Showing papers on "Sperm plasma membrane published in 2008"

Mammalian sperm metabolism: oxygen and sugar, friend and foe

Abstract: Mammalian spermatozoa expend energy, generated as intracellular ATP, largely on motility. If the sperm cell cannot swim by use of its flagellar motion, it cannot fertilize the egg. Studies of the means by which this energy is generated span a period of six decades. This review gives an overview of these studies, which demonstrate that both mitochondrial oxidative phosphorylation, for which oxygen is friend, and glycolysis, for which sugar is friend, can provide the energy, independent of one another. In mouse sperm, glycolysis appears to be the dominant pathway; in bull sperm, oxidative phosphorylation is the predominant pathway. In the case of bull sperm, the high activity of the glycolytic pathway would maintain the intracellular pH too low to allow sperm capacitation; here sugar is enemy. The cow's oviduct has very low glucose concentration, thus allowing capacitation to go forward. The choice of the pathway of energy generation in vivo is set by the conditions in the oviduct of the conspecific female. The phospholipids of the sperm plasma membrane have a high content of polyunsaturated fatty acids represented in their acyl moieties, rendering them highly susceptible to lipid peroxidation; in this case oxygen is enemy. But the susceptibility of the sperm membrane to lethal damage by lipid peroxidation allows the female oviduct to dispose of sperm that have overstayed their welcome, and so keep in balance sperm access to the egg and sperm removal once this has occurred.

Characterization of Sperm Plasma Membrane Properties after Cholesterol Modification: Consequences for Cryopreservation of Rainbow Trout Spermatozoa

Abstract: During cryopreservation, the cell plasma membrane faces severe perils, including lipid phase separation, solute effects, and osmotic stresses associated with ice crystallization. How the initial biophysical properties of the plasma membrane can be modulated before cryopreservation in order to influence cellular resistance to the freeze-thaw stress is addressed in this study. Rainbow trout (Oncorhynchus mykiss) spermatozoa were chosen because the lack of an acrosome in this species suppresses potential interactions of cryopreservation with capacitation. Methyl-beta cyclodextrin-induced modulation of membrane cholesterol revealed the presence of a significant cholesterol exchangeable pool in the trout sperm plasma membrane, as membrane cholesterol content could be halved or doubled with respect to the basic composition of the cell without impairing fresh sperm motility and fertilizing ability. Biophysical properties of the sperm plasma membrane were affected by cholesterol changes: membrane resistance to a hypo-osmotic stress increased linearly with membrane cholesterol whereas membrane fluidity, assessed with DPH (1,6-diphenyl-1,3,5-hexatriene) and with several spin-labeled analogues of membrane lipids, decreased. Phosphatidyl serine translocation between the bilayers was slowed at high cholesterol content. The increased cohesion of fresh trout sperm plasma membrane as cholesterol increased did not improve the fertilizing ability of frozen-thawed sperm whereas the lowest cholesterol contents impaired this parameter of sperm quality. Our study demonstrated that cholesterol induced a stabilization of the plasma membrane in rainbow trout spermatozoa, but this stabilization before cryopreservation brought no improvement to the poor freezability of this cell.

Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model.

Abstract: Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be approximately 10-70 and approximately 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.

Effect of docosahexaenoic acid on quality of cryopreserved boar semen in different breeds.

Abstract: During the cryopreservation process, the level of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), in the sperm plasma membrane decreases significantly because of lipid peroxidation, which may contribute to sperm loss quality (i.e. fertility) of frozen-thawed semen. The aim of this study was to investigate the effect of supplementation of DHA (fish oil) in freezing extender II on frozen-thawed semen quality. Semen from 20 boars of proven motility and morphology, were used in this study. Boar semen was split into four groups, in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various levels of fish oil to reach DHA level of 1X (group I, control, no added fish oil), 6X (group II), 12X (group III) and 18X (group IV). Semen solutions were frozen by using a controlled rate freezer. After cryopreservation, frozen semen was thawed and evaluated for progressive motility, viability by using SYBR-14/Ethidiumhomodimer-1 (EthD-1) staining and acrosome integrity by using FITC-PNA/EthD-1 staining. There was a significantly higher (p < 0.001) percentage of progressive motility, viability and acrosome integrity in DHA (fish oil) supplemented groups than control group. Generally, there seemed to be a dose-dependent effect of DHA, with the highest percentage of progressive motility, viability and acrosome integrity in group-III. In conclusion, supplementation of the LEY extender with DHA by adding fish oil was effective for freezing boar semen as it resulted in higher post-thaw plasma membrane integrity and progressive motility.

Physiological action of oestradiol on the acrosome reaction in human spermatozoa.

Abstract: The acrosome is a secretory vesicle located in the sperm head. The acrosome reaction consists in the fusion of the sperm plasma membrane with the external acrosomal membrane. It has been observed that this reaction does not take place in spermatozoa incubated in cervical mucus, hydrogel that contains high concentrations of oestradiol in the peri-ovulatory period. The objective of the present study was to analyse the influence of oestradiol on the acrosome reaction in human spermatozoa to evaluate the possible inhibitory effect of this hormone. Spermatozoa were incubated in progesterone (10.1 nmol l(-1)); oestradiol plus progesterone (oestradiol at 840 pmol l(-1) and progesterone at 10.1 nmol l(-1)), oestradiol (840 pmol l(-1)) and control (without steroidal hormones) for 30 min, 60 min, 240 min and 24 h. The acrosome reaction was evaluated by stain with Hoechst 33258 and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin lectin. Progesterone-incubated spermatozoa showed the highest percentage of acrosome reaction (P < 0.05). Spermatozoa incubated with oestradiol and oestradiol plus progesterone showed the lowest percentage of acrosome reaction. The present study demonstrates the inhibitory role of oestradiol on the acrosome reaction, stimulated by progesterone in human spermatozoa under physiological conditions.

Ganglioside GM1 Mediates Decapacitation Effects of SVS2 on Murine Spermatozoa

Abstract: Prior to fertilization, mammalian spermatozoa need to acquire fertilizing ability (capacitation) in the female reproductive tract. On the other hand, capacitated spermatozoa reversibly lose their capacitated state when treated with seminal plasma (decapacitation). Previously, we demonstrated that a mouse seminal plasma protein, SVS2, is a decapacitation factor and regulates sperm fertilizing ability in vivo. Here, we examined the mechanisms of regulation of fertilizing ability by SVS2. Capacitation appears to be mediated by dynamic changes in lipid rafts since release of the cholesterol components of lipid rafts in the sperm plasma membrane is indispensable for capacitation. When the ejaculated spermatozoa were stained with a cholera toxin subunit B (CTB) that preferably interacts with ganglioside GM1, another member of the lipid rafts, the staining pattern of the sperm was the same as the binding pattern of SVS2. Interestingly, SVS2 and CTB competitively bound to the sperm surface with each other, suggesting that the binding targets of both molecules are the same, that is, GM1. Molecular interaction studies by the overlay assay and the quartz crystal microbalance analysis revealed that SVS2 selectively interacts with GM1 rather than with other gangliosides. Furthermore, external addition of GM1 nullified SVS2-induced sperm decapacitation. Thus, ganglioside GM1 is a receptor of SVS2 and plays a crucial role in capacitation in vivo.

Characterization of human sperm N-acetylglucosaminidase.

Abstract: N-acetylglucosaminidase (NAG) is particularly active in mammalian spermatozoa and appears to be involved in fertilization. Although it is assumed that this enzyme is acrosomal, previous results from our laboratory suggest the presence of NAG at the sperm plasma membrane level. The present study attempted to analyse the subcellular distribution of this enzyme in human spermatozoa. Sperm were incubated under different conditions and NAG activity measured in the soluble extracts and cell pellets using a specific fluorometric substrate. A significant proportion of NAG activity was released when sperm were incubated in culture medium, suggesting a weak association with the plasma membrane. This location was confirmed by western blot analysis of plasma membrane fractions and immunofluorescence on non-permeabilized sperm, which showed a positive signal mainly on the acrosomal domain. The distribution of NAG activity between plasma membrane and acrosome was analysed after cell disruption by freezing and thawing. Triton X-100 stimulated sperm and epididymal NAG activity but not the enzyme obtained from other sources. In addition, biotinylated human recombinant NAG was able to bind to human sperm. Finally, after sperm incubation under capacitating conditions, NAG total activity increased and the sperm enzyme lost its ability to be stimulated by Triton X-100. The possible connection of these results with sperm maturation, capacitation and NAG participation in primary binding to the zona pellucida, was discussed.

Involvement of sperm proteases in the binding of sperm to the vitelline envelope in Xenopus laevis.

Abstract: Sperm binding to the vitelline envelope in dejellied Xenopus laevis eggs was effectively inhibited by inhibitors for trypsin (soybean trypsin inhibitor and p-toluenesulfonyl-L-lysine chloroethyl ketone) and aminopeptidase B (o-phenanthroline, bestatin, and arphamenine B). Likewise, synthetic 4-methylcoumaryl-7-amide (MCA) substrates (t-butoxycarbonyl-GlyArgArg-MCA, benzyloxycarbonyl-ArgArg-MCA, and Arg-MCA) inhibited binding. Consistently, when jellied eggs were inseminated in the presence of these substrates or inhibitors for proteases, fertilization was effectively blocked. The medium in which live sperm or the sperm membrane fraction were suspended exhibited hydrolyzing activities against the synthetic substrates mentioned above, and these activities were effectively inhibited by the protease inhibitors. Ultracentrifugal fractionation of the sperm suspension following induction of the acrosome reaction by a calcium ionophore, A23187, indicated that a considerable amount of the total tryptic and aminopeptidase B activity was released into the medium. On this occasion, part of the tryptic and aminopeptidase B activity was definitely estimated to be discharged in association with a vesiculated membrane, supporting the notion that the proteases involved in binding to the vitelline envelope are present on the sperm plasma membrane.

Antifreezer for diluting boar semen

Abstract: The invention relates to an antifreeze agent for diluting pig sperm, pertaining to the technical field of animal breeding. The antifreeze agent is prepared by N-acetyl-D-dextrosamine, hyaluronic acid, amido-sodium-lauryl sulfuric ester, glucose, trihydroxymethyl aminomethane, sodium citrate, sodium bicarbonate, potassium chloride, bactericide, distilled water and the like, wherein, the N-acetyl-D-dextrosamine can stimulate the emulsification function of the amido-sodium-lauryl sulfuric ester, enhances the protection function of the amido-sodium-lauryl sulfuric ester and yolk, the hyaluronic acid and the amido-sodium-lauryl sulfuric ester can effectively inhibit the damage rate of freezing and thawing sperm plasma membrane and DNA and improve the quality of freezing and thawing sperm, the glucose is the energy source for metabolism in vitro of the sperm, and the sodium citrate can maintain the osmotic pressure balance between diluted antifreeze agent environment and the internal environment of the sperm. The antifreeze agent for diluting pig sperm can effectively prolong the survival time of pig sperms for ultra-low temperature cryopreservation, and improve the liveliness of the freezing and thawing pig sperms, the integrity of plasma membrane and the integrity of acrosome.

Surfactants as Microbicidal Contraceptives: A Calorimetric Study of Partitioning and Translocation in Model Membrane Systems

Abstract: Surfactant partitioning into lipid vesicles was studied using isothermal titration calorimetry (ITC), comparing the behavior of four surfactants with current or potential application in contraception and the prevention of sexually transmitted diseases: nonoxynol-9 (N-9), the amphoteric mixture known as C31G, benzalkonium chloride (BZK), and sodium dodecyl sulfate (SDS). Membranes varied in composition from a single-component system, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, to a complex lipid mixture that models the sperm plasma membrane. The partitioning of N-9 into the membranes was found to be endothermic in contrast to the other surfactants studied. For all four surfactants, the partition coefficient decreased as the membrane cholesterol content increased. Surfactant translocation across the membrane leaflets was also determined, with SDS being the only surfactant of the four not to exhibit “flip-flop” on experimental time scales. The results of these studies shed light on the process of surf...

Methods and compositions for intracytoplasmic sperm injection-mediated transgenesis

Abstract: Methods and compositions for the generation of transgenic animals by intracytoplasmic sperm injection (ICSI) are provided herein In some embodiments, such methods can include: treating isolated spermatozoa with one of the group consisting of: lysolecithin, digitonin, sonication, and piezo pulses to disrupt or remove the sperm plasma membrane; contacting a sperm thus treated with a nucleic acid mixture that includes a nucleic acid containing a transgene to form a composition; and introducing the composition into an unfertilized oocyte of the same species to form a transgenic embryo In some embodiments, the nucleic acid mixture includes a nucleic acid containing a transgene flanked by two terminal repeats and one of the group consisting of: a transposase polypeptide and a nucleotide sequence encoding a transposase In some embodiments, the transposase is a piggyBac transposase

Effects of LDL on Cryopreservation of Sheep Spermatozoa

Abstract: The fertility test showed that pregnant rate of AI with frozen semen were significantly lower than that of natural mating. One of the reasons might be the sperm plasma membrane was serious injured during freezing. Therefore,the paper studied on sheep spermatozoa during freezing which was protected with egg yolk(EY) and local fine wool sheep was used as the experimental animals. Then low density lipoprotein(LDL) was used in diluents instead of egg yolk to study on the effect of LDL on sperm motility,acrosome intact rate and plasma membrane integrity. The optimal LDL dilution formula was screened to improve sheep semen quality,reduce the damage sperm,and provide theoretical basis for improving fertility of ram sperm after cryopreservation.

223 enzymatic activity level of different glycosidases in intact and acrosome-reacted porcine sperm

Abstract: The sperm–egg interactions are species-specific forms of cell recognition and the binding event which are a necessary prerequisite for fertilization (Park et al 2002 Anim Reprod Sci 72, 83–94) Glycosidase enzymes that remove carbohydrates could play an important role in the reproductive tract, modulating decisive physiological events mediated by carbohydrates, which play a key role in sperm–oocyte recognition The aim of this study was to analyze the presence of the glycosidases α-D-mannosidase, α-L-fucosidase, β-D-glucosaminidase, and β-D-galactosaminidase in intact and acrosome-reacted sperm from fertile matured boars Sperm were washed three times in PBS by centrifugation at 800g for 10 min The pelleted sperm were resuspended in the same buffer to obtain a final concentration of 250 × 106 spermatozoa mL–1 The acrosome reaction was induced by incubation of the sperm with 10 µm of calcium ionophore A23187 at 37°C for 30 min Different enzymes were detected by incubating 8 µL (for α-Dmannosidase) or 80 αL (for the rest of the enzymes) of sperm sample with the corresponding substrate conjugated to 4-methylumbelliferil for 2 h at 37°C in PBS at pH 73 Fluorescences were read on a Fluostar Galaxy fluorimeter (BMG LabTech GmbH, Offenburg, Germany), using wavelengths of 340 and 450 nm for excitation and emission, respectively, and were corrected by subtracting tissue and substrate blanks The results were analyzed using a one way ANOVA An average of fluorescence units of 968586 ± 108175, 739463 ± 87429, 315417 ± 51410, and 166640 ± 11786 was detected in the intact sperm sample for the α-D-mannosidase, α-L-fucosidase, β-D-glucosaminidase, and β-D-galactosaminidase, respectively For the acrosome-reacted sperm sample (60–65% acrosome-reacted sperm in the samples measured by fluorescence microscope), an average of 975614 ± 101145, 702693 ± 77148, 118570 ± 27751, and 111160 ± 17670 for α-D-mannosidase, α-L-fucosidase, β-D-glucosaminidase, and β-D-galactosaminidase, respectively Statistically significant differences (P < 005) between intact and acrosome-reacted sperm were detected only for the β-D-glucosaminidase and β-D-galactosaminidase These results suggest that the four different enzymes detected are mainly present in the sperm plasma membrane Under the conditions used in this study, α-D-mannosidase is the main enzyme activity present in the sperm Importantly, β-D-glucosaminidase and β-D-galactosaminidase activity detected in the intact sperm is decreased after the induction of the acrosome reaction

Cryopreservation of Kangaroo Spermatozoa

Abstract: Kangaroo spermatozoa have proven extremely difficult to cryopreserve such that despitenumerous empirical studies using high concentrations of glycerol and/or DSMO, the bestpost-thaw motility has only been in the order of 10%. The efficacy of glycerol as acryoprotectant for marsupial spermatozoa is somewhat paradoxical, being necessary athigh concentrations for adequate cryopreservation, but at the same time “cytotoxic.” Theunderlying objectives of this project were to understand the causes of cryoinjury tokangaroo spermatozoa and to apply this information in an attempt to develop a reliablemethod for cryopreservation. These objectives were addressed through strategically linkedstudies incorporating the documentation of cryoinjury, hypothesis driven investigations intothe causes of cryoinjury and the application of novel cryopreservation protocols.The ultrastructure and freeze-fracture of caput and cauda epididymal Eastern greyKangaroo (EGK) spermatozoa at 35°C, 4°C and following cryopreservation with andwithout 20% glycerol was investigated. The addition of 20% glycerol resulted in significantdamage to the sperm plasma membrane and mitochondria compared to no glycerol at thesame temperatures (P < 0.05). Following cryopreservation, 20% glycerol significantlyimproved the preservation of the cauda epididymal sperm plasma membrane andmitochondria and reduced the incidence of axonemal damage and periaxonemal spaces.For caput epididymal spermatozoa, glycerol only improved the preservation of the plasmamembrane following cryopreservation (P < 0.05).Freeze fracture microscopy revealed a pattern of helically wound intramembranousparticles in the plasma membrane over the fibre network of the mid piece of the sperm tail.After thawing, the plasma membrane was damaged such that this structure was missing iniipatches, and the helical rows of particles were mal-aligned. On the principal piece,particles were arranged randomly at physiological temperatures; however, upon cooling to4°C with 20% glycerol, the particles become aggregated. Once re-warmed (35°C),particles over the principal piece resumed their random organisation. This finding is furtherevidence of a reversible phase transition of the macropod sperm plasma membrane duringcooling that is not associated with a loss of motility or membrane integrity.In attempt to investigate cryoinjury caused by alternative cryoprotectants the next studiesexamined the effect of cryoprotectants (20% DMSO, a 10% DMSO / 10% glycerol mixture,20% glycerol and 1 M sucrose solution) on kangaroo sperm structure and function, alongwith the effect of varying concentrations of glycerol on sperm mitochondrial function.Eastern grey kangaroo cauda epididymidal spermatozoa were incubated for 10 mins at35°C in each cryoprotectant and the plasma membrane integrity (PMI) and motilityassessed using light microscopy. The same samples were fixed for TEM and theultrastructural integrity of the spermatozoa examined. To investigate the effect of glycerolon the kangaroo sperm mitochondrial function, epididymidal spermatozoa were incubatedwith JC-1 in Tris-citrate media at 35°C for 20 mins in a range of glycerol concentrations (0,5, 10, 15 and 20%) and the mitochondrial membrane potential (MMP) and plasmamembrane integrity determined. As expected, incubation of spermatozoa in 20% glycerolfor 10 mins resulted in a significant reduction in motility, PMI and ultrastructural integrity.Interestingly, incubation in 20% DMSO resulted in no significant reduction in motility orPMI but a significant loss of structural integrity when compared to the control spermatozoa(0% Glycerol). However, 20% DMSO was overall less damaging to sperm ultrastructurethan glycerol, 10% glycerol and 10% DMSO, and sucrose. While all glycerolconcentrations had an adverse effect on mitochondrial function, the statistical modelspresented for the relationship between MMP and glycerol predicted that sperm, wheniiiadded to 20% glycerol, would loose half of their initial MMP immediately at 35°C and after19.4 mins at 4°C. Models for the relationship between PMI and glycerol predicted thatsperm would loose half of their initial PMI after 1.8 mins at 35°C and 21.1 mins at 4°C.These results suggest that if glycerol is to be used as a cryoprotectant for kangaroo spermthen it is best administered at 4°C and that mitochondrial function is more sensitive toglycerol than PMI. Further studies were directed at investigating strategies that reduceexposure of spermatozoa to glycerol during processing and evaluating the cryoprotectiveproperties of 20% DMSO for kangaroo sperm.The first hypothesis to be tested with respect the causes of cryopathology examined thepossibility that the ultrastructural changes that occur to the kangaroo sperm duringepididymidal maturation reduced the tolerance of the sperm cell organelles to respond toosmotic flux, glycerol cytotoxicity and ice-crystal damage. Caput and cauda epididymidalspermatozoa were recovered from red-necked wallabies (Macropus rufogriseus) and EGK.In Experiment 1, caput and cauda epididymal spermatozoa were frozen and thawed usinga standard cryopreservation procedure in Tris-citrate buffer with or without 20% glycerol.Although cryopreservation of caput epididymidal spermatozoa resulted in a significantincrease in sperm plasma membrane damage, they were more tolerant of the procedurethan spermatozoa recovered from the cauda epididymidis (P < 0.05). In Experiment 2,caput and cauda epididymidal spermatozoa were diluted into phosphate-buffered salinemedia of varying osmolarity and their osmotic tolerance was determined. Plasmamembranes of caput epididymidal spermatozoa were clearly more tolerant of hypo-osmoticmedia than were cauda epididymidal spermatozoa (P < 0.05). In Experiment 3, caput andcauda epididymidal spermatozoa were incubated in Tris-citrate buffer with and without20% glycerol at 35 and 4°C to examine the cytotoxic effects of glycerol. At bothtemperatures, caput epididymidal spermatozoa showed less plasma membrane damageivcompared with cauda epididymidal spermatozoa when exposed to 20% glycerol (P < 0.05).The results from these experiments clearly indicate that epididymal maturation of kangaroospermatozoa resulted in a decreased ability to withstand the physiological stressesassociated with cryopreservation.The second hypothesis to be tested with respect the causes of cryopathology examinedwhether filamentous (F) actin associated with the complex cytoskeleton of the kangaroosperm head and tail may be contributing to lack of plasma membrane plasticity and aconsequent loss of membrane integrity during cryopreservation. In the first study, thedistribution of G and F actin within EGK cauda epididymidal spermatozoa was successfullydetected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam),respectively. G-actin staining was most intense in the acrosome but was also observedwith less intensity over the nucleus and mid-piece. F-actin was located in the spermnucleus but was not discernable in the acrosome or sperm tail. To investigate whethercytochalsin D (a known F-actin depolymerising agent) was capable of improving theosmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypoosmoticmedia (61 and 104 mOsm) containing a range of cytochalasin D concentrations(0-200μM). Cytochalsin D had no beneficial effect on the plasma membrane integrity ofsperm incubated in hypo-osmotic media. The results of this study indicated that the F-actindistribution in cauda epididymidal spermatozoa of the EGK was surprising different to thatof the Tammar Wallaby (M. eugenii) and that cytochalsin-D does not appear to improvethe tolerance of EGK cauda epididymidal sperm to osmotic induced injury.The aims of the final study in this thesis were to investigate alternative techniques for thecryopreservation of kangaroo spermatozoa that reduce or eliminate the need for glycerol.This was investigated by (1) freezing sperm with 20% glycerol in pre-packaged 0.25 mL"Cassou" straws in such a way as to enable rapid dilution of the glycerol post-thaw, (2)vinvestigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide(DMA - 10, 15 and 20% v/v) as cryoprotectants and (3) to evaluate the potential use ofultra-rapid small volume vitrification cooling of kangaroo spermatozoa with or withoutcryoprotectant (20% v/v Glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-strawpost-thaw dilution (1: 20) of 20% glycerol produced no significant improvement in postthawviability of kangaroo spermatozoa. Cryopreservation of kangaroo spermatozoa in20% DMSO resulted in only 2% motile sperm and 5% of sperm with an intact plasmamembrane. While kangaroo spermatozoa frozen by ultra-rapid freezing techniquesshowed no evidence of post-thaw viability irrespective of whether cryoprotectant was usedor not, sperm frozen in 10 - 20% DMA showed post-thaw motility and plasma membraneintegrity of 11 - 13% and 18 - 23% respectively. Although still modest compared to othermarsupial and eutherian spermatozoa, these results represent a significant improvementin the development of a cryopreservation procedure for kangaroos and provide the basisfor further investigation using other amide-based cryoprotectants.

The effects of oxidative stress and age on human spermatozoa

Abstract: Male infertility is now recognised as a significant factor in couples having difficulty conceiving. The impact of maternal age has long been known as a limiting factor, however recent research indicates that advancing paternal age can also negatively impact on a couple's chances of conception. One of the major contributing causes of male infertility has now been linked to spermatozoa exposure to reactive oxygen species (ROS). Such exposure induces oxidative stress when coupled with reduced total antioxidant capacity (TAC). Measures of both ROS and TAC are used as tests of oxidative stress status (OSS) which are used together to give further insight into male fertility status. Current research has revealed that the level of DNA fragmentation increases with age and, due to their composition, the sperm plasma membrane, DNA double helix and single stranded DNA are highly susceptible to ROS attack. ROS cause high levels of lipid peroxidation (LPO) that ultimately damages the plasma membrane and interferes with its vital functions. ROS also readily attack the purine and pyrimidine bases of DNA resulting in DNA damage. It is hypothesized that advancing paternal age will impact negatively of levels of ROS, TAC and DNA fragmentation in ageing men. The aim of this study was to determine the relationship between ROS, TAC and DNA damage on spermatozoon viability in relation to age. Ejaculated semen samples from 54 men undergoing infertility assessment were collected and divided into men aged ~40 years (n=16) and men aged ::;;39 years (n=38). Samples were examined for their level of ROS and TAC as a possible indication of oxidative stress status. Samples were also assessed for DNA fragmentation and damage using TUNEL. Statistical analysis consisted of independent t-tests, chi-square tests and multivariate logistic regression. Analysis found no significant associations between ROS, TAC, TUNEL and age. Significant differences were observed between abnormal sperm motility and age (p<0.05; t-test and

Effect of cryopreservation on the efficiencies of goat sperm in picking up exogenous DNA and resuted transgeneic embryos

Abstract: The efficiency and the ability of fresh and frozen-thawed sperm in picking up exogenous DNA were investigated in this study.The fresh and frozen-thawed sperm was incubated with linearized end-labeled pEGFP-N_1 plasmid DNA,and detected by in situ hybridization method.The in vitro producted embryos were screened by PCR assay.The ultrastructure of activated spermatozoa were observed by transmission electron microscope(TEM),and the integrity of sperm plasma membrane was evaluated with a combination of fluorescent probes-carboxifluorescein diacetate and propidium iodide(PI).The sperm genomic DNA damage was determined by single cell gel electricity assay(SCGE)methods.The results showed that the frozen-thawed treated goat sperm cells were more efficient and more reliable than untreated sperm in picking up exogenous DNA and subsequently internalizing the DNA into sperm nuclei(81.60%±16.59% vs 32.95%±2.93%,t=4.873,P=0.003;41.80%±6.26% vs 27.89%±8.64%,t=2.634,P=0.039).The exogenous DNA retained its integrity in sperm as demonstrated with PCR and Southern Blotting assay methods.The rate of transgenesis embryos produced by exogenous DNA incubated sperm was enhanced significantly(45.45%±10.87% vs 24.44%±6.06%,t=1.750,P=0.013).The semen cryopreservation reduced the integrity of sperm plasma membrane(8.34%±4.21% vs 65.67%±6.46%,t=12.492,P0.001)and enhanced the sperm genomic DNA strand breakage significantly.These results suggest that semen cryopreservation could enhance the sperm ability in picking up foreign DNA.The cryopreservation-induced changes in the sperm plasma membrane facilitate the internalization exogenous DNA.