The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.     

Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro fertilizing ability of frozen-thawed bull spermatozoa

In this study, we evaluated the effects of glutathione (l-gamma-glutamyl-l-cysteinylglycine; GSH) supplementation of the thawing extender on bull semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To address these questions fully, we used a set of functional sperm tests. These included tests of sperm motility assayed by computer-assisted semen analysis, membrane lipid packing disorder, spontaneous acrosome reaction, free radical production [reactive oxygen species (ROS) generation], sperm chromatin condensation, DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling and acridine orange staining measured by flow cytometry. Finally, the in vitro penetrability of in vitro matured oocytes and the in vitro production of embryos were evaluated. The main findings emerging from this study were that addition of GSH to the thawing medium resulted in: (i) a higher number of non-capacitated viable spermatozoa; (ii) a reduction in ROS generation; (iii) lower chromatin condensation; (iv) lower DNA fragmentation; (v) higher oocyte penetration rate in vitro and (vi) higher in vitro embryo production compared with control group. Nevertheless, GSH had no significant effect on motion parameters or the occurrence of the spontaneous acrosome reaction. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen bull spermatozoa.     

Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase

In the present study the potential benefit of reactive oxygen species (ROS)-scavenging enzymes superoxide dismutase (SOD) and catalase (CAT) when cryopreserving boar spermatozoa was evaluated. Pooled ejaculate sperm-rich fractions collected from 3 fertile boars were frozen in a split design, after being extended in a conventional freezing extender (control) or the same extender supplemented with SOD (150 or 300 IU/mL, experiment 1), CAT (200 or 400 IU/mL, experiment 2), or SOD + CAT in combination (150 + 200 or 300 + 400 IU/mL, experiment 3). Irrespective of the concentration used, SOD and CAT, alone or in combination, significantly improved postthaw sperm survival, in terms of total sperm motility (assessed with CASA) and viability (assessed with a triple stain; propidium iodide/R123/fluorescein isothiocyanate-labeled peanut agglutinin). Moreover, CAT alone, at a concentration of 400 IU/mL, or in combination with SOD, at concentrations of 200 and 400 UI/mL, improved the ability of frozen-thawed spermatozoa to produce embryos in vitro (zygote cleavage and blastocyst formation as end points). Additional data of ROS generation (luminol- and lucigenin-dependent chemiluminescence) and membrane lipid peroxidation (malondialdehyde [MDA] production) indicated that SOD and CAT reduced postthaw ROS generation by boar spermatozoa, without any influence on MDA production.     

Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function

In this study, we evaluated the effects of glutathione (L-gamma-glutamyl-L-cysteinylglycine; GSH) supplementation of the freezing extender on semen parameters during the cooling (2 hours at 5 degrees C) and freezing phases of the cryopreservation process to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we incorporated a new set of functional sperm tests. These included tests of mitochondrial function, inducibility of the acrosome reaction, in vitro penetration (IVP) of oocytes, changes in sulfhydryl group content in membrane proteins, and capacitation status. The main findings emerging from this study were that the addition of GSH to the freezing media resulted in 1) an improvement in percent motility (%MOT) and motion parameters of thawed spermatozoa, as measured by both microscopic analysis and computer-assisted semen analysis (CASA); 2) a higher number of total viable spermatozoa; 3) a higher number of noncapacitated viable spermatozoa; and 4) a decrease in the number of spermatozoa with changes in the sulfhydryl groups in membrane proteins. This protective effect on sperm function was more pronounced with 1 mM of GSH than with 5 mM of GSH.     

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 °C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 μ m Fe²⁺/1 m m ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA®) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 ± 2.9%) and OXI (11.6 ± 7.6%) ( p  < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI ( p  < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 ± 0.8% OXI vs. 17.4 ± 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa.     

The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas

This experiment documented the incidence and variability of sperm characteristics found in freshly collected and ex vivo-manipulated semen samples from a population of disease-free captive koalas with a special emphasis on the dynamic aspects of DNA fragmentation. These changes were analyzed in light of the putative negative effect of iatrogenic damage after chilled storage and cryopreservation with respect to different semen extender compositions to maximize sperm longevity. Sperm DNA fragmentation (SDF) dynamics (SDF assessment after a varying period of time) was investigated with the sperm chromatin dispersion assay after either dilution in tris-citrate media and chilled preservation at 4°C for upward of 16 days or cryopreservation in either glycerol or dimethylacetamide (DMA) tris-citrate-based cryoprotectant media; corresponding data on progressive sperm motility, plasma membrane integrity, and the proportion of koala sperm with relaxed chromatin were also recorded. SDF analysis of the captive koala population revealed a low mean (±SEM) basal level of only 6.7% ± 0.9%. The percentage of progressive sperm motility, percentage of intact sperm plasma membranes, and the percentage of relaxed chromatin did not correlate significantly with that of basal SDF. Moreover, despite the absence of cysteine residues in marsupial protamines, koala spermatozoa showed remarkable stability in terms of their DNA integrity after the incubation of either fresh, chilled, or frozen-thawed semen samples; observations of progressive motility (P < .05) and plasma membrane integrity (P < .05) revealed that chilled koala spermatozoa declined after 4 days, whereas the incidence of relaxed chromatin increased after 8 days. Although koala SDF increased significantly (P < .05) with the period of chilled storage, these values remained less than 16% after 16 days storage and subsequent incubation at 35°C for a further 48 hours. Survivorship of prefreeze sperm DNA damage was not different when compared with sperm frozen in DMA or between sperm frozen in DMA or glycerol; however, spermatozoa frozen in glycerol showed a higher (P = .042) rate of DNA fragmentation than prefreeze spermatozoa. This result differed from that of observations of progressive motility, plasma membrane integrity, and relaxed chromatin, which were all adversely affected (P < .05) after cryopreservation in either glycerol or DMA; however, the postthaw characteristics of sperm cryopreserved in either glycerol or DMA were not different. After thawing, koala sperm chromatin tended to decondense; however, the incidence of sperm DNA fragmentation was not correlated with the incidence of sperm chromatin relaxation after glycerol (R = .2) or DMA (R = -.04) cryopreservation.     

Catalase improves motility,vitality and DNA integrity of cryopreserved human spermatozoa

Cryopreservation of human spermatozoa offers a pre‐therapeutic possibility of preserving progenity in patients with testicular tumours. We aimed to investigate effects of cryopreservation and addition of catalase on sperm motility, vitality and DNA integrity in fresh and swim‐up spermatozoa. Semen samples were collected from 50 fertile men. Each sample was divided into two parts. First part was subdivided into two equal aliquots: both cryopreserved with and without catalase. The second part was subdivided into two equal aliquots: both processed by swim up and then cryopreserved with or without catalase. Semen analyses, sperm vitality and sperm DNA integrity were performed. Sperm concentration showed significant decrease while percentage of progressive motility, sperm vitality and % of DNA damage showed significant increase in processed and cryopreserved processed samples when compared with fresh and cryopreserved fresh samples. There was no significant difference in sperm concentration while there was significant increase in % of progressive motility and sperm vitality and % of DNA damage showed significant decrease in samples with catalase when compared with samples without catalase (either fresh or processed). Catalase supplementation (fresh and processed) during cryopreservation results in better post‐thawing percentage of progressive motility and percentage of sperm vitality and improved DNA integrity.     

Protective effect of fallopian tubal fluid against activated leucocyte-induced sperm DNA fragmentation: preliminary results

The integrity of the paternal genome is of paramount importance in the initiation and maintenance of a viable pregnancy. Oxygen radicals (ROS) have been identified as one of the main factors responsible for the induction of sperm DNA damage. Spermatozoa are mainly protected against ROS-induced damage by seminal plasma. However, this protective effect disappears once spermatozoa enter the female genital tract. The fallopian tube mucosa may play a protective role against ROS-induced sperm damage. The main objective of this study was to determine whether human tubal explants and tubal fluid exert a protective effect on ROS-induced sperm DNA damage. Spermatozoa were exposed to tubal explants and/or tubal fluid in the presence of phorbol myristate acetate (PMA)-activated polymorphonuclear leucocytes or control medium and sperm DNA fragmentation was measured using the TdT-mediated dUTP-biotin nick end labelling (TUNEL) test. Exposure of human spermatozoa to PMA-activated leucocytes resulted in a 2-fold increase in sperm DNA fragmentation. Co-incubation of spermatozoa with tubal explants did not reduce this damage. However, pre-incubation of spermatozoa with tubal fluid resulted in a statistically significant reduction in sperm DNA fragmentation levels, comparable to those observed in control. In conclusion, tubal fluid appears to protect against activated leucocyte-induced sperm DNA fragmentation, thus preserving the integrity of the paternal genome.     

异黄酮辅剂对解冻精子特征的影响

本研究评价了使用异黄酮作为辅剂的解冻剂对解冻精子精液参数的影响。我们分析了辅剂在解冻过程中对精子活力、精子获能能力（膜脂功能紊乱）、活性氧生成、精子核浓缩及DNA损害的影响。根据初步的数据,辅剂可能会改善解冻过程,减少细胞损伤。我们已经证实异黄酮在精子解冻过程中有抗氧化作用、能够减少活性氧的生成,有利于精子活力轻微提高,降低膜脂功能紊乱和由冷冻而引起的DNA破坏。结果显示异黄酮作为辅剂有助于提高精子功能,这对于辅助生育中使用解冻精子很有意义。我们的发现有必要进一步研究来确证并评价临床应用的可能性。     

N‐acetyl‐L‐cysteine pre‐treatment protects cryopreserved bovine spermatozoa from reactive oxygen species without compromising the in vitro developmental potential of intracytoplasmic sperm injection embryos

Excess of reactive oxygen species (ROS) on in vitro embryo production systems negatively affects the quality and developmental potential of embryos, as result of a decreased sperm quality and increased DNA fragmentation. This issue is of major importance in assisted fertilisation procedures such as intracytoplasmic sperm injection (ICSI), because this technique does not allow the natural selection of competent spermatozoa, and therefore, DNA‐damaged spermatozoa might be used to fertilise an egg. The aim of this study was to investigate a new strategy to prevent the potential deleterious effect of ROS on cryopreserved bovine spermatozoa. We evaluated the effect of a sperm pre‐treatment with different concentrations of N‐acetyl‐L‐cysteine (NAC) on ROS production, viability and DNA fragmentation and assessed the effect of this treatment on the in vitro developmental potential and quality of embryos generated by ICSI. The results show a strong scavenging effect of 1 and 10 mm NAC after exposure of spermatozoa to a ROS inducer, without compromising the viability and DNA integrity. Importantly, in vitro developmental potential and quality of embryos generated by ICSI with spermatozoa treated with NAC were not affected, confirming the feasibility of using this treatment before an ICSI cycle.     

Cryoprotective effect of l‐carnitine on motility,vitality and DNA oxidation of human spermatozoa

Successful cryopreservation for human spermatozoa markedly influences the reproductive outcomes of assisted reproductive technologies. But in spite of its usefulness, cryopreservation significantly decreases sperm quality. l ‐carnitine has been found to improve the quality of spermatozoa in selected cases with male infertility. Here, we examined the efficacy of l ‐carnitine in improving sperm motility and vitality and reducing sperm DNA oxidation during cryopreservation. Semen samples from infertile patients (n = 22) were collected and analysed. Cryopreservation medium supplemented with l ‐carnitine was mixed with the semen at a ratio of 1 : 1 (v/v). The final l ‐carnitine concentration in each cryovial was 0.5 mg ml^?1 per 5 × 10⁶ cell ml^?1. Controls were cryopreserved without addition of l ‐carnitine. After 24 h of cryopreservation, thawed sperm samples were analysed for motility, vitality and DNA oxidation. Sperm vitality was assessed by the eosin–nigrosin test, while sperm DNA oxidation was measured by flow cytometry. Addition of l ‐carnitine significantly improved sperm motility and vitality (P < 0.05) compared with the control. The flow cytometry experiment showed no statistical difference (P > 0.05) in the levels of DNA oxidation between samples and controls. In conclusion, l ‐carnitine improves human sperm motility and vitality, but has no effect on sperm DNA oxidation after cryopreservation.     

DNA integrity in human spermatozoa: relationships with semen quality

The literature contains conflicting evidence regarding the existence of DNA damage in spermatozoa from infertile male patients. To examine this phenomenon, we have studied ejaculated spermatozoa from normozoospermic semen donors and from a group of the unselected male partners of couples attending an infertility clinic for initial investigation. Classical semen analysis according to World Health Organization (WHO) guidelines was undertaken with computer-assisted sperm analysis (CASA). Spermatozoa were prepared by sequential washing and centrifugation and were analyzed for DNA fragmentation using three assays: 1) a single-cell gel electrophoresis (comet) assay, 2) in situ nick translation with prior chemical decondensation (ISNT-decondensed), and 3) in situ nick translation without prior chemical decondensation (ISNT-condensed). In addition, reactive oxygen species (ROS) generation by spermatozoa was measured, and seminal plasma was analyzed for its total reactive antioxidant potential (TRAP). When the donor and patient groups were compared, the latter had lower levels of semen quality and higher levels of DNA damage, which was particularly apparent using the comet assay. Highly significant negative correlations were observed between DNA fragmentation, detected by all three assays, and semen quality, particularly sperm concentration. In addition, multiple regression analysis indicated that other attributes of semen quality, such as sperm movement and ROS generation, were also related to DNA damage. We conclude that a significant proportion of infertile men have elevated levels of DNA damage in their ejaculated spermatozoa.     

The relationship between sperm morphology and chromatin integrity in the koala (Phascolarctos cinereus) as assessed by the Sperm Chromatin Dispersion test (SCDt)

Koala (Phascolarctos cinereus) sperm nuclei show a tendency to swell after cryopreservation, but it is uncertain whether this phenomenon is associated with DNA fragmentation. In this study, we validated a modified version of the sperm chromatin dispersion test (SCDt) for use with koala spermatozoa, which is the first use of the test for a marsupial. Cryopreserved spermatozoa (multiple straws) from a single koala were used to explore the relationship between sperm morphology, viability, chromatin dispersion, and DNA fragmentation. A SCDt prototype kit (Sperm Halomax) was specifically developed for koala spermatozoa with the use of a lysing solution that did not contain dithiothreitol. DNA fragmentation of lysed and nonlysed spermatozoa was examined in microgel slides and validated by means of in situ nick translation (ISNT). The SCDt was then applied to the analysis of extended and frozen-thawed semen samples of 3 different koalas. Spermatozoa were classified into 3 distinct koala sperm morphotypes (KSMs) after the SCDt: 1) KSM-1, rod-shaped cells with no halo of DNA; 2) KSM-2, rounded nuclei with various degrees of halo formation about a dense chromatin core; and 3) KSM-3, rod-shaped or rounded nuclei consisting of an inner chromatin core but with large dispersed halos of stellar chromatin. Although ISNT after the SCDt did not label KSM-1, both KSM-2 and KSM-3 stained positively for DNA fragmentation. ISNT was not able to differentiate between KSM-2 and KSM-3. Although application of the SCDt to the spermatozoa of another 3 koalas showed no difference in the percentage of the 3 sperm morphotypes found between extended and frozen-thawed semen, thawed spermatozoa incubated at 35 degrees C for 2 hours showed an increase in the incidence of KSM-3 and a corresponding decrease in KSM-2. We propose that KSM-1 and KSM-2 represent nuclei that show either no, or only limited, sperm DNA fragmentation, respectively. It is likely that the halos formed around KSM-2 are from DNA that is damaged as part of the normal processing of the spermatozoa and is a consequence of the lack of cysteine residues and associated stabilizing disulfide bonds in marsupial sperm DNA. "True" sperm DNA damage is most likely associated with KSM-3, which shows a massive dispersion of chromatin similar to that described in other species. A model of koala sperm chromatin structure is proposed to explain the behavior of the sperm nuclei after the SCDt. Further studies are required to determine whether DNA damage found in KSM-2 is indicative of single-stranded DNA breakage associated with an inherent lack of cysteine residues in marsupial sperm chromatin. Conversely, it will also be important to establish whether KSM-3 is caused by an increased incidence of double-stranded DNA breakage and whether this abnormality is correlated with impaired fertility as it is in other species.     

Effects of long-term in vitro incubation of human spermatozoa: functional parameters and catalase effect

Prolonged incubation of human spermatozoa can have deleterious effects on sperm function. The aim of this paper was to describe the effects of a prolonged in vitro incubation, under similar conditions to those employed in human assisted reproduction, on various sperm functional parameters, and to investigate the effect of an antioxidant (catalase) on this system. Freshly collected ejaculates from 20 healthy donors were studied. Samples were divided into two aliquots: the first was incubated with Ham's F10 containing 3.5% HAS, and the second was incubated in the same medium plus catalase (100 units ml-1). All experiments were carried out with spermatozoa isolated using the swim-up technique. Spermatozoa recovered from the supernatant after 1 h (T1) of incubation in 5% CO2 in air at 37 degrees C, and after 5 h (T6), 23 h (T24) and 47 h (T48), were evaluated for concentration, motion parameters including hyperactivation (computer-assisted analysis), viability, ATP concentration, reactive oxygen species (ROS) generation, DNA integrity (acridine orange), and acrosome reaction (AR). The major alteration observed in sperm function during the prolonged in vitro incubation was a reduction in the number of motile spermatozoa, together with an impairment in the quality of sperm movement. ROS levels increased with the incubation time. No substantial modifications of sperm viability, chromatin condensation and AR inducibility were observed. The addition of catalase to the medium, while keeping ROS values within baseline levels, did not prevent the loss of motility or the corresponding increase in ATP.     

Small‐volume vitrification for human spermatozoa in the absence of cryoprotectants by using Cryotop

Cryotop is a carrier that has been used successfully in the cryopreservation of human spermatozoa. Here, we explored a novel method to vitrify human spermatozoa without cryoprotective agents (CPAs) using Cryotop. Spermatozoa from 21 Normozoospermic patients were collected and vitrified without CPAs or with sucrose in small volume using Cryotop. The sperm recovery rate, motility, viability, chromatin damage and DNA fragmentation were assessed. No significant difference was observed in the sperm recovery rate and motility rate between the spermatozoa cryopreserved without CPAs and with sucrose. The post‐thawed spermatozoa cryopreserved without CPAs had a higher viability and lower damage to sperm chromatin and DNA than those cryopreserved with sucrose. These results suggest that small numbers of human spermatozoa can be successfully vitrified without CPAs using Cryotop.     

The effect of vitamin B12 supplement on post-thaw motility,viability and DNA damage of human sperm

Sperm cryopreservation may lead to adverse effects on sperm structure and function. Cyanocobalamin (vitamin B12) has antioxidant potential and can protect DNA from free radical-induced damages. Recent studies have shown that vitamin B12 preserves glutathione that leads to modulate oxidative stress responses. Also, vitamin B12 might act directly as a scavenger of ROS. The aim of this study was to investigate the effects of vitamin B12 supplementation on human sperm parameters during the cryopreservation process. Thirty semen samples were obtained from normozoospermic men. Using cryopreservation medium supplemented with different concentrations of vitamin B12 (0, 0.5, 1, 2, 2.5 mg/ml), the semen samples were cryopreserved. After thawing, all samples were evaluated for motility and viability. Based on results, 2 mg/ml was considered as the optimal concentration of vitamin B12 for evaluating sperm DNA fragmentation. The results showed that 1 and 2 mg/ml vitamin B12 significantly increased post-thawing motility and viability compared with the 0 mg/ml vitamin B12 (p < .05). Also, by supplementing with 2 mg/ml vitamin B12, DNA fragmentation decreased when compared to the control. The present study showed that cryopreservation medium supplemented with vitamin B12 at 2 mg/ml could improve sperm quality after freeze–thaw process.     

Canthaxanthin protects human sperm parameters during cryopreservation

Different antioxidants have been introduced to reduce oxidative stress during the cryopreservation. The main goal of this study was to evaluate the effects of canthaxanthin on human sperm parameters during the freeze‐thaw process. This study was performed on 25 normozoospermic semen samples dividing into five groups including 0, 0.1, 1, 10, and 25 µM of canthaxanthin. The prepared spermatozoa were cryopreserved by rapid freezing technique. Sperm motility, viability (eosin‐nigrosin), morphology (Papanicolaou), acrosome reaction (double staining), DNA denaturation (acridine orange), chromatin packaging (aniline blue and toluidine blue), and DNA fragmentation (sperm chromatin dispersion test) were evaluated before freezing and after thawing. All sperm parameters after thawing significantly were decreased compared to before freezing. Twenty‐five micromolar canthaxanthin could significantly improve the progressive and total motility, viability, normal morphology, chromatin packaging, acrosome integrity and DNA denaturation and fragmentation. Ten micromolar canthaxanthin significantly improved total motility, viability, normal morphology, chromatin packaging, acrosome integrity and DNA denaturation and fragmentation. Whereas, in 1 µM group, there were significant differences only in improvement of acrosome integrity, chromatin packaging (toluidine blue) and DNA denaturation and fragmentation. But, in 0.1 µM group, there were no significant differences in any of measured parameters. It seems that canthaxanthin ameliorates detrimental effects of cryopreservation on human sperm parameters.     

Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes.

Mammalian spermatozoa are sensitive to oxygen-induced damages mediated by lipid peroxidation of the cell membrane. The aim of this study was to evaluate whether reactive oxygen species (ROS) could also induce axonemal damage. When Percoll-separated spermatozoa were treated with hydrogen peroxide, or the combination xanthine and xanthine oxidase (X + XO), there was a progressive decrease, leading to a complete arrest, in sperm flagellar beat frequency. Once demembranated in a medium containing magnesium adenosine triphosphate (Mg.ATP), ROS-immobilized spermatozoa still reactivated motility; however, the percentage and duration of motility obtained in these tests gradually decreased to zero in the next hour. In 50% of the cases, motility of intact spermatozoa spontaneously reinitiated after 6 to 24 hours of immobilization due to ROS treatment, although with percentages and beat frequencies lower than those of untreated spermatozoa. Studies using ROS scavengers (such as catalase, superoxide dismutase, and dimethylsulfoxide) indicated that hydrogen peroxide was the most toxic of the ROS involved, but that .O2- and .OH probably also played a role in immobilization of spermatozoa by ROS. The data suggest that ROS induce a chain of events leading to sperm immobilization, that axonemes are affected, and that limited endogenous repair mechanisms exist to reverse these damages.     

Protective effects of exogenous gangliosides on ROS-induced changes in human spermatozoa

This article summarizes the available evidence on the efficacy of gangliosides to reduce the degree of reactive oxygen species (ROS)-mediated damage. The antioxidative efficacy of exogenous gangliosides in protecting different cells encouraged us to examine their ability to protect human spermatozoa. Gangliosides are sialic acid-containing glycosphingolipids with strong amphiphilic character due to the bulky headgroup made of several sugar rings with sialic acid residues and the double-tailed hydrophobic lipid moiety. The amphiphilicity of gangliosides allows them to exist as micelles in aqueous media when they are present at a concentration above their critical micellar concentration. The protective effect of ganglioside micelles on spermatozoa is believed to stem from their ability to scavenge free radicals and prevent their damaging effects. In our study, we particularly focused our attention on the protective effect of ganglioside micelles on DNA in human spermatozoa exposed to cryopreservation. The results indicate that ganglioside micelles can modulate the hydrophobic properties of the sperm membrane to increase tolerance to DNA fragmentation, thus protecting the DNA from cryopreservation-induced damage. Further actions of ganglioside micelles, which were documented by biochemical and biophysical studies, included (i) the modulation of superoxide anion generation by increasing the diffusion barrier for membrane events responsible for signal translocation to the interior of the cell; (ii) the inhibition of iron-catalysed hydroxyl radical formation due to the iron chelation potential of gangliosides; and (iii) inhibition of hydrogen peroxide diffusion across the sperm membrane.     

ATM/H2AX与冷冻精子DNA损伤修复

精液冷冻保存是辅助生殖领域中的一项重要技术,但冷冻本身可引起精子DNA损伤,其中过量活性氧(reactive oxygen species,ROS)的生成起主要作用;而DNA损伤精子仍可以受精形成胚胎,影响生殖结局。蛋白激酶ATM(ataxia-telangiectasia mutated)是DNA损伤的信号感受器,可启动级联反应磷酸化多种蛋白分子调控DNA损伤修复和细胞周期检查点。在ATM的下游分子中,组蛋白H2AX(histone H2AX)被磷酸化激活后参与了DNA损伤精子受精后的修复,与ATM相互作用共同调控胚胎发育过程的细胞周期。ATM/H2AX参与DNA损伤修复以及细胞周期检查点信号转导,以及在氧化应激性DNA损伤修复中的潜在作用,提示其可能参与了冷冻精子氧化应激性DNA损伤的修复过程。