Acridine orange-induced precipitation of mouse testicular sperm cell DNA reveals new patterns of chromatin structure

Precipitate resulting from interaction between certain intercalators, such as acridine orange (AO), and nucleic acids can be detected by electron microscopy. Formation of precipitate in nuclei of live cells is modulated by chromatin structure. Susceptibility of in situ DNA to precipitation was studied in mouse testicular germ cells during various stages of sperm maturation. DNA in round spermatid chromatin, similar to somatic cell euchromatin, was rather resistant to precipitation; the electron-dense precipitate was granular and randomly distributed. DNA in elongated spermatids was more susceptible to precipitation; the products were in the form of fibers. At early stages of spermatid maturation these fibers were distributed uniformly throughout the entire nucleus. At later stages, the products appeared as approximately 25-nm-thick fibers arranged longitudinally in arrays within the nucleus. With further cell maturation, fibers in the anterior portion of the nucleus appeared to fuse, forming homogeneously dense product. These fibrous products likely represent AO interactions with DNA in chromatin in which transition proteins had replaced histones. Changing patterns of these precipitated fibers likely reflect progressive stages of chromatin condensation, which starts at the center and anterior portion of the nucleus where the fibers coalesce. Mature sperm cell DNA, known to be complexed with protamines, was more resistant to AO-induced precipitation. The data suggest that precipitation induced by AO and monitored by electron microscopy may be a useful probe of nuclear chromatin structure.     

Trout sperm chromatin. II. Ultrastructural aspects after salt dissociation of proteins

The ultrastructural organization of the trout sperm nucleus was studied in ultrathin sections and spread preparations after partial decondensation of the nucleus with increasing NaCl concentrations. The obtained results suggest that the organization of the trout sperm chromatin is much more complex than a pure nucleoprotamine. Three types of complexes were observed. The first one results from the association of DNA with protamines. This complex appears as a fibrous network when partially decondensed nuclei are digested with DNase I indicating that at least a part of DNA remains protected by protamines and favours models accepting a colinear alignment of the latter on the DNA molecules. The second type of structures represent the DNA-protamine fibers compacted into dense clumps which appear as separate compaction units seen upon partial decondensation of the sperm nucleus. A third type are complexes of the ring-shaped granular bodies tightly associated with DNA and resisting high salt-urea and detergent treatment.     

Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection

Successful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1-4) did not increase, even at 180 min (0.7-4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120-180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0-60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39. The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.     

Single-chain antibody against human lipocalin-type prostaglandin D synthase: Construction, expression, purification, and activity assay

An active form of single-chain antibody (ScFv) from murine monoclonal antibody 4A7, which is specific for lipocalin-type prostaglandin D synthase (L-PGDS), was produced in Escherichia coli. The complementary DNA fragments encoding the variable regions of heavy chain (VH) and light chain (VL), which amplified from hybridoma 4A7 producing a monoclonal antibody (IgG1) against L-PGDS, were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The resultant ScFv were cloned into the vector pGEM and expressed in E. coli as inclusion bodies. The expressed ScFv fusion proteins were purified by Ni2+-nitrilotriacetic acid chromatography. The purity and activity of purified ScFv were confirmed by SDS-PAGE and ELISA. The result revealed that 4A7 ScFv conserved the same characteristics of specific recognition and binding to sperm as the parental 4A7 monoclonal antibody.     

Microscopic, biochemical, and molecular characteristics of the Chilean Blob and a comparison with the remains of other sea monsters: nothing but whales

We have employed electron microscopic, biochemical, and molecular techniques to clarify the species of origin of the "Chilean Blob," the remains of a large sea creature that beached on the Chilean coast in July 2003. Electron microscopy revealed that the remains are largely composed of an acellular, fibrous network reminiscent of the collagen fiber network in whale blubber. Amino acid analyses of an acid hydrolysate indicated that the fibers are composed of 31% glycine residues and also contain hydroxyproline and hydroxylysine, all diagnostic of collagen. Using primers designed to the mitochondrial gene nad2, an 800-bp product of the polymerase chain reaction (PCR) was amplified from DNA that had been purified from the carcass. The DNA sequence of the PCR product was 100% identical to nad2 of sperm whale (Physeter catadon). These results unequivocally demonstrate that the Chilean Blob is the almost completely decomposed remains of the blubber layer of a sperm whale. This identification is the same as those we have obtained before from other relics such as the so-called giant octopus of St. Augustine (Florida), the Tasmanian West Coast Monster, two Bermuda Blobs, and the Nantucket Blob. It is clear now that all of these blobs of popular and cryptozoological interest are, in fact, the decomposed remains of large cetaceans.     

Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster

Eggs of the newt, Cynops pyrrhogaster, arrested at the second meiotic metaphase are activated by sperm at fertilization and then complete meiosis to initiate development. We highly purified a sperm factor for egg activation from a sperm extract with several chromatographies. The purified fraction containing only a 45 kDa protein induced egg activation accompanied by an intracellular Ca2+ increase when injected into unfertilized eggs. Although injection of mouse phospholipase C (PLC) zeta-mRNA caused a Ca2+ increase and egg activation, partial amino acid sequences of the 45 kDa protein were homologous to those of Xenopus citrate synthase, but not to PLCs. An anti-porcine citrate synthase antibody recognized the 45 kDa protein both in the purified fraction and in the sperm extract. Treatment with the anti-citrate synthase antibody reduced the egg-activation activity in the sperm extract. Injection of porcine citrate synthase or mRNA of Xenopus citrate synthase induced a Ca2+ increase and caused egg activation. A large amount of the 45 kDa protein was localized in two lines elongated from the neck to the middle piece of sperm. These results indicate that the 45 kDa protein is a major component of the sperm factor for egg activation at newt fertilization.     

Comparison of human and mouse sperm chromatin structure by flow cytometry

Human and mouse sperm nuclei obtained by sonication or mechanical agitation of freshly isolated sperm in the presence of anionic detergent were purified through a sucrose gradient and stained with acridine orange (AO); their fluorescence intensity was measured by flow cytometry. The green fluorescence, characteristic of AO binding to DNA by intercalation, was twice lower per unit of DNA for human sperm nuclei than for human peripheral blood lymphocytes. After extraction of basic proteins with 0.08 N HCl, AO binding to DNA increased 3.2-fold for lymphocytes and only 1.3-fold for sperm indicating that, in contrast to somatic cells, the proteins restricting AO binding to DNA are essentially non-extractable from sperm at that low pH. Treatment of human and mouse nuclei with dithiothreitol (DTT), a sulfhydryl reducing agent, and trypsin, removed constraints responsible for the restriction of AO binding. Specifically, as a result of DTT treatment alone there was up to a 20–30% increase of AO binding; upon subsequent addition of trypsin there was a further rapid rise in AO binding up to a final level of approximately 5 times the original AO binding to isolated sperm nuclei. Electron microscopy of DTT-treated human sperm nuclei showed that the reducing agent caused chromatin decondensation to a level whereby 20–30 Å diameter fibers interconnecting chromatin bodies about 30–75 nm in diameter were revealed. Trypsin digestion in the presence of DTT converted the chromatin bodies into a network of fibrous structures about 150 Å in diameter. Both electron microscopy and flow cytometry demonstrated an extremely large intercellular variation among human sperm nuclei in response to DTT and trypsin treatment indicating heterogeneity of chromatin structure. In contrast, AO staining of mouse sperm nuclei increased homogeneously in response to DTT and trypsin treatment.     

The influence of the storage temperature and cryopreservation conditions on the extent of human sperm DNA fragmentation

We explored the influence of different storage temperature conditions, including different methods of cryopreservation, on the structure of DNA organization in human sperm using a direct labeling procedure for detecting DNA fragmentation. Nineteen sperm samples that were obtained from healthy men with normozoospermia (according to the criteria of the World Health Organization) were used for the investigation. A significant increase in human sperm DNA fragmentation was observed 8 h after the incubation at +39°C (by 76.7%) and at +37°C (by 68.9%). It was found that cooling the sperm with a cryoprotectant immediately after thawing did not produce a significant difference in the extent of DNA fragmentation; however, the samples that contained cryoprotectants showed a sharp increase in the DNA fragmentation 24 h after the incubation, which could suggest cryoprotectant cytotoxicity.     

Fragmentation dynamics of frozen-thawed ram sperm DNA is modulated by sperm concentration

This study investigated the hypothesis that post-thaw incubation of ram sperm at high concentrations results in a faster rate of DNA fragmentation than when sperm are incubated at a lower concentration. Ejaculates from 10 rams were frozen-thawed, prepared in sperm concentrations of 100, 50, 25, 12, and 6 × 10⁶ sperm/mL, and incubated for 6 h at 37 °C. Sperm DNA fragmentation was assessed using the sperm chromatin dispersion test (Sperm-Halomax®) at 1, 3, 4, and 6 h of incubation at 37 °C. On fitting a binary logistic regression with a cubic over time and treating ram and dilution levels as factors, there were significant effects with respect to the ram, dilution and time (all P-values were very much smaller than 0.001). Therefore, DNA fragmentation dynamics of incubated frozen-thawed ram sperm were not only dependent on the inherent sperm DNA fragmentation expressed immediately after thawing, but also on the concentration of sperm incubated in the sample. Although there was evidence of individual ram variation in SDF during the incubation period, the general finding of the current study was that lower sperm concentrations resulted in a slower rate of DNA fragmentation These findings have important implications for the post-thaw manipulation of ram sperm used for AI and advanced reproductive procedures that use sperm at low concentrations. Our data also emphasised the highly dynamic nature of sperm DNA fragmentation and the importance of conducting the procedure in a standardised manner.     

Effect of Sperm DNA Fragmentation on Clinical Outcome of Frozen-Thawed Embryo Transfer and on Blastocyst Formation

During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ≤30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ≤30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles.     

Meiotic DNA synthesis during mouse spermatogenesis

The incorporation of radioactivity into various cells in the sequence of spermatogenesis was measured by preparing highly purified spermatozoan nuclei from the cauda epididymidis of mice at daily intervals after injection of (3H)thymidine. The stages of differentiation of these sperm at the time of thymidine administration were calculated from the kinetics of spermatogenesis. The procedure for purification of sperm nuclei included sonication, mechanical shearing, and treatment with trypsin, DNase, Triton X-100, 2M NaC1, and sodium dodecyl sulfate. DNA was isolated from these nuclei by treatment with dithiothreitol and pronase, followed by phenol extraction and ethanol precipitation. The levels of radioactivity in the epididymal sperm head preparations were low (less than 13 dpm/mouse) for 27 days after injection, and then rose dramatically to over 4 times 104 dpm/mouse. Further experiments demonstrated that the 11 dpm of 3H radioactivity contained in sperm heads at 21 or 26 days after injection of (3H)TdR was significantly above background and contamination levels from other cells or other sources. Most of the radioactivity was in the sperm DNA and represented incorporation of tritium from (3H)TdR into the nuclear DNA of meiotic cells at 0.002 percent of the rate of incorporation into S-phase cells. Little, if any, (3H)TdR was incorporation into the DNA of spermatids. The levels of DNA synthesis during the meiotic prophase in the mouse appear to be much lower than those reported for other organisms.     

The major component of the rat sperm fibrous sheath is a phosphoprotein

The fibrous sheath from rat epididymal sperm was isolated by sequential extraction, first with Triton X-100 and dithiothreitol, and then with 6 M urea and dithiothreitol. The latter extraction procedure solubilized most of the sperm components, leaving the head and the fibrous sheath as the only intact structures. This material was purified by sucrose gradient centrifugation. Electron microscopy confirmed the purity of the isolated material and revealed the characteristic structural features of the fibrous sheath. Polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) of the fibrillar material, showed a complex polypeptide composition. The polypeptides with molecular weights of 80,000, 24,000, and 11,500 accounted for about 65% of the total protein of the fibrous sheath. Peptide map analyses indicated that the components of molecular weights of 80,000 and 24,000 are unrelated to the polypeptides of similar size of the outer dense fibers. On the other hand, it appears that the fibrous sheath and the outer dense fibers share the polypeptide of 11,500 daltons. The component of 80,000 daltons contains on the average about 3 mol of phosphoserine per mol of polypeptide, indicating that the most abundant polypeptide of the fibrous sheath is a phosphoprotein.     

A new method to analyze boar sperm DNA fragmentation under bright-field or fluorescence microscopy

We present a new, rapid and simple method to study DNA Fragmentation Index (DFI) in sperm samples from boar under bright-field and fluorescence microscopy. Discrimination of sperm cells containing fragmented DNA relies on the extreme peripheral diffusion of their chromatin fragments, whereas those sperm nuclei without DNA fragmentation do not disperse or show very restricted spreading of DNA loops close to the flagellum. The basic methodology provided in the commercial kit Sperm-Sus-Halomax allows, in addition to a direct estimation of DFI in a sperm sample under bright field microscopy, a direct visualization of DNA breaks by incorporation of labelled nucleotides using the DNA polymerase I following the in situ nick translation assay (ISNT methodology not provided in the kit). An external control using DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) on human and boar sperm samples was used in this experiment. The results obtained show (i) low levels of background DNA fragmentation (from 0.7 to 10%), (ii) no significant differences for DFI after the application of Sperm-Sus-Halomax and ISNT, with a tendency to be underestimated after using DBD-FISH and (iii) a characteristic chromatin organization in boar sperm nucleus, with a particular response to chromatin loop relaxation and preferential DNA labelling by ISNT at the proximal nuclear area, close to the flagellum. This methodology allows the routine assessment of boar sperm samples for DFI, as well as basic and clinical research on this relevant topic in any laboratory of semen analysis.     

Poor centrosomal function of cat testicular spermatozoa impairs embryo development in vitro after intracytoplasmic sperm injection

In the domestic cat, morula-blastocyst formation in vitro is compromised after intracytoplasmic sperm injection (ICSI) with testicular compared to ejaculated spermatozoa. The aim of this study was to determine the cellular basis of the lower developmental potential of testicular spermatozoa. Specifically, we examined the influence of sperm DNA fragmentation (evaluated by TUNEL assay) and centrosomal function (assessed by sperm aster formation after ICSI) on first-cleavage timing, developmental rate, and morula-blastocyst formation. Because the incidences of DNA fragmentation were not different between testicular and ejaculated sperm suspensions, DNA integrity was not the origin of the reduced developmental potential of testicular spermatozoa. After ICSI, proportions of fertilized and cleaved oocytes were similar and not influenced by sperm source. However, observations made at 5 h postactivation clearly demonstrated that 1) zygotes generally contained a large sperm aster after ICSI with ejaculated spermatozoa, a phenomenon never observed with testicular spermatozoa, and 2) proportions of zygotes with short or absent sperm asters were higher after ICSI with testicular spermatozoa than using ejaculated spermatozoa. The poor pattern of aster formation arose from the testicular sperm centrosome, which contributed to a delayed first cleavage, a slower developmental rate, and a reduced formation of morulae and blastocysts compared to ejaculated spermatozoa. When a testicular sperm centrosome was replaced by a centrosome from an ejaculated spermatozoon, kinetics of first cell cycle as well as embryo development quality significantly improved and were comparable to data from ejaculated spermatozoa. Results demonstrate for the first time in mammals that maturity of the cat sperm centrosome (likely via epididymal transit) contributes to an enhanced ability of the spermatozoon to produce embryos that develop normally to the morula and blastocyst stages.     

Mouse spermatozoa contain a nuclease that is activated by pretreatment with EGTA and subsequent calcium incubation

We demonstrated that mouse spermatozoa cleave their DNA into approximately 50 kb loop-sized fragments with topoisomerase IIB when treated with MnCl(2) and CaCl(2) in a process we term sperm chromatin fragmentation (SCF). SCF can be reversed by EDTA. A nuclease then further degrades the DNA in a process we term sperm DNA degradation (SDD). MnCl(2) alone could elicit this activity, but CaCl(2) had no effect. Here, we demonstrate the existence of a nuclease in the vas deferens that can be activated by ethylene glycol tetraacetic acid (EGTA) to digest the sperm DNA by SDD. Spermatozoa were extracted with salt and dithiothreitol to remove protamines and then incubated with EGTA. Next, the EGTA was removed and divalent cations were added. We found that Mn(2+), Ca(2+), or Zn(2+) could each activate SDD in spermatozoa but Mg(2+) could not. When the reaction was slowed by incubation on ice, EGTA pretreatment followed by incubation in Ca(2+) elicited the reversible fragmentation of sperm DNA evident in SCF. When the reactions were then incubated at 37 degrees C they progressed to the more complete degradation of DNA by SDD. EDTA could also be used to activate the nuclease, but required a higher concentration than EGTA. This EGTA-activatable nuclease activity was found in each fraction of the vas deferens plasma: in the spermatozoa, in the surrounding fluid, and in the insoluble components in the fluid. These results suggest that this sperm nuclease is regulated by a mechanism that is sensitive to EGTA, possibly by removing inhibition of a calcium binding protein.     

Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation

In this paper, DNA laddering analysis and single-cell gel electrophoresis (SCGE) or Comet assay, were used to detect DNA damage in response to a cryopreservation process in sea bass spermatozoa. The results obtained demonstrate that the cryopreservation protocol used to cryopreserve the sea bass sperm cause significantly damage at DNA level. In fact, the degree of DNA damage in frozen-thawed sperm (%DNAT=38.2+/-11.2, MT=498.9+/-166.4, n=3) was different (P<0.01) from that measured in fresh sperm (%DNAT=32.7+/-11.1, MT=375.2+/-190.7, n=3). Data here reported also demonstrated the fundamental role played by cryoprotectants (BSA and Me2SO) in reducing fish sperm DNA fragmentation. Finally, from our results, the ability of SCGE to reveal DNA fragmentation in fish sperm is also confirmed.     

Sperm DNA fragmentation in boars is delayed or abolished by using sperm extenders

The semen quality of seven young adult boars was assessed for percentages of sperm motility, normal acrosomes, abnormal sperm, cells positive to sHOST (short Hipoosmotic Swelling Test), HPNA cells (sHOST Positive with Normal Acrosome cells) and the percentage of sperm heads, which exhibited DNA fragmentation using the Sperm Chromatin Dispersion test (SCD). These parameters were analysed in sperm samples both undiluted and diluted using a commercial extender and stored at 15 degrees C for 21 days. Results showed that semen quality decreases faster in the undiluted semen samples from day 0 to day 7 compared to diluted semen samples that remained with a high quality up to day 11. The undiluted semen exhibited a low DNA fragmentation index (DFI) during the first days and then a significant increase from day 7 up to day 21. This increase in the DFI coincided with the lowest levels of the other semen quality parameters. On the contrary, the samples diluted in the commercial extender showed very low levels of DNA fragmentation in all boars during the preservation period. When the evolution of DNA fragmentation was analysed in the undiluted samples, differences were found among boars. These differences were not shown in the samples diluted in the extender where the basal DFI remained stable during the 21 days. The main conclusion of this study was that some sperm extenders delay or partially prevent sperm DNA fragmentation.     

Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility

Extended semen doses from some boars used for AI have been shown to develop high levels of sperm DNA fragmentation during storage. Studies in other animals and humans have shown that if DNA damage is present in a certain percentage of the sperm cells the fertility potential of the semen sample is reduced. The objectives of the present study was to determine the relationship between sperm DNA fragmentation measured using the sperm chromatin structure assay (SCSA) in extended stored semen and field fertility in the boar. Three ejaculates from each of 145 boars were collected. Preparation of the semen doses included dilution with an EDTA extender and storage for up to 72 h post collection. The semen doses were assessed using flow cytometric methods for the percentage of viable sperm (PI/SYBR-14) and sperm DNA fragmentation (SCSA) at 0, 24, 48, and 72 h. A total of 3276 experimental inseminations in Danish breeding herds were conducted. The results showed that for 11 (7.6%) of the boars at least one of the three samples showed a value of DNA fragmentation index (DFI) above 20% within the storage period. Total number of piglets born (litter size) for Hampshire, Landrace and Danish Large White boars was, respectively, 0.5, 0.7 and 0.9 piglets smaller per litter when DFI values were above 2.1% as opposed to below this value. In conclusion the SCSA technique appears to be able to identify individuals with lower fertility with respect to litter size, and could in the future be implemented by the pig industry after a cost-benefit analysis.     

Effects of UV irradiation and hydrogen peroxide on DNA fragmentation, motility and fertilizing ability of rainbow trout (Oncorhynchus mykiss) spermatozoa

Preservation of DNA integrity is essential for protection of sperm quality. This study examined, with the use of comet assay, DNA fragmentation of rainbow trout (Oncorhynchus mykiss) spermatozoa subjected to UV irradiation (2,075 microW/cm(2), 0-15 min) or oxidative stress induced by hydrogen peroxide (0-20mM). Sperm motility and fertilizing ability were also measured. A dramatic increase in DNA fragmentation was recorded after 5 min UV irradiation but no significant changes in sperm motility were observed at this time. Longer irradiation resulted in a decrease in motility parameters and further increase of DNA fragmentation. UV irradiation caused a clear decrease in the percentage of eyed embryos and most of the embryos did not hatch. When highly diluted sperm suspensions (50,000-fold) were exposed to 0.1mM H(2)O(2) evident increase in DNA fragmentation was observed. On the other hand, when more concentrated sperm suspensions (diluted only 40-fold) were employed (in order to conduct motility and fertilization measurements at the same time) 1-20mM H(2)O(2) caused only moderate increase in DNA fragmentation and dose-dependent decline in sperm motility and fertilizing ability. This suggests that toxic effects of H(2)O(2) were primarily related to inhibition of sperm motility. Our results demonstrate that comet assay can be used for monitoring the effectiveness of fish sperm DNA inactivation by UV irradiation. Therefore, the comet assay together with sperm motility analysis can be applied in optimization works of gynogenetic procedures in fish. Lack of effectiveness of H(2)O(2) in inducing major DNA fragmentation suggests presence of mechanisms of antioxidative defense in rainbow trout spermatozoa.     

Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia

Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.