Nuclear Decondensation Research Articles

227 THE EFFECTS OF SEXED SEMEN ON EMBRYONIC DEVELOPMENT TO THE BLASTOCYST STAGE

Sexed semen (SS) exhibits approximately 80% of the fertilizing ability of conventional semen (CS), and studies have shown that this continues through the 8-cell stage of bovine embryo development. At the time of this study, no information could be found that, when used for IVF and intracytoplasmic sperm injection (ICSI) development, had been carried to the blastocyst stage. In addition, questions have arisen regarding which of the measured sperm parameters are responsible for the difference between the SS and CS and contribute to this decline in fertility. The goals of this project were to evaluate the effects of using sexed sperm as it relates to embryonic development and to determine if any of the differences in sperm parameters affect embryonic development. A preliminary project evaluated SS and CS from 5 bulls for IVF and ICSI. One bull was selected to provide the sperm (both SS and CS) for the trial, and 1752 oocytes were assigned to either IVF or ICSI. The SS and CS were divided among the available oocytes used for IVF and ICSI. Straws were thawed for 30 s at 37°C, and sperm were then evaluated for motility (provided by CASA, SpermVision, MiniTube of America, Verona, WI), morphology, acrosomal integrity (Coomassie and Pope stains), viability, and nuclear decondensation (SYBR Green and HALO). Results for SS v. CS were as follows: motility, 8 v. 26%; viability, 40.6 v. 30%; nuclear decondensation, 40 v. 30%; normal morphology and acrosomal integrity, no differences. Oocytes were obtained from Applied Reproductive Technologies, LLC (Madison, WI). The fertilization rate was consistently lower (Table 1) for both IVF and ICSI when SS were used (Z = 3.65; P = 0.0003), and there was no evidence that this decline in fertilization rate differed for the 2 methods (Z = 0.18; P = 0.86). Nor was there any evidence that the method affected the fertilization rate in general (Z = 0.75; P = 0.45). Thus, the difference was specific for fertilization rate and had no effect on Day 3 cells or Day 7 blastocysts. A higher fertility rate using ICSI would have indicated that a surface membrane factor may have been decreasing the fertility rate with SS because of the elimination of binding factors associated with ICSI. Thus, it may not be the sperm surface membrane that is distorted in the sexing procedure, but likely the integrity of the spermal DNA, as indicated by the increased nuclear decondensation of SS. Table 1.Comparison of sexed sperm with conventional sperm when used for IVF and intracytoplasmic sperm injection (ICSI) Partial funding for this project was made available by the California State University Agricultural Research Institute (ARI). Appreciation is extended to Sexing Technologies, Inc. (Navasota, TX) for donating the semen.

Sperm of the wasted mutant are wasted when females utilize the stored sperm in Drosophila melanogaster

Females of many animal species store sperm after copulation for use in fertilization, but the mechanisms controlling sperm storage and utilization are largely unknown. Here we describe a novel male sterile mutation of Drosophila melanogaster, wasted (wst), which shows defects in various processes of sperm utilization. The sperm of wst mutant males are stored like those of wild-type males in the female sperm storage organs, the spermathecae and seminal receptacles, after copulation and are released at each ovulation. However, an average of thirteen times more wst sperm than wild type sperm are released at each ovulation, resulting in rapid loss of sperm stored in seminal receptacles within a few days after copulation. wst sperm can enter eggs efficiently at 5 hr after copulation, but the efficiency of sperm entry decreases significantly by 24 hr after copulation, suggesting that wst sperm lose their ability to enter eggs during storage. Furthermore, wst sperm fail to undergo nuclear decondensation, which prevents the process of fertilization even when sperm enter eggs. Our results indicate that the wst gene is essential for independent processes in the utilization of stored sperm; namely, regulation of sperm release from female storage organs, maintenance of sperm efficiency for entry into eggs, and formation of the male pronucleus in the egg at fertilization.

The disruption in actin-perinuclear theca interactions are related with changes induced by cryopreservation observed on sperm chromatin nuclear decondensation of boar semen

The perinuclear theca (PT) is a cytoskeletal structure that surrounds the mammal sperm nucleus which must be disrupted once the sperm has penetrated the oocyte to permit normal chromatin decondensation and formation of male pronucleus. F-actin is a thermo sensitive protein found in the equatorial segment which is involved in the stability of PT. It has been reported that cryopreservation induces alterations in nuclear decondensation of spermatozoa, which have been interpreted as an over condensation. The aims of the present study were identified the presence of changes in sperm sPT integrity of frozen–thawed boar spermatozoa and its effect in sperm nuclei decondensation; and whether changes in the actin cytoskeleton are involved using an in vitro model to test probably differences in a chemical decondensation (DTT/heparin) between fresh (FS) and frozen–thawed (TS) spermatozoa. Results showed an increase on sPT damage in TS ( P < 0.001), and significant changes in sperm chromatin nuclear decondensation ( P < 0.05). In same way differences on the swelling degree was found assessed by measures in equatorial region of head sperm ( P < 0.05). Evaluation with rodamine-labeled actin (0.2 μM) showed two different patterns with differences in percentages before and after cryopreservation ( P < 0.001). F-actin stabilization constrained the equatorial segment of FS while this was not observed in TS. The data showed that the presence of early changes in sPT integrity and changes in the F-actin localization on TS may suggest the participation in F-actin in decondensation process and probably that the disruption of actin-PT interaction during freezing–thawing process could have far-reaching consequences for the subsequent fertility of spermatozoa.

255 SPERM NUCLEAR DECONDENSATION ABILITY OF IN VITRO MATURED CANINE OOCYTES

The sperm chromatin decondensation occurs when a spermatozoon enters into an oocyte during fertilization, and the effectiveness of this process is connected to the grade of oocyte cytoplasmic maturation. In this study chromatin sperm decondensation was evaluated after IVF of canine oocytes matured in vitro (IVM), comparing different durations of maturation. Cumulus–oocytes complexes (COC) for IVM were obtained from bitch ovaries after ovariohysterectomy, selecting those COC with compact cumulus cells and a homogeneous dark cytoplasm. The COC were matured in vitro for 0, 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM HEPES, 10% FCS, 0.25 mM pyruvate, 10 IU mL–1 of hCG, 300 IU mL–1 of penicillin, and 20 mg mL–1 of streptomycin at 38.5°C and 5% CO2. Fresh ejaculates from 3 adult dogs were centrifuged, and the sperm pellet was resuspended in fert-TALP medium. In each replicate, 100-μL fert-TALP drops containing 10 to 12 IVM oocytes after each culture time were co-culture with 2.5 × 106 spermatozoa mL–1 for 24 h under culture conditions. Soon after co-culture, all oocytes were denuded from cumulus cells and fixed in 3% paraformaldehyde. The nuclear stage of the oocytes and the appearance of the sperm nucleus were determined by 4′,6-diamidino-2-phenylindole staining under a fluorescence inverted microscope. For each treatment, at least 4 replicates were performed, and the data were compared statistically by chi-square test, using InfoStat Professional Program. A total of 800 oocytes were evaluated, and the percentages of oocytes with sperm penetration were 58% (138/238), 61% (108/177), 72% (118/165), and 70% (153/220) at 0, 48, 72, and 96 h of IVM, respectively. The percentage of sperm nuclear decondensation at each time point significantly increased up to 72 h of culture, showing 12, 34, 81, and 85% of sperm nuclei deconsated, respectively. The percentages of nuclear maturation also increased (P ≤ 0.05) with time, showing 0, 8, 20, and 27% of oocytes at second metaphase (MII) stage at 0, 48, 72, and 96 h of culture. The percentage of MII stage was much lower than that of chromatin decondensation in all maturing groups. These results suggest that canine oocytes matured in vitro are able to decondense the sperm chromatin during IVF, and this ability increases up to 72 h of culture. Nevertheless, cytoplasmic maturation, as evaluated by sperm chromatin decondensation, in canine oocytes matured in vitro may not be completely connected with nuclear development. This work was supported by Grant FONDECYT 1080618.

Gold nanoparticles disturb nuclear chromatin decondensation in mouse sperm in vitro

The effect of gold nanoparticles on mouse epididymal sperm has been studied using the model system of nuclear chromatin decondensation in vitro. It is shown that the treatment of gametes, preliminary membrane-freed by sodium dodecyl sulfate, in the mediums containing gold nanoparticles (with diameter ∼2.5 nm) in concentrations 1.0 × 1015 or 0.5 × 1015 particles/ml and following incubation in dithiothreitol solution (DTT) resulted in failure of chromatin decondensation process and nucleus structure. We conclude that gold nanoparticles possess spermatotoxicity. The mechanism of cytotoxic effect of gold nanoparticles may be related with their interaction with molecules of double-helix DNA. The model system studied in this research is applicable for further investigations of cytotoxic effects of nanoparticles of different origin and made of different metals.

Kinetics of human male pronuclear development in a heterologous ICSI model

To evaluate human sperm nuclear chromatin decondensation in a heterologous ICSI system using hamster ova injected with human sperm. Frozen hamster oocytes were injected with Triton X-100 treated sperm and fixed at different time points post ICSI. Oocytes injected with non-treated sperm served as controls. Male pronuclear decondensation was evaluated after staining with DAPI. Sperm cells with partially destroyed membranes and depletion of the acrosome decondense more rapidly and to a greater extent than membrane/acrosome intact cells. Marked variability in pronuclear size was observed for any time point post ICSI, which most probably reflects the heterogeneity in the mature human sperm population. Remodeling of male gamete nuclei in this heterologous ICSI mimics events that occur during natural fertilization in humans and therefore this approach may be used for studies of human sperm chromosomes transformations.

Abstract C66: A novel technique to quantify condensation/decondensation status in the nuclear subdomains by QD-FRET

Abstract Recent studies have reported that control of nuclear decondensation at specific nuclear subdomains (i.e. euchromatin) is highly desired for the efficient transfection efficiency. Here, we have established a novel technology for the imaging of nuclear condensation/decondensation status in the nuclear subdomains (i.e. heterochromatin and euchromatin) by based on the three key technologies. One is imaging of fluorescence resonance energy transfer (FRET) between quantum dots (QDs)-labeled plasmid DNA (pDNA) as a donor, and acceptor-labeled polycation, which can monitor a specific molecular interactions at the nanometre scale. The second technology is a multilayered lipid envelope-type nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA—polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are designed for nuclear delivery via a stepwise fusion. It is required to evaluate the nuclear dispositions in relation to the transgene expression, to avoid a situation that intracellular trafficking processes rate-limit transfection activity. The last one is confocal image-assisted 3-dimensionally integrated quantification (CIDIQ) method. In this method, fluorophore-labeled pDNA was transfected in HeLa cell line, and then intracellular distribution was distinguished by staining organelle. Z-series of confocal images were captured by confocal laser scanning microscopy (CLSM), and thereafter, pDNA was quantified based on the pixel area of the signals derived from the fluorophore on pDNA in nuclear subdomains (heterochromatin and euchromatin). As a model of the analysis, QD-labeled DNA was condensed with reductive environment-dependently cleavable polyrotaxane (DMAE-ss-PRX), an artificial condenser, and encapsulated in T-MEND. As a result, transfection activity depends on the number of cationic DMAE of cyclodextrin (α-CD), in conjunction with a drastic change in decondensation efficiency in euchromatin region. These data collectively indicate that this imaging technology is highly advantageous to clarify the dynamics of plasmid DNA in post-nuclear delivery process of cancer cells. Citation Information: Cancer Res 2009;69(23 Suppl):C66.

Possible participation of calmodulin in the decondensation of nuclei isolated from guinea pig spermatozoa

The guinea pig spermatozoid nucleus contains actin, myosin, spectrin and cytokeratin. Also, it has been reported that phalloidin and/or 2,3-butanedione monoxime retard the sperm nuclear decondensation caused by heparin, suggesting a role for F-actin and myosin in nuclear stability. The presence of an F-actin/myosin dynamic system in these nuclei led us to search for proteins usually related to this system. In guinea pig sperm nuclei we detected calmodulin, F-actin, the myosin light chain and an actin-myosin complex. To define whether calmodulin participates in nuclear-dynamics, the effect of the calmodulin antagonists W5, W7 and calmidazolium was tested on the decondensation of nuclei promoted by either heparin or by a Xenopus laevis egg extract. All antagonists inhibited both the heparin- and the X. laevis egg extract-mediated nuclear decondensation. Heparin-mediated decondensation was faster and led to loss of nuclei. The X. laevis egg extract-promoted decondensation was slower and did not result in loss of the decondensed nuclei. It is suggested that in guinea pig sperm calmodulin participates in the nuclear decondensation process.

Improvement in buffalo ( Bubalus bubalis) spermatozoa functional parameters and fertility in vitro: Effect of insulin-like growth factor-I

The aim of the current study was to assess the effect of insulin-like growth factor-I (IGF-I; 100 ng/mL) on buffalo ( Bubalus Bubalis) sperm functional parameters related to in vitro fertilization. The acrosin activity (the mean diameter of halo formation in micrometers) was significantly higher in the IGF-I group (14.17 ± 1.51) compared with that in the control group (9.50 ± 0.36) at 2 h incubation. The mitochondrial membrane potential (per cent) was significantly higher in the IGF-I group compared with that in the control group at 30 min (33.27 ± 2.62 vs. 26.71 ± 1.02), 60 min (24.24 ± 3.45 vs. 18.77 ± 2.09), and 90 min (22.86 ± 3.02 vs. 16.92 ± 1.24) incubation. The percentage of spermatozoa positive for sperm nuclear chromatin decondensation (NCD) differed significantly between the groups at 90 and 120 min incubation. The comet length was significantly lower in the IGF-I group compared with that in the control group at 2 h incubation. The percentage of fragmented DNA in the tail did not differ significantly between the groups at 2 h incubation. The percentage of acrosomal-reacted spermatozoa did not differ significantly between the IGF-I and the control groups at 4 h (41.12 ± 6.44 vs. 43.53 ± 5.05) incubation. The cleavage rate (per cent) was significantly higher in the IGF-I–treated group (56.73 ± 3.70) compared with that in the control group (44.85 ± 2.15). The current study suggests that the addition of IGF-I prevents deterioration of sperm functional parameters and fertility.

Comparative study on the spermicidal activity of organic solvent fractions from hydroethanolic extracts of Achyranthes aspera and Stephania hernandifolia in human and rat sperm

Background This study was conducted to determine the most effective fraction of the hydroethanolic (water:ethanol, 1:1) extracts of Stephania hernandifolia leaves and Achyranthes aspera roots (in a composite manner at a ratio of 1:3, respectively) that will provide maximum spermicidal activity in human and rat spermatozoa out of five different ratios (1:1, 1:3, 1:7, 3:1 and 7:1) that have been studied in pilot experiments. Study Design n-Hexane, chloroform and ethyl acetate fractions of the hydroethanolic (1:1) extracts of S. hernandifolia and A. aspera were mixed at 1:3. Different concentrations were tested for sperm immobilization, sperm viability, acrosome status, 5′-nucleotidase activity and nuclear chromatin decondensation using human and rat spermatozoa for the selection of the most effective concentration. Results Out of three fractions of the hydroethanolic (1:1) extracts of the said plants, the n-hexane fraction was most effective, and the chloroform fraction exhibited minimum activity for this purpose. At a concentration of 0.1 g/mL hexane fraction, all sperm of the human sample were immobilized immediately (within 20 s). In case of the rat sample, all epididymal spermatozoa were immobilized immediately (within 20 s) by treatment with hexane fraction at a concentration of 0.004 g/mL. All human sperm were found to be nonviable within 20 min. The activity of acrosome enzymes was reduced, and significant release of 5′-nucleotidase (a plasma membrane marker) into the surrounding medium was noted after treatment with 0.1 g/mL hexane fraction, indicating that the hexane fraction affected the cytoarchitecture of the sperm plasma membrane. The maximum number of human sperm failed to decondense when treated with 0.1 g/mL hexane fraction, and sperm motility was also irreversible. The hexane fraction was tested in rats as vaginal contraceptive and showed 100% efficacy, indicating its potential for development as vaginal contraceptive. Conclusion The findings indicate that, among the different fractions, the hexane fraction of the hydroethanolic extracts of the two plants produced the most effective spermicidal activity and can be considered as vaginal contraceptive.

Vesicular stomatitis virus inhibits mitotic progression and triggers cell death

Vesicular stomatitis virus (VSV) infects and kills a wide range of cell types; however, the mechanisms involved in VSV-mediated cell death are not fully understood. Here we show that VSV infection interferes with mitotic progression, resulting in cell death. This effect requires the interaction of VSV matrix (M) protein with the Rae1-Nup98 complex in mitosis, which is associated with a subset of ribonucleoproteins (RNPs). VSV displaced Rae1 from spindle poles, caused spindle abnormalities and triggered substantial cell death during metaphase. These effects were attenuated in cells infected with VSV expressing a mutant M protein that does not bind efficiently to the Rae1-Nup98-RNP complex. In cells that progressed to late mitosis, M protein prevented proper nuclear formation and chromatin decondensation. VSV is an oncolytic (anti-tumour) agent as it preferentially replicates and kills tumour cells. As tumour cells have a high mitotic index, VSV-mediated mitotic cell death probably contributes to its oncolytic activity.

Study of nuclear decondensation of the rat spermatozoa by reducing agents during epididymal transit.

Chromatin packing, mostly due to disulfide bond formation, is one important step in epididymal sperm maturation. In this study the in vitro effect of a reducing agent such as thioglycolate (Tg) was tested. Thioglycolate was assayed on rat sperm obtained from caput, corpus and cauda epididymides. Changes were verified via light microscopy (and area measurements) and both transmission and scanning electron microscopy using standard techniques. Due to the low molecular weight of Tg, it does not require detergent to enter sperm. The nuclear decondensation elicited by Tg and its effect on sperm surface, differ from caput to cauda, although the final nuclear area is not significantly different. The pattern of sperm nuclear decondensation suggests that it starts at the caudal segment of the head, perhaps due to the abundance of nuclear pores in this region. Under the experimental conditions described below the perforatorium was not affected. Thus, the nature and role in the rat sperm of the perforatorium deserves more detailed analysis.

Association of human sperm nuclear decondensation and in vitro penetration ability.

Sperm nuclear decondensation is an integral step in fertilization which leads to the formation of the male pronucleus. The association between the in vitro spontaneous nuclear decondensation of human sperm and its fertilizing ability was studied in infertile male patients. The ability of sperm to fertilize an egg using the discontinuous two-layer Percoll method was significantly correlated to the percentage of decondensed swollen head (r = 0.43; P less than 0.005). The fertilizing ability of sperm processed with Test-Yolk buffer was correlated with the percentage of sperm at the fully decondensed stalk stage (r = 0.51; P less than 0.05). There were insignificant correlations for the whole-wash centrifugation, cryopreserved-thawed and swim-up methods. Samples of sperm that were positive (greater than 0% fertilization) in the sperm penetration assay had a higher percentage of decondensed sperm heads (66.7% vs. 20.6%) after Percoll wash or whole-wash centrifugation (60.5% vs. 44.3%) treatments compared with samples with no fertilization. Treatments that included Test-Yolk resulted in high percentages of decondensed swollen heads. The results suggest a positive association between sperm nuclear decondensation and the fertilizing ability of sperm, and affirm the importance of nuclear decondensation to the study of fertilization events.

Sperm head ultramorphology and chromatin stability of males with unexplained infertility who fail to fertilize normal human ova in vitro.

An in vitro nuclear chromatin decondensation test, and quantitative nuclear ultramorphology analysis, were performed on 18 males judged to be infertile, by two failures in in vitro fertilization, and 16 fertile males. These two clinical groups only differed significantly in (1) the direction of their chromatin stability change, which took place 30-120 min post-ejaculation while stored in the seminal plasma, and (2) in the incidence of the hypoelongated sperm-head. Generally, the fertile male group exhibited positive chromatin stability change after prolonged storage, and low incidence of hypoelongated sperm heads, and vice versa in the unexplained infertile group. When the nuclear chromatin decondensation test and quantitative ultramorphology analysis were performed in step-wise fashion, it was possible to correctly classify 94% of the fertile cases with 6% of false-negative, and 89% of the unexplained infertile cases with 11% of false-positive. Therefore, it appears that these tests might be of benefit clinically for identifying functional properties of sperm-cells in unexplained infertile males, which cannot be detected by routine semen analysis.

Ultrastructure of human spermatozoa in the presence of the spermicide nonoxinol-9 and a vaginal contraceptive containing nonoxinol-9.

The effect of nonoxinol-9, a potent non-ionic, surface-active spermicide and of the vaginal contraceptive Patentex oval containing this spermicide, on human sperm ultrastructure were studied by transmission electron microscopy. Pooled human semen was mixed either with nonoxinol-9 to give a final concentration of 1.25 or 0.05% or with Patentex oval (final concentration of the spermicide nonoxinol-9 1.25%). Saline containing no spermicidal agent served as control. After incubation at 37 degrees C for 10 min., the washed sperm pellet was processed for electron microscopy. Human spermatozoa from control experiments showed a normal ultrastructure whereas significant changes occurred after the addition of nonoxinol-9 or Patentex oval to human semen. The plasma membrane and the acrosomal membrane complex were removed. The nuclear membrane was swollen, showed breaks, discontinuities and an enlarged submembranous space. Different stages of nuclear chromatin decondensation were observed. In the middle piece, the bi-laminar membrane of the mitochondria appeared as a mono-layer membrane. The normal cristae were destroyed, the mitochondriae were empty or contained fine granular material. The observed irreversible severe membrane alterations cause an immediate devitalization of the spermatozoa and support the effectiveness of the spermicide nonoxinol-9 and the vaginal contraceptive Patentex oval.

Metallothionein binding zinc inhibits nuclear chromatin decondensation of human spermatozoa.

Nuclear chromatin decondensation (NCD) of the human spermatozoa was induced by 1% sodium dodecyl sulphate (SDS). NCD of the spermatozoa induced in healthy and fertile men was significantly stronger and at higher rates than that in infertile men. In 1% SDS with 6 mM zinc chelating EDTA, metallothionein (MT) significantly enhanced NCD in a healthy man. In contrast MT alone significantly inhibited sperm NCD. Sperm NCD rate induced by 1% SDS in 10 infertile men was significantly inhibited by adding 75 or 750 micrograms ml-1 of MT. By adding 1.5 mM zinc, MT at concentrations of 0.75, 7.5, 75, or 750 micrograms ml-1, enhanced the inhibitory effect of 1.5 mM zinc. This suggested that thiols in the MT could, when liberated from zinc by zinc-chelating EDTA, induce sperm decondensation by cleaving stabilizing S-S bridges and that zinc bound to MT could exert a chromatin stabilizing effect mediated by the zinc dependent type of chromatin stability. The present study suggested that zinc bound to MT, which is secreted mainly from the prostate gland, is one factor that contributes to the chromatin stabilizing effect of human prostatic fluid.

Relationship between the nuclear morphology of the sperm of 10 bulls and their fertility

The relationships between the fertility and nuclear morphology, chromatin maturity and chromatin condensation of the sperm of three bulls with a calving rate over a year of more than 65...

Estrogenic Compounds Induce Oxidative Stress in Human Spermatozoa: Implications for DNA Integrity and Fertilization Success.

Unusually high levels of DNA damage in the male germ line are, unfortunately, characteristic of our species. A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the mechanisms responsible for creating this stress have not yet been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure to estrogenic xenobiotics. We have undertaken a detailed analysis of the ability of these compounds to trigger oxidative stress and/or DNA damage in human spermatozoa. This investigation was completed using a range of techniques validated for use in these highly specialized cells. DNA integrity was assessed using the Comet and TUNEL assays, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens (reactive metabolites of estrogen), quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity in human spermatozoa while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17β-estradiol, nonylphenol, bisphenol A and 2,3- dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation and potentially preventing fertilization. These findings raise fundamental questions about the importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line.

The presence of heparan sulfate in the mammalian oocyte provides a clue to human sperm nuclear decondensation in vivo

Previous results from our laboratory have led us to propose heparan sulfate (HS) as a putative protamine acceptor during human sperm decondensation in vivo. The aim of this paper was to investigate the presence of glycosaminoglycans in the mammalian oocyte in an effort to better support this contention. Two experimental approaches are used: oocyte labeling to identify the presence of HS and analysis of sperm decondensing ability of fresh oocytes in the presence or absence of specific glycosidases. Staining of mouse zona-intact oocytes with the fluorescent cationic dye, Rubipy, at pH 1.5 allowed for the detection of sulfate residues in the ooplasm by confocal microscopy. HS was detected in the ooplasm by immunocytochemistry. A sperm decondensation microassay using heparin and glutathione was successfully developed. The same level of sperm decondensation could be attained when heparin was replaced by mouse zona-free oocytes. Addition of heparinase to the oocyte/glutathione mixture significantly reduced sperm decondensation (P = 0.0159), while there was no effect following addition of either chondroitinase ABC or hyaluronidase. The results presented in this paper demonstrate for the first time that HS is present in the mammalian oocyte and show that HS is necessary for fresh oocytes to express their sperm decondensing ability in vitro.

Impact of estrogenic compounds on DNA integrity in human spermatozoa: Evidence for cross-linking and redox cycling activities

A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (β-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17β-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male infertility is warranted.