DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa

Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies.     

Application of sperm sorting and associated reproductive technology for wildlife management and conservation

Efforts toward the conservation and captive breeding of wildlife can be enhanced by sperm sorting and associated reproductive technologies such as sperm cryopreservation and artificial insemination (AI). Sex ratio management is of particular significance to species which naturally exist in female-dominated social groups. A bias of the sex ratio towards females of these species will greatly assist in maintaining socially cohesive groups and minimizing male-male aggression. Another application of this technology potentially exists for endangered species, as the preferential production of females can enable propagation of those species at a faster rate. The particular assisted reproductive technology (ART) used in conjunction with sperm sorting for the production of offspring is largely determined by the quality and quantity of spermatozoa following sorting and preservation processes. Regardless of the ART selected, breeding decisions involving sex-sorted spermatozoa should be made in conjunction with appropriate genetic management. Zoological-based research on reproductive physiology and assisted reproduction, including sperm sorting, is being conducted on numerous terrestrial and marine mammals. The wildlife species for which the technology has undergone the most advance is the bottlenose dolphin. AI using sex-sorted fresh or frozen-thawed spermatozoa has become a valuable tool for the genetic and reproductive management of captive bottlenose dolphins with six pre-sexed calves, all of the predetermined sex born to date.     

Elamipretide as a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation

As a technique widely used in assisted reproduction, human spermatozoa cryopreservation makes it possible to conserve functional sperm for a long time, but the impact of cryodamage on sperm during the process could not be ignored. The objective of the present study was to investigate the efficacy of Elamipretide, a novel small mitochondrial targeting short cytoprotective peptide, in attenuating cryodamage during spermatozoa cryopreservation. Semen samples were collected and cryopreserved in freeze solution containing different concentrations (0.0, 0.1, 1, and 10 μM) of Elamipretide. Sperm motility, viability, membrane integrity, mitochondrial membrane potential, DNA fragmentation, antioxidant profiles, and acrosome reaction were measured and analyzed. The results showed that supplementation of the freeze media with Elamipretide (1 and 10 μM) significantly improved post-thaw sperm parameters including motility and viability, stability of the plasma membrane, and mitochondria and chromosomes. In addition, by adding Elamipretide, excessive oxidation and acrosome dysfunction in sperm cells undergoing freeze-thaw were also significantly attenuated. Therefore, Elamipretide may be a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation.     

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage.The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2 h (fresh) or 5 days at 4 °C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.     

DNA integrity of fresh and frozen canine epididymal spermatozoa

The aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1 mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3 ± 3.6) and samples frozen with (4.2 ± 3.8) or without (3.6 ± 3.7) melatonin. Sperm motility was significantly (p < 0.0001) higher in fresh compared to frozen samples. The presence of melatonin in the freezing extender did not affect the sperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (p < 0.01) by cryopreservation, and melatonin did not exert any beneficial effects. In conclusion, DNA fragmentation of canine epididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques.     

Mito-Tempo alleviates cryodamage by regulating intracellular oxidative metabolism in spermatozoa from asthenozoospermic patients

As the largest proportion of male infertility population, asthenozoospermia patients often resort to sperm cryopreservation to preserve fertility as well as to enrich motile sperm for assisted reproductive techniques (ART), although it may cause some cryodamage during the freezing–thawing process. The objective of this study was to investigate whether mitochondrial antioxidant Mito-Tempo was effective in preventing cryodamage of asthenozoospermic spermatozoa. Asthenozoospermic semen samples were collected and cryopreserved in media supplemented with different concentrations (0.0, 1.0, 10 and 100 μM) of Mito-Tempo. We measured sperm motility, viability, membrane integrity, DNA fragmentation, mitochondrial membrane potential, oxidation product, and antioxidant enzymes activities. Supplementation of the cryopreservation media with Mito-Tempo (10 and 100 μM) induced a significant improvement in sperm viability, motility, membrane integrity, mitochondrial membrane potential and chromatin integrity (P < 0.05). Significant enhancement of antioxidant enzymes activities accompanied by the decreased formation of oxidation products (ROS and MDA) was also observed in groups supplemented with Mito-Tempo (10 and 100 μM). It is concluded that mitochondria targeted antioxidant Mito-Tempo alleviates cryodamage by regulating intracellular oxidative metabolism in spermatozoa from asthenozoospermic patients after cryopreservation.     

Influence de la congélation sur le taux de fragmentation de l’ADN des spermes normaux à sévèrement altérés

Ejaculated sperm cryopreservation can be proposed in the course of anART procedure, particularly in the case of severe oligozoospermia likely to deteriorate. The aim of this study was to evaluate the influence of the freezing-thawing process on sperm DNA fragmentation (analysed by the TUNEL technique). The first step of this work consisted of adapting the TUNEL technique to perform this analysis on very poor quality sperm. A study was then performed on 72 patients divided into 4 groups according to their spermatic characteristics: group 1 [n=20] (“normal” parameters according to WHO), group 2 [n=24] (normal sperm count associated with asthenospermia and/or teratospermia), group 3 [n=16] (total sperm count between 5 and 20 M) and group 4 [n=12] (total sperm count below 5 M). Spermatic parameters and DNA fragmentation (performed by TUNEL in situ technique, 400 spermatozoa read per slide) were evaluated on raw semen - for all patients -, raw migrated sperm - for patients of group 1 and 2 -, migrated frozen-thawed sperm - for all patients-. A TUNEL technique adapted to oligospermic samples was developed, manipulating spermatozoa directly on the slide rather than in suspension, to limit spermatic sample loss. After the whole migration-freezing-thawing process, the mean DNA fragmentation rate decreased for patients in group 1 (2.9 vs 5.1%, p<0.0001) whereas this rate increased for patients in groups 2 (10.5 vs 6.8%, p<0.0001), 3 (10.7 vs 7.6%, p<0.05) and 4 (15.2 vs 8.7%, p<0.005). DNA fragmentation rates from thawed samples were also correlated with initial spermatic parameters. At the intermediary step, migration decreased DNA fragmentation rate in comparison with raw semen rate in both groups (1.9 vs 4.7% [p<0.05] in group 1; 2.5 vs 5.4% [p<0.05] in group 2). DNA fragmentation rate decreases after migration and then increases after freezing-thawing so that this rate is lower than the raw semen rate for “normal“ sperms and higher than the raw semen rate for altered sperms. Nevertheless, this DNA damage induced by cryopreservation on altered sperms remains moderate. Sperm “resistance” to cryopreservation also appears to depend on spermatic parameters. Cryopreservation may positively select spermatozoa, accelerating elimination of senescent spermatozoa by necrosis, so that early apoptotic spermatozoa from fresh ejaculate are not found in thawed samples. These results, that need to be completed by a study on a larger sample of oligospermic patients, encourage us to continue cryopreserving severely altered sperms.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

Dynamics of sperm DNA fragmentation in domestic animals II. The stallion

The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. This study reports on the validation of a sperm DNA fragmentation test based on the sperm chromatin dispersion test (SCD) for stallion spermatozoa and on its application to semen that was chilled (4 degrees C; n=10) or frozen-thawed (n=13). Semen samples were collected by artificial vagina and the proportion of sperm with fragmented DNA determined. Seminal plasma was then removed by centrifugation and the sperm pellet re-suspended in commercial extenders prior to being chilled or cryopreserved using standard industry protocols. Chilled semen was cooled slowly to 4 degrees C and stored for 1h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 degrees C and analyzed for SCD after 0, 4, 6, 24 and 48 h storage. The results of this investigation revealed that there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation. However, within 1h of incubation at 37 degrees C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage. While the sDFI of individual stallions at equivalent times of incubation was variable, an analysis of the rate of change of sDFI revealed no difference between stallions or the way in which the semen was preserved. In terms of sperm DNA fragmentation dynamics, the highest intensity of sperm DNA damage occurred in the first 6h of incubation. We suggest that the SCD test can be used as a routine assessment tool for the development and refinement of preservation protocols designed to reduce stallion sperm DNA damage.     

Dynamics of sperm DNA fragmentation in domestic animals III. Ram

From a biological viewpoint spermatozoa are ejaculated by the male and received into the female while maintaining roughly constant temperature, which in most mammals is below the temperature of the soma. When ejaculated spermatozoa are used for artificial reproductive purposes a temperature excursion episode is produced, because the spermatozoa are often stored as frozen or chilled samples and the biological temperature is only recovered after insemination. In this study we have analyzed the effects of cooling (to 15 degrees C) and freezing ram spermatozoa on the subsequent sperm DNA fragmentation index (sDFI) during a varying period of storage at 37 degrees C. The aim was to emulate in vivo processes that cooled or frozen-thawed spermatozoa experience after insemination. The study was performed using commercial semen samples derived from rams regularly used for reproductive purposes. Semen samples were studied after a cooling or cryopreservation episode followed by biological temperature recovery and incubation up to 48h. The results indicated that when spermatozoa experience a severe (frozen) or mild (cooled) temperature excursion episode, major effects on sperm viability and DNA fragmentation are induced and cause the subsequent rapid decline of ram sperm quality. This effect could be detected just at the onset of the biological temperature recovery. Sperm DNA damage in cooled samples was observed after 5h of incubation at 37 degrees C, while this time was reduced to less than 60min in frozen-thaw samples. The dynamics of sDFI in different animals, analyzed under the same experimental conditions, was different from one sample to another, regardless of the method used for storage. Sperm viability was better preserved in cooled rather than in frozen samples. While for the frozen-thawed samples sperm viability was almost abolished after 5h of incubation, a stable proportion of viable spermatozoa (ranging from 20% to 60%) was observed in the cooled samples at the corresponding time points. Finally, with respect to the prevalence of sDFI in ram, the level commonly found was lower than 5% at the onset of the experiment. However, sDFI was higher than 5% in 25% of the samples and in 15% of rams this index exceeded 10%.     

Mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality

Human spermatozoa cryopreservation is an important means of assisted reproductive technology and male fertility preservation. Although this technique is particularly useful, sperm cryopreservation significantly reduces the quality of spermatozoa after freezing and thawing. The objective of the study is to examine the efficacy of mitochondria-targeted antioxidant MitoTEMPO in improving sperm quality during semen cryopreservation processes. Semen samples were collected and cryopreserved in extenders containing different concentrations (0.0, 0.5, 5, 50, and 500 μM) of MitoTEMPO. Sperm motility, viability, membrane integrity, mitochondrial membrane potential and antioxidant activities were measured and analyzed. The results showed that the addition of MitoTEMPO (5–50 μM) significantly improved post-thaw sperm motility, viability, membrane integrity and mitochondrial membrane potential (P < .05). Meanwhile, antioxidant enzymes activities were enhanced and MDA content were decreased in the group supplemented with MitoTEMPO. In conclusion, mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality and antioxidant enzymes profile.     

The role of antioxidants in sperm freezing: a review

Cryopreservation of spermatozoa is becoming more important because of new clinical requirements and current clinical practice. Despite the success of sperm cryopreservation this routinely used procedure induces serious detrimental changes in sperm function. Some researchers believe that cryopreservation is associated with DNA fragmentation and DNA single strand breaks in sperm. Mechanisms of cryodamage to human spermatozoa are thought to be multifactorial including: cold shock, osmotic stress, intracellular ice crystal formation, oxidative stress, and combinations of these conditions. Additives showing antioxidative properties reported to reduce the impact of ROS-induced and cold shock damages. Many studies exist as regards the effects of antioxidants on the cryopreservation aimed at improving the quality of post-thaw semen. Hence, this review will clarify results of recent applications of various antioxidants used in numerous research efforts to improve cryopreservation of spermatozoa. This review is to increase the understanding of the roles of these antioxidants concerning mechanisms which enhance resistance to cryodamage of spermatozoa.     

Intérêt de l’analyse de la morphologie fine et de la qualité nucléaire des spermatozoïdes dans le cadre des techniques d’AMP

Routine semen examination does not identify minor malformations of the sperm nucleus and chromatin architectural defects, which may be associated with ART outcome and cannot be detected by the embryologist even at 1000x magnification. Recent publications have demonstrated the advantages, compared to routine analysis, of a new method of real-time detailed morphological evaluation of motile spermatozoa: motile sperm organellar morphology examination (MSOME). MSOME is performed with an inverted light microscope equipped with high-power differential interference contrast optics enhanced by digital imaging to achieve a magnification of 10000x. To be considered morphologically normal, a sperm nucleus must have both a normal shape and a normal chromatin content. The aim of the present study was to combine MSOME and sperm DNA fragmentation characteristics to assess reproductive outcome. The study population consisted of the male partners of 52 couples referred for conventional IVF or split cycles (half IVF-half ICSI cycles) and exhibiting normal routine sperm parameters. Spermatozoa were analysed by examining the fine nuclear morphology and DNA integrity using the sperm chromatin dispersion test (SCD test), based on the principle that the deproteinized nuclei of spermatozoa with nonfragmented DNA show extended halos of DNA dispersion that are either absent or only minimally present in sperm nuclei with fragmented DNA. Fertilization rates were significantly lower in the group showing less than 8% of normal spermatozoa according to MSOME criteria, but early embryo development was not affected. Fine sperm morphology correlated with DNA fragmentation rate. These results demonstrate that the assessment of sperm nuclear normality by MSOME analysis and SCD test improves characterization of the semen sample and should be evaluated as a tool for allocating patients to specific assisted reproduction treatments.     

DNA methylation stability in fish spermatozoa upon external constraint: Impact of fish hormonal stimulation and sperm cryopreservation

Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2‐propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.     

Short-term storage of human spermatozoa in electrolyte-free medium without freezing maintains sperm chromatin integrity better than cryopreservation

Previous attempts to maintain human spermatozoa without freezing were based on short-term storage in component-rich medium and led to fast decline in motility and increased incidence of chromosome breaks. Here we report a new method in which sperm are maintained without freezing in an electrolyte-free medium (EFM) composed of glucose and bovine serum albumin. Human sperm were stored in EFM or human tubal fluid medium (HTFM) or were cryopreserved, and their motility, viability, and DNA integrity were examined at different intervals. Cryopreservation led to significant decline in sperm motility and viability and induced DNA fragmentation. Sperm stored in EFM maintained motility and viability for up to 4 and 7 wk, respectively, much longer than sperm stored in HTFM (<2 and <4 wk, respectively). DNA integrity, assessed with comet assay, was also maintained significantly better in EFM than in HTFM. One-week storage in EFM yielded motility and viability similar to that of cryopreserved sperm, but DNA integrity was significantly higher, resembling that of fresh sperm. After several weeks of storage in EFM, sperm were able to activate oocytes, undergo chromatin remodeling, and form normal zygotic chromosomes after intracytoplasmic sperm injection. This study demonstrated that human spermatozoa can be stored in EFM without freezing for several weeks while maintaining motility, viability, and chromatin integrity and that 1-wk storage in EFM offers better protection of sperm DNA integrity than cryopreservation. Sperm storage in EFM may become a viable option for the physicians working in assisted reproduction technology clinics, which would avoid cryodamage.     

Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa

We investigated the impact of cryopreservation and thawing on levels of caspases-3, -8, and -9 activity, intact mitochondrial membrane potential (Deltapsim), and DNA fragmentation in human spermatozoa. Eleven pools of cryopreserved and eight pools of fresh semen samples were examined. Mature and immature fractions were separated on a two-layer density gradient (47% and 90%) and further subdivided based on the externalization of phosphatidylserine and its binding to annexin V-labeled superparamagnetic microbeads (ANMB). Levels of activated caspases were assessed using fluorescein-labeled inhibitors of caspases (FLICA), Deltapsim using a lipophilic cationic dye, and DNA fragmentation by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Cryopreservation was significantly associated with activation of caspases-3, -8, and -9, as well as disruption of the mitochondrial membrane potential but no significant changes were observed in DNA fragmentation. In mature sperm, caspase activation was only detected in the ANMB+ fraction, whereas in immature sperm, both ANMB+ and ANMB- fractions showed activated caspase levels. In ANMB+ immature sperm, apoptosis seemed to be triggered by a surface ligand-receptor mechanism as well as by disruption of mitochondria, whereas in ANMB- immature sperm, apoptosis was induced by activation of caspase-9 following loss of intact Deltapsim. These results demonstrate that selection of annexin V-negative mature spermatozoa might be of clinical relevance for fertility preservation, as this sperm fraction shows no activated apoptosis during the cryopreservation process.     

Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level

Sperm quality seems to be one of the reasons for the reproduction constraints faced by Senegalese sole (Solea senegalensis) aquaculturists. Previous studies in this species indicated that the sperm quality of individuals kept in culture varies throughout the year and that different sperm subpopulations can be identified in ejaculates according to the motility pattern of spermatozoa. Aiming to better understand factors affecting sole sperm quality in captivity, sperm of 11 males was assessed during the reproductive season using different parameters: motility characteristics using CASA analysis; cell plasma membrane resistance to seawater hyperosmolarity; DNA fragmentation with single-cell gel electrophoresis; and early apoptosis, labeled with Annexin-V FITC. Computer-assisted sperm analyses motility data were treated using multivariate analysis to identify the presence of different spermatozoa subpopulations according to their motility pattern. Four distinct sperm subpopulations were obtained: Subpop1, which includes fast linear spermatozoa; Subpop2, made up of fast nonlinear spermatozoa; Subpop3, which includes slow linear spermatozoa; and Subpop4, which contains slow nonlinear spermatozoa. The sperm subpopulation structure varied with time after activation and with male. Low cell resistance to the seawater hyperosmotic conditions was noticed. The Annexin-V assay allowed the identification of an apoptotic population ranging from 6% to 20%. A high percentage of cells (64.1%) showed a DNA fragmentation level below 30%, but these values varied significantly between males. DNA fragmentation appears to be related to cell membrane resistance to hyperosmotic conditions faced by the cells when in contact with seawater. This condition seems to modulate the composition of the motile sperm population and performance after activation. This phenomenon could be related to the spermatozoa maturation process.     

Apoptose dans le sperme éjaculé: Revue

It has become clear in recent years that programmed cell death occurs spontaneously in the cycle of the seminiferous epithelium. Although apoptosis is a key phenomenon in the control of sperm production, the existence and role of apoptosis in ejaculated sperm cells remain controversial. Apoptosis — as determined by DNA fragmentation and ultrastructural analysis — is abnormally frequent in the sperm cells of the ejaculate of sterile men. In this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa and the consequences of this DNA damage when apoptotic spermatozoa are used for ICSI. Percentages of DNA fragmentation in human ejaculated sperm are correlated with fertilization rates after IVF or ICSI assay. Detection of DNA fragmentation in human sperm could provide additional information about the biochemical integrity of sperm and may be used in future studies for fertilization failures not explained by conventional sperm parameters. However, the analysis of new markers of apoptosis (Fas, ANNEXINE V) and molecular mechanisms is now necessary to assess the role of apoptosis in human ejaculated sperm cells.     

Cryopreservation induces an apoptosis-like mechanism in bull sperm

Cryopreservation induces many changes in sperm cells, including membrane disorders and cell death. We tested the hypothesis that apoptosis, a form of programmed cell death, can contribute to the fatal effect of cryopreservation on sperm cells. A multiparametric study of apoptosis on bovine sperm is proposed, using flow cytometry, including mitochondrial membrane potential (DeltaPsi(m)), caspase activation, membrane permeability, nucleus condensation, DNA fragmentation, and phosphatidylserine (PS) externalization. The relevance of each test was first validated on a human somatic cell line, U937. Cryopreservation and/or thawing induced significant changes in all apoptotic markers in living bull sperm cells except those concerning the nucleus. After cryopreservation, 44.9% +/- 17% (vs. 11.3% +/- 10.6% before cryopreservation) of sperm cells showed low DeltaPsi(m), 12% +/- 6.3% (vs. 2.2% +/- 1.0% before) contained active caspases, and 10.8% +/- 5.8% (vs. 1.4% +/- 1.1% before) exhibited high membrane permeability. However, cryopreservation had no effect on DNA fragmentation (9.1% +/- 7.7% before vs. 11.1% +/- 5.7% after cryopreservation) or on nucleus condensation (46% +/- 12.7% before vs. 43.8% +/- 13.1% after). Cryopreservation acts as an apoptotic mechanism inducer in bovine sperm cells, where the earliest but not the latest features of cells undergoing apoptosis occur. We have named this abortive process an apoptosis-like phenomenon.