potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa were acquired, proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine.
Study on comparative analysis of differential metabolites in Guanzhong dairy goat semen before and after freezing
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In order to explore the differential metabolites between fresh and frozen-thawed semen of Guanzhong dairy goats, semen samples were collected by artificial vagina method, and divided into fresh and frozen-thawed semen groups, with six replicates in each group. Liquid Chromatography-mass spectrometry (LC-MS) technology was used to detect semen metabolites in both groups. The metabolites were analyzed and identified by the combination of multidimensional statistical analysis, namely principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA) and univariate statistical analysis, Differential metabolites were identified according to One-step Solution for Identification of Small Molecules in Metabolomics Studies (OSI/SMMS) combined with Human Metabolome Database (HMDB), Lipidmaps and Metlin and the metabolic pathways of different metabolites were enriched and analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The results showed that a total of 53 differential metabolites were detected in fresh and frozen-thawed semen groups, of which 10 metabolites were significantly up-regulated (P < 0.05) and 43 were significantly down-regulated (P < 0.05). Most of the metabolites belonged to lipids and lipid-like molecules, organic acids and their derivatives, organic oxygen compounds, etc. According to the functional enrichment analysis of the top twenty differential metabolites in the KEGG database, significant changes occurred in linoleic acid metabolism pathway out of total eleven pathways observed. These differential metabolites can be used as metabolic markers of sperm cryo-injury in dairy goats.
Proteomic analysis of rabbit fresh and cryopreserved semen provides an important insight into molecular mechanisms of cryoinjuries to spermatozoa
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Cryoinjury and protein changes are a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, viability and fertilizing ability. However, potential proteomic changes in rabbit semen throughout the cryopreservation process have never been previously investigated. The aim of the present study was to compare the whole proteome of fresh and cryopreserved rabbit semen (spermatozoa and extracellular fluid), to examine the effects of freeze-thawing on proteins changes in semen. Comparative analysis and identification of proteins was carried out using 2-dimensional difference in-gel electrophoresis coupled with a matrix-assisted laser desorption/ionization mass spectrometry. Proteomic raw data are available via ProteomeXchange with identifier PXD034832 for spermatozoa and PXD034853 for extracellular fluid. Respectively, 107 and 28 proteins differed in abundance in spermatozoa and extracellular fluid between fresh and frozen groups. Most of these proteins were involved in pathways related to energy metabolism and protein quality control under stress conditions, reproductive processes and mechanisms of cell death/survival regulation, resulting in a significant decrease of motility and viability of post-thawing rabbit sperm and its potential fertilizing ability. These results broaden the understanding of the effects of cryopreservation on rabbit semen and represent a new starting point for the development of improved freezing procedures.
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Based on cytometric analysis, cryopreservation significantly increased the amounts of N-acetyllactosamine, N-acetylglucosamine, and galactose on the sperm surface of viable rooster and ram [18,19]. In addition, the glycocalyx of sperm was changed by excessive ROS produced during cryopreservation [8,20]. However, to date, there have been no reports on the effect of semen freezing and thawing on large-scale spermatozoa glycoproteome, especially for bovine spermatozoa.
Cryopreservation of bovine semen plays a vital role in accelerating genetic improvement and elite breeding, but it has a detrimental effect on sperm quality, resulting in the decline of the reproductive efficiency. The glycosylation modification of protein has irreplaceable roles in spermatozoa. Herein, the effect of cryopreservation on glycoproteins of bovine spermatozoa has been studied for the first time using a tandem mass tag (TMT)-labeled quantitative glycoproteome. A total of 2598 proteins and 492 glycoproteins were identified, including 83 different expression proteins (DEPs) and 44 different expression glycosylated proteins (DEGPs) between fresh and frozen spermatozoa. Thirty-three DEPs are glycoproteins, which demonstrates that glycoproteins of bovine sperm were seriously affected by cryopreservation. Moreover, the effects include glycoprotein expression, glycosylation modification, and substructure localization for proteins such as glycoproteins TEX101, ACRBP, and IZOMU4. The biologic functions of the 115 changed proteins are mainly involved in sperm capacitation, migration in female genitalia, and sperm–egg interaction. Mostly key regulators were identified to be glycoproteins, which confirms that glycosylated proteins played important roles in bovine sperm. This comprehensive study of sperm glycoproteins helps to unravel the cryoinjury mechanisms, thus implying that glycoprotein protection should be an effective way to improve the quality of frozen sperm.
Bimodal interplay of reactive oxygen and nitrogen species in physiology and pathophysiology of bovine sperm function
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In addition, cryopreservation induces changes in the metabolome profile of bovine semen both at plasmatic and cellular compartments [14]. The stressors primarily involved are, sudden osmotic and temperature changes [15], deleterious effects of media components and cryoprotectants, generation of free radicals and depletion of antioxidants [16]. Elevated intracellular calcium in cryopreserved viable human and bull sperm reflects impaired mitochondrial membrane fluidity and subsequently increases mitochondrial membrane potential to induce ROS release [17]; therefore, cryopreservation serves to be a common root cause of oxidative stress.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the mediators of redox activity and are known to perform concentration-specific bimodal roles. At lower concentrations, serves as a molecular messenger and signaling molecule while at higher concentrations induces stress which in turn alters the sperm's functional characteristics. Production of ROS and RNS cannot be prevented entirely and should not be followed as a pragmatic approach as they are involved in numerous sperm physiological functions. When the antioxidants defense armory is meager, excess generation of these species cross the physiological limits and inactivates essential metabolic enzymes and disrupts signal transduction altering normal sperm functions. As per the available literature, oxidants mostly arise as a result of pathological conditions or cryopreservation-induced injury. Dead and debilitated or abnormal spermatozoa and associated leukocytes release free radicals in an excess amount which elicits oxidative and nitrosative stressors that are potentially toxic to cryosurviving sperm. ROS plays a double edge sword effect on sperm function, as regulators of physiological mechanisms at low levels and as toxicants when produced at high concentrations. Recently nitric oxide (NO^.) has emerged as a potential regulator of sperm physiology, in addition, found to mediate homeostasis of the seminal plasma microenvironment when semen samples are incubated with optimal concentrations of NO^. compounds. The NO^. compounds can provide some resistance to future stresses which are not usually harnessed by using the defensive strategy of supplementing antioxidants. Therefore, through the optimized addition of NO^. donor and inhibitor in extender, the free radical-induced damage can be avoided without inhibiting their essential physiological effects on fertilization and subsequent embryo development. This article is intended to describe the role of reactive oxidants in the physiology and pathophysiology of spermatozoa and their relationship with various seminal attributes.

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