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A series of six experiments was conducted to determine the fundamental cryobiological properties of boar spermatozoa to develop optimal approaches for cryopreserving this important cell type. In the first experiments, boar spermatozoa samples were diluted in various osmolalities of experimental solutions (185-900 mOsmol kg-1) to provide hypo-, iso-, and hyperosmotic conditions. Equilibrium cell volumes (Expts 1 and 2) were measured after exposure for 3 min and the change in cell volume was measured over time using an electronic particle counter (Expt 3). The isosmotic cell volume was found to be 26.3 +/- 0.39 microns 3 (mean +/- SEM; n = 5). Over this range of osmolalities, boar spermatozoa behaved as linear osmometers (a linear volume versus 1/osm plot, r2 = 0.99) with an osmotically inactive cell fraction of 67.4 +/- 4.5%. The rate of water permeability (Lp) was determined to be 1.03 +/- 0.05 microns min-1 atm-1, which was consistent within and among donors (P > 0.130). A second series of experiments was performed to determine the effect of temperature and osmolality on boar sperm motility (Expt 4), and the effect of osmolality on the integrity of the sperm plasma membrane and its temperature dependence. Plasma membrane integrity was measured before and after boar spermatozoa were returned to an isosmolality (Expt 6). Motility was not affected at 30 degrees C, relative to that at room temperature, but was significantly decreased (P < 0.05) at 8 degrees C and 0 degree C (yielding a relative reduction to 85% and 35% of original motility, respectively; n = 6). Sperm motility was not significantly decreased (P > 0.05) until the osmolality reached 210 mOsmol kg-1, at which time motility began to decrease from 95% to 10% of the original value at 90 mOsmol kg-1. The integrity of the plasma membrane of boar spermatozoa was found to be dependent on temperature, donor and osmolality, decreasing significantly (P < 0.05) below room temperature, and below 185 mOsmol kg-1 (P < 0.05). There was no significant difference (P > 0.10) in the integrity of the plasma membrane of the samples before and after returning to 290 mOsmol kg-1, indicating that osmotic damage occurs during the initial change from isosmotic to hyposmotic media. These osmotic characteristics could be used to determine optimal conditions for cryopreservation of boar spermatozoa.
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PMID:Osmotic properties of boar spermatozoa and their relevance to cryopreservation. 869 39

Human spermatozoa from healthy donors (n = 7) were washed in Tyrode's medium containing 0.6 mg/ml freeze-dried egg yolk, filtered through glass wool and exposed to 0.5, 5, 50, 500 or 1000 microM hydrogen hexachloroplatinate. Viability, membrane integrity and the acrosome reaction were examined using trypan blue exclusion, hypo-osmotic swelling test and fluoresceinated Pisum sativum agglutinin, respectively, after incubation for 3 or 6 h at 37 degrees C. While sperm motility, viability and membrane integrity were not affected by the platinum compound after incubation for 3 h, the number of acrosome-reacted spermatozoa increased from 16.0 +/- 6.4% (control, mean +/- SEM) to 21.0 +/- 3.3 (0.5 microM), 22.3 +/- 4.3% (5 microM), 28.0 +/- 4.3% (50 microM, p < 0.01), 29.3 +/- 3.9% (500 microM, p < 0.01) and 43.9 +/- 7.4% (1 mM, p < 0.001); a further increase was detected after incubation for 6 h. However, the percentages of dead and immotile spermatozoa and those with defective membranes were also higher, suggesting that the acrosome reaction was caused by degenerative processes after long-term incubation. In conclusion, hydrogen hexachloroplatinate does not affect sperm motility, membrane integrity or viability, but it does induce the acrosome reaction after incubation for 3 h before cytotoxic effects are measurable. Similar effects of halide salts of platinum on receptor-mediated exocytosis have been described in other cells such as mast cells and basophils in vivo and in vitro.
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PMID:Hydrogen hexachloroplatinate induces the acrosome reaction in human spermatozoa. 871 48

A simple technique has been established for the purpose of characterizing heparin binding to boar sperm. Binding experiments were performed using [3H]heparin and extended boar semen. [3H]Heparin binding to boar sperm was effectively displaced by increasing concentrations of heparin. [3H]Heparin binding was linear at least between 50,000 and 10(6) spermatozoa and was stable for at least 120 min. Binding was sensitive to fucoidan, chondroitin sulfate B, chondroitin sulfate C, and chondroitin sulfate A, while keratan sulfate had only a marginal effect on binding. The [3H]heparin binding was saturable. Assuming a 13,000 M(r) for the [3H]heparin used, binding to boar spermatozoa showed an apparent equilibrium dissociation constant (Kd) of 23.6 +/- 2.5 nM and a maximum binding capacity (Bmax) of 6.0 +/- 1.1 pmol/10(6) spermatozoa (average values from 6 boars, means +/- SEM). Interejaculate variations in binding parameters were dependent on the male. Thus, with respect to Bmax variation, 4 of 6 boars studied exhibited an interejaculate coefficient of variation of less than 0.33 (0.09, 0.11, 0.11, and 0.29, respectively, for 3 consecutive ejaculates), while in the case of Kd interejaculate variation, only 2 of the 6 boars studied showed acceptable variation coefficients (0.16 and 0.28). No seasonal effect was observed in either of the binding parameters, with the variations following boar-specific patterns. Kd and Bmax intermean differences for different boars during the course of the study period (April-June) were not significant (p > 0.05). Correlations of mean boar binding parameters (Kd and Bmax) with conventional semen quality parameters showed a correlation between Bmax values and those of the osmotic resistance test (r = 0.8990, p < 0.01), normal acrosomes (r = 0.7946, p < 0.05), and bended tails (r = -0.8632, p < 0.02). Kd values were correlated with cytoplasmic distal droplet (r = -0.7992, p < 0.05). The glycosaminoglycans and polysulfated boar sperm binding sites reported in the present work should be regarded as binding sites until further studies elucidate their physiological role. The results obtained suggest that this technique be given a role as a research tool.
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PMID:[3H]heparin binding in boar spermatozoa: characterization and correlation with routine semen quality parameters. 887 1

Male C57B1/6 lacI transgenic mice were used to evaluate germ cell mutagenesis in vivo as part of a collaborative study. Groups of 10 mice were administered single intraperitoneal doses of ethylnitrosourea (ENU; 150 mg/kg), isopropyl methanesulfonate (IPMS; 200 mg/kg), methyl methanesulfonate (MMS; 40 mg/kg) or vehicle. Epididymal spermatozoa and testes were recovered 3 days later and DNA isolated subsequently from epididymal spermatozoa and seminiferous tubules were analyzed for lacI mutations. The mutant frequency in seminiferous tubules (average +/- SEM) increased significantly compared with untreated controls (7.2 +/- 0.7 x 10(-5) following treatment with ENU (11.7 +/- 0.8 x 10(-5), p = 0.003) or with IPMS (9.6 +/- 0.5 x 10(-5), p = 0.018) but not following treatment with MMS (8.1 +/- 0.8 x 10(-5), p = 0.213). Group mutant frequencies were not determined for epididymal spermatozoa from MMS- or IPMS-treated mice because of poor DNA recoveries. As another indicator of the genotoxicity of these alkylating agents, the frequencies of micronuclei were determined in the peripheral blood 48 h after carcinogen administration in the same transgenic mice. The micronuclei frequencies were elevated significantly (p < 0.05) by each treatment (IPMS: 1.0%; MMS: 0.94%) compared to vehicle controls (0.3%). In a separate experiment, 40 mg/kg ENU was previously found to increase the frequency of micronuclei in peripheral blood of lacI transgenic mice 48 h after treatment (3.2%; Gibson et al., 1995). These results demonstrate that the lacI transgenic mouse male germ cells are sensitive to some, but not all, mutagens under the conditions used in this experiment. Investigation of other experimental designs would offer additional perspective on the usefulness of this transgenic model for routine mutagenicity testing in germ cells.
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PMID:Evaluation of lacI mutation in germ cells and micronuclei in peripheral blood after treatment of male lacI transgenic mice with ethylnitrosourea, isopropylmethane sulfonate or methylmethane sulfonate. 905 80

The acrosome reaction is a prerequisite for zona pellucida penetration by mammalian spermatozoa. In some species, the sperm undergo the acrosome reaction before binding to the zona pellucida, and in other species only acrosome-intact sperm can initiate binding to the zona. In the present investigation, we addressed the question whether acrosome-intact or acrosome-reacted boar sperm initiate binding to the pig zona pellucida by studying the acrosomal status of sperm bound to zonae pellucidae. Our approach was to vary the percentage of acrosome-intact sperm in suspension by long preincubation before incubation with hemizonae for 1 min. We hypothesized that if only acrosome-intact sperm are able to initiate binding to the zona pellucida, the majority of the sperm on the zona surface would be acrosome-intact regardless of the percentage of acrosome-reacted sperm in suspension. Fluorescein isothiocyanate-conjugated peanut agglutinin (Arachis hypogaea; FITC-PNA) in combination with optical sectioning by confocal laser-scanning microscopy was used to study the acrosomal status of sperm bound to the hemizona. Electron microscope studies showed that the FITC-PNA binding site is mainly limited to the outer acrosomal membrane of boar sperm, thus validating the use of FITC-PNA as an accurate probe for studying boar sperm acrosome reaction. Over 90% of the sperm bound to a hemizona were acrosome-intact irrespective of whether the majority of sperm in the suspension were acrosome-intact, acrosome-reacting, or acrosome-reacted. There was a significant difference (Kruskal-Wallis test, p < 0.05) in the mean +/- SEM number of sperm bound to the outer side, inner side, and edge of a hemizona (48 +/- 8, 14 +/- 3, and 7 +/- 2; n = 58; respectively). The acrosomal status of sperm bound to the various surfaces of hemizonae was similar. Taking the respective zona pellucida surface area into consideration, it was calculated that an average of 1.9 +/- 0.3, 1.0 +/- 0.2, and 1.5 +/- 0.3 spermatozoa were bound per 1000 microm2 of outer side, inner side, and edge of a hemizona, respectively (mean +/- SEM, n = 38). These observations indicate that acrosome-intact boar spermatozoa initiate binding to the pig zona pellucida. A gradient of sperm binding sites also exists, decreasing from the outside to the inside of the zona pellucida.
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PMID:Acrosome-intact boar spermatozoa initiate binding to the homologous zona pellucida in vitro. 911 43

The characteristics of the glass wool filtration technique for the preparation of semen samples in an intracytoplasmic sperm injection programme (ICSI) were compared with a two-layer Percoll density gradient procedure. Half of each of 25 semen samples were prepared by each technique. The oocytes from each patient were randomly injected, half with spermatozoa prepared by glass wool filtration and half with Percoll-separated spermatozoa from the same semen sample. The percentage of recovered motile spermatozoa, the total motile sperm count, the percentage of morphologically normal forms, the fertilization rate, the cleavage rate and embryo quality obtained with both preparations were analysed. The 95% confidence intervals obtained through the Altman-Bland analysis showed that the percentage of motile spermatozoa recovered was 3.5-9.9% higher with the glass wool filtration technique, and this was highly correlated (r = 0.92, P = 0.0001) to the method. Similarly, the total number of spermatozoa available for ICSI was 0.18 x 10(6)-2.44 x 10(6) higher with the glass wool column and was highly correlated to the method (r = 0.956, P = 0.0001). Also, the percentage of normal forms was 1.25-3.31% higher after glass wool filtration but was poorly correlated to the method (r = 0.47, P = 0.017). Out of 100 metaphase II oocytes injected with glass wool-extracted spermatozoa, 77 fertilized and 72 cleaved. Out of 97 metaphase II oocytes injected with Percoll-selected spermatozoa, 71 fertilized and 69 cleaved. These results were not statistically different. The mean +/- SEM embryo quality score for the glass wool group (2.90 +/- 0.27) was the same as that for the Percoll group (2.80 +/- 0.24). No fertilization failures occurred and 11 patients (44% per oocyte retrieval) became pregnant. It was concluded that glass wool filtration for semen preparation in an ICSI programme offers higher sperm recovery and sperm morphology of superior quality than with the classic two-layer Percoll gradient method, without affecting the fertilization rate and embryo quality.
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PMID:Glass wool column filtration, an advantageous way of preparing semen samples for intracytoplasmic sperm injection: an auto-controlled randomized study. 913 Jul 52

Collection of semen using a modified ovine artificial vagina was attempted on 90 occasions from 25 Queensland koalas (Phascolarctos cinereus adustus). Complete ejaculates consisting of a copulatory plug and sperm fraction were collected on 36 occasions (40%) from 19 animals. Seventeen of the males produced a complete ejaculate on their first or second service. Failure to collect semen (38%) and the collection of partial ejaculates (14.5%) was attributed to lack of sexual interest, aggressive behaviour towards the female by the male, the use of non-compliant teaser females or a distinct dislike of the artificial vagina. Only a few ejaculates were contaminated with urine (4.5%) or obtained after ejaculation behaviour was terminated (3%). The mean (+/-SEM) values for the seminal characteristics of 19 complete ejaculates were: mass of copulatory plug fraction 0.78 +/- 0.10 g, sperm fraction volume 0.73 +/- 0.10 ml, sperm concentration 165.1 +/- 26.7 x 10(6) ml-1, pH of sperm fraction 6.7 +/- 0.2, osmolarity of sperm fraction 315.0 +/- 5.4 mOsm, percentage forward motility 70.7 +/- 1.8%, rate of sperm movement 4.0 +/- 0.1 and percentage of abnormal spermatozoa 26.9 +/- 2.5%. Percentages of sperm head morphotypes and tail abnormalities were documented. Although the artificial vagina technique is limited by the need for access to oestrous females, the procedure described has been shown to be a simple, reliable method of collecting semen from captive koalas.
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PMID:Characteristics of koala (Phascolarctos cinereus adustus) semen collected by artificial vagina. 915 42

Hydrosalpinges were created to collect adequate volumes of fluid during pre-, peri- and postovulatory intervals from the ampulla, ampullary-isthmic junction and the isthmic-utero-tubal junction of the oviducts from Large White gilts that had exhibited at least two natural oestrous cycles. The accumulated fluids, follicular fluid and Butschwiler's medium were compared for their effects on various parameters of boar sperm motility using the CellSoft, computer-assisted, digital image analysis system. Sperm velocity (micron s-1 +/- SEM) was significantly higher (P < 0.05) in follicular fluid (84 +/- 3; n = 5) than in fluids from the ampulla during peri- and early postovulatory intervals, and from the isthmic-utero-tubal junction during pre- and early postovulatory intervals. It was also higher (P < 0.05) than in the fluid from the ampullary-isthmic junction during pre- and early postovulatory intervals; however, sperm velocity in follicular fluid was not significantly different from that in the periovulatory fluid from the ampullary-isthmic junction. The mean lateral head displacement (ALHmean) of spermatozoa was significantly greater in follicular fluid (3.9 +/- 0.3 microns; n = 5) than in fluid from the ampulla during peri- and early postovulatory intervals and from the isthmic-utero-tubal junction during pre- and early postovulatory intervals, and was also higher (P < 0.05) than in fluid from the ampullary-isthmic junction during the preovulatory period, but was not different from the peri- and postovulatory ampullary-isthmic junction fluids. The proportion of spermatozoa exhibiting circular motion was significantly higher (P < 0.05) in the periovulatory fluid from the ampullary-isthmic junction (24 +/- 3%) compared with fluids obtained during preovulatory and early postovulatory periods. Follicular fluid had no effect on the proportion of spermatozoa exhibiting circular motion. The average radius of sperm movement in circular trajectories was higher in follicular fluid than in the periovulatory fluids from the ampulla and ampullary-isthmic junction (P < 0.05). In hydrosalpingeal fluids collected 2-5 days after ovulation, the average radius of movement was greater in the ampulla fluid and ampullary-isthmic junction fluid than in fluid from the isthmic-utero-tubal junction (P < 0.05). These results demonstrate that follicular fluid and oviductal fluids have considerable influences on boar sperm motility. Furthermore, the immediate effect of periovulatory ampullary-isthmic junction fluid in increasing the percentage of spermatozoa swimming in circles (hyperactivated) is relevant, since it is at this time and within this region that fertilization occurs.
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PMID:Motility of spermatozoa in hydrosalpingeal and follicular fluid of pigs. 922 60

Recombinant human ZP3 (rhuZP3) generated by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA was used to study the acrosome reaction (AR) and intracellular calcium fluxes in capacitated human spermatozoa. Conditioned medium containing rhuZP3 significantly induced the AR (P < or = 0.005) in 59.4 +/- 4.7% of spermatozoa (control = 8.5 +/- 3.1%) and caused complete acrosomal loss in a further 17.2 +/- 3.8% of cells (control = 3.7 +/- 0.7%; mean +/- SEM, n = 5). Sperm motility was not affected and acrosomal exocytosis in response to rhuZP3 was also shown to be time-dependent. Basal concentrations of sperm intracellular calcium were measured (82 +/- 7 nM; mean +/- SEM, n = 9). A transient increase in intracellular calcium (typically up to 400-450 nM) occurred within 1 min of rhuZP3 addition and was followed by sustained lower values of calcium (200-400 nM). These responses were dependent on the amount of rhuZP3. This is the first report of zona protein-induced changes in intracellular calcium levels in human spermatozoa. The results support the premise that ZP3 is an agonist of the human sperm AR and that rhuZP3 generated in a eukaryotic cell is effective in this respect.
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PMID:Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa. 923 70

The objective of this study was to examine the effects of reactive oxygen species (ROS) on bovine sperm function and on the developmental competence of in vitro-matured bovine oocytes. In a first series of experiments, spermatozoa were exposed to ROS generated through the use of the hypoxanthine-xanthine oxidase system +/- catalase prior to the conduct of in vitro fertilization (IVF). Reactive oxygen species exposure reduced significantly (P < 0.001) the rates of oocyte penetration (control: 56% +/- 4 SEM; ROS: 16 +/- 2-23% +/- 7 SEM), and this effect was reversed by adding catalase (ROS+catalase: 67% +/- 0.3 SEM). During IVF, addition of superoxide dismutase (SOD: 1, 10, or 100 U/ml) had no effect on penetration rates. However, increasing concentrations of catalase (0.1 or 1 mg/ml) reduced these rates significantly (control: 70% +/- 3 SEM; treated: 45% +/- 5 and 1% +/- 1 SEM; P < 0.001). In a second series of experiments, when oocytes were matured in vitro in the presence of exogenous antioxidants (SOD: 10, 100, or 1000 U/ml; beta-mercaptoethanol: 0.01, 0.1, or 0.5 mM; ascorbic acid: 0.05 mg/ml), the developmental competence of the oocytes after IVF was not significantly improved. On the other hand, presumed production of ROS using the hypoxanthine-xanthine system at the beginning of the in vitro maturation period did improve subsequent developmental competence of the oocytes under some conditions and when catalase was present (control: 14% +/- 4 SEM and treated: 23% +/- 9 and 27% +/- 8 SEM; P < 0.05). These observations demonstrate that ROS may be beneficial to gamete function under specific conditions.
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PMID:The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation. 928 60


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