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Find video protocols related to scientific articles indexed in Pubmed.
Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.
Sci Rep
PUBLISHED: 04-14-2014
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Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (<1?hr) P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.
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Suprazero cooling rate, rather than freezing rate, determines post thaw quality of rhesus macaque sperm.
Theriogenology
PUBLISHED: 08-05-2013
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Sperm become most sensitive to cold shock when cooled from 37 °C to 5 °C at rates that are too fast or too slow; cold shock increases the susceptibility to oxidative damage owing to its influence on reactive oxygen species (ROS) production, which are significant stress factors generated during cooling and low temperature storage. In addition, ROS may be a main cause of decreased motility and fertility upon warming. They have been shown to change cellular function through the disruption of the sperm plasma membrane and through damage to proteins and DNA. The objective of this study was to determine which cryopreservation rates result in the lowest degree of oxidative damage and greatest sperm quality. In the rhesus model, it has not been determined whether suprazero cooling or subzero freezing rates causes a significant amount of ROS damage to sperm. Semen samples were collected from male rhesus macaques, washed, and resuspended in TEST-yolk cryopreservation buffer to 100 × 10(6) sperm/mL. Sperm were frozen in 0.5-mL straws at four different combinations of suprazero and subzero rates. Three different suprazero rates were used between 22 °C and 0 °C: 0.5 °C/min (slow), 45 °C/min (medium), and 93 °C/min (fast). These suprazero rates were used in combination with two different subzero rates for temperatures 0 °C to -110 °C: 42 °C/min (medium) and 87 °C/min (fast). The different freezing groups were as follows: slow-med (SM), slow-fast (SF), med-med (MM), and fast-fast (FF). Flow cytometry was used to detect lipid peroxidation (LPO), a result of ROS generation. Motility was evaluated using a computer assisted sperm motion analyzer. The MM and FF treated sperm had less viable (P < 0.0001) and motile sperm (P < 0.001) than the SM, SF, or fresh sperm. Sperm exposed to MM and FF treatments demonstrated significantly higher oxidative damage than SM, SF, or fresh sperm (P < 0.05). The SM- and SF-treated sperm showed decreased motility, membrane integrity, and LPO compared with fresh semen (P < 0.001). Slow cooling from room temperature promotes higher membrane integrity and motility post thaw, compared with medium or fast cooling rates. Cells exposed to similar cooling rates with differing freezing rates were not different in motility and membrane integrity, whereas comparison of cells exposed to differing cooling rates with similar freezing rates indicated significant differences in motility, membrane integrity, and LPO. These data suggest that sperm quality seems to be more sensitive to the cooling, rather than freezing rate and highlight the role of the suprazero cooling rate in post thaw sperm quality.
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Oxidative damage to rhesus macaque spermatozoa results in mitotic arrest and transcript abundance changes in early embryos.
Biol. Reprod.
PUBLISHED: 01-01-2013
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Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of metaphase II (MII) oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO? in air for 2.25 h. Sperm were then assessed for motility, viability, and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the eight-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in two-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to four- and eight-cell stage was significantly lower for embryos generated with ROS-treated sperm than for controls. All embryos produced from ROS-treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of two-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS-induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the two-cell stage of development.
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Loss of fertilization potential of desiccated rhesus macaque spermatozoa following prolonged storage.
Cryobiology
PUBLISHED: 02-03-2011
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Desiccation provides a novel spermatozoal preservation technique because it eliminates the need to store spermatozoa in liquid nitrogen, resulting in decreased opportunities for cross-contamination of samples and lower costs of spermatozoal banking, storage, and shipping. The objective of this study was to desiccate rhesus macaque spermatozoa and to evaluate the fertility following storage. Semen from four male rhesus macaques (Macaca mulatta) were collected using electroejaculation, washed through a Percoll gradient, and resuspended to 100×10(6) spermatozoa/mL in a simple vacuum drying buffer containing the disaccharide trehalose (10mM HEPES, 5mM KCl, 65mMNaCl, 150mMtrehalose, 5.7% bovine serum albumin, BSA). Cells were desiccated in 50?l drops under vacuum (22inHg) at ambient temperature until the water content was less than 1g H(2)O/g dry weight. Initial motility was high (90-70%) and was reduced by desiccation (0%). Membrane integrity was investigated using the two fluorochromes, SYBR 14 and propidium iodide (PI, Molecular Probes, Inc.), with flow cytometry. After desiccation, 100% of the spermatozoa were stained red with PI indicating plasma membrane compromise. Samples were stored in air-tight polyvinyl plastic bags purged with N(2) gas for 10s and vacuum sealed. The samples were protected from light and either stored at room temperature (treatment 1) or at -80°C (treatment 2). Samples were rehydrated 7-10 days post desiccation in 150?l BWW containing 0.5% BSA and used for intracytoplasmic spermatozoa injection (ICSI) to compare fertilization and embryo development to freshly collected samples. For control embryos, one freshly ejaculated motile sperm with normal morphology was immobilized by scoring a small incision in the plasma membrane over the sperm tail before injecting into an oocyte. For the dried sperm treatments, one sperm with normal morphology was selected and scored before injecting into an oocyte. After injection, the embryos were individually cultured in CMRL medium with 10% fetal bovine serum (FBS) media on pre-plated buffalo rat liver (BRL) cells at 37°C and 5% CO(2). Fertilization was assessed at 14, 16, 22, and 24h. Embryo development was evaluated daily from day 3 to day 11. The fertilization rate was 68%, 73%, and 45% for the control, treatment 1, and treatment 2 groups, respectively. The blastocyst rate was 40%, 5%, and 0% for control, treatments 1, and 2, respectively. Treatment group 1 had comparable fertilization rates with control group (73% vs. 68%) and was not significantly different (P<0.05), but as development progressed, fewer embryos developed beyond the morula stage. Treatment 2 had a lower fertilization rate than control (45% vs. 68%), although not significantly different, and embryos did not develop past the morula stage. This study demonstrates that while the vacuum dried spermatozoa were immotile and had compromised plasma membrane integrity, they were capable of fertilization using ICSI and could support embryo development to the morula stage.
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Antioxidant treatment in the absence of exogenous lipids and proteins protects rhesus macaque sperm from cryopreservation-induced cell membrane damage.
Theriogenology
PUBLISHED: 01-26-2011
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Osmotic stress caused oxidative stress in rhesus macaque sperm, which was alleviated by antioxidant supplementation. The objective of the present study was to demonstrate that cryopreservation of rhesus macaque sperm also induces reactive oxygen species (ROS) production, and to determine whether ROS have an important role in cryopreservation-induced membrane. Additionally, we evaluated the antioxidant capacity of TEST (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid-Tris) buffer (with 20% egg yolk and 13% skim milk) and supplementation with antioxidants, superoxide dismutase (SOD), catalase (CAT), and ?-tocopherol. There was a substantial level of ROS production in both the presence (15% increase in superoxide, P < 0.01; 14% increase in hydrogen peroxide, P < 0.01) and absence of egg yolk (EY) and skim milk (SM; 33% increase in superoxide, P < 0.001; 48% increase in hydrogen peroxide, P < 0.001). Superoxide dismutase provided little membrane protection against ROS, but increased postthaw total and progressive motility by 10% (P < 0.01) and 15% (P < 0.05), respectively. Supplementation with CAT and ?-tocopherol in the presence of EY and SM decreased H(2)O(2) by 55% (P < 0.01) and 49% (P < 0.001), whereas supplementation with CAT and ?-tocopherol in the absence of EY and SM reduced the level of lipid peroxidation by 61% (P < 0.05) and 28% (P < 0.01). In conclusion, this is apparently the first report that cryopreservation of rhesus macaque sperm induced a significant increase in ROS and that antioxidant supplementation (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid-Tris) can significantly decrease the extent of ROS-induced membrane damage.
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Osmotic stress stimulates phosphorylation and cellular expression of heat shock proteins in rhesus macaque sperm.
J. Androl.
PUBLISHED: 11-18-2010
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The cryosurvival of sperm requires cell signaling mechanisms to adapt to anisotonic conditions during the freezing and thawing process. Chaperone proteins heat shock protein 70 (HSP 70) and heat shock protein 90 (HSP 90; recently renamed HSPA and HSPC, respectively) facilitate some of these cell signaling events in somatic cells. Sperm were evaluated for their cellular expression and levels of phosphorylation of both HSP 70 and HSP 90 under anisotonic conditions as a potential model for cell signaling during the cryopreservation of macaque spermatozoa. In order to monitor the level of stress, the motility and viability parameters were evaluated at various time points. Cells were then either prepared for phosphoprotein enrichment or indirect immunocytochemistry. As controls, the phosphoserine, phosphothreonine, and phosphotyrosine levels were measured under capacitation and cryopreservation conditions and were compared with the phosphoprotein levels expressed under osmotic conditions. As expected, there was an increase in the level of tyrosine phosphorylation under capacitation and cryopreservation conditions. There was also a significant increase in the level of all phosphoproteins under hyperosmotic conditions. There was no change in the level of expression of HSP 70 or 90 under osmotic stress conditions as measured by Western blot. The enrichment of phosphoproteins followed by Western immunoblotting revealed an increase in the phosphorylation of HSP 70 but not HSP 90 under osmotic stress conditions. Indirect immunofluorescence localized HSP 70 to the postacrosomal region of sperm, and the level of membrane expression of HSP 70 was significantly affected by anisotonic conditions, as measured by flow cytometry. Taken together, these results suggest a differential role for HSP 70 and HSP 90 during osmotic stress conditions in rhesus macaque sperm.
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Osmotic stress induces oxidative cell damage to rhesus macaque spermatozoa.
Biol. Reprod.
PUBLISHED: 10-21-2009
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Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa survival. Additionally, evidence indicates that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa as well. Our objective was to determine the effect of reactive oxygen species (ROS) on rhesus macaque (Macaca mulatta) sperm function and to determine whether osmotic stress elicits the production of ROS. In the first experiment, the xanthine-xanthine oxidase (X-XO) system was used to generate the ROS superoxide anion (O(2)(-.)) and hydrogen peroxide (H(2)O(2)) in the presence or absence of the ROS scavengers superoxide dismutase and catalase, respectively. In the second experiment, osmotic stress was introduced by incubation of spermatozoa in a series of anisosmotic media ranging from 100 to 1000 mOsmol/kg in the presence or absence of the antioxidant alpha-tocopherol. Treatment with the X-XO system resulted in a significant increase in the generation of O(2)(-.) and H(2)O(2) that was detectable using flow cytometry. The ROS generated by the X-XO system was dose dependent, and as the concentration of ROS increased, motility decreased and lipid peroxidation increased while no affect was observed on viability. Incubation of spermatozoa in anisosmotic media also resulted in an increase in O(2)(-.) generation and lipid peroxidation that was significantly decreased in the presence of the powerful antioxidant alpha-tocopherol. These results clearly indicate that osmotic stress causes oxidative stress in rhesus macaque spermatozoa, which strongly supports the hypothesis that cryopreservation-induced osmotic stress may lead to oxidative cell damage.
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New insights into the mechanisms of fertilization: comparison of the fertilization steps, composition, and structure of the zona pellucida between horses and pigs.
Biol. Reprod.
PUBLISHED: 07-08-2009
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The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was to develop a comparative strategy between these two divergent models. First, we compared the role of equine and porcine gametes in the following five functions using intraspecific and interspecific IVF: ZP binding, acrosome reaction, penetration through the ZP, gamete fusion, and pronucleus formation. Under in vitro conditions, we showed that the ZP is a determining element in sperm-ZP attachment and penetration, whereas the capacity of the spermatozoa is of less importance. In contrast, the capacity of the spermatozoa is a key component of the acrosome reaction step. Second, we compared the composition and structure of the equine and porcine ZP. We observed differences in the number and localization of the ZP glycoproteins and in the mesh-like structure of the ZP between equine and porcine species. These differences might correlate with the differences in spermatozoal attachment and penetration rates. In conclusion, our comparative approach allows us to identify determining elements in the mechanism of fertilization.
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Rhesus macaque blastocysts resulting from intracytoplasmic sperm injection of vacuum-dried spermatozoa.
J. Med. Primatol.
PUBLISHED: 04-07-2009
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Sperm desiccation is an attractive approach for sperm preservation. In this study, we examined the feasibility and efficiency of intracytoplasmic sperm injection using vacuum-dried rhesus macaque sperm in CZB medium supplemented with 10% fetal bovine serum.
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Cooling rate affects rhesus monkey sperm survival.
J. Med. Primatol.
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The rate at which lethal intracellular ice formation occurs during cryopreservation is highly dependent on several variables. The objective of this study was to determine the optimal rate at which rhesus sperm can be cooled.
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Oxidative stress in zebrafish (Danio rerio) sperm.
PLoS ONE
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Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line.
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Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral.
PLoS ONE
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Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (?5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had?50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems.
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