彗星电泳检测 12C6+离子束辐照人类肝L02细胞的DNA损伤效应

以传能线密度为30 keV/μm的12C6+离子束辐照人类肝L02细胞, 利用彗星电泳技术检测了以DNA链断裂为生物终点的DNA辐射损伤效应。 CASP软件分析彗星图像, 主要检测尾部DNA（TDNA％）、 彗星全长（CL）、 尾长（TL）、 尾矩（TM）和Olive尾矩（OTM）等指标, SPSS 11.5软件进行统计学分析, 绘制并拟合TM\|剂量曲线。 结果显示, 辐照以剂量依赖的方式引起L02细胞彗星图像各指标的增大, 且TM值与剂量线性正相关。 说明12C6+离子束对DNA有较强的致损伤效应, 且与剂量正相关。 研究为正确评价重离子对人体正常组织的辐射风险及危害提供了一定的基础数据和依据。     

Production of normal offspring from partially zona-incised vitrified mouse oocytes fertilized with cryopreserved spermatozoa using an optimized protocol

The present study was designed to investigate the optimized conditions for cryopreservation of Kunming (KM) mice spermatozoa (Experiment 1) and to compare the developmental potential of IVF embryos produced from fresh oocytes (Group 1), vitrified-warmed oocytes without (Group 2) or with partial zona pellucida incised by a piezo manipulator (ZIP) (Group 3) fertilized with frozen-thawed spermatozoa (Experiment 2). In experiment 1, spermatozoa were cryopreserved with the medium containing raffinose and egg yolk with different concentrations (0 to 60 percent) and then followed by fertilization with fresh oocytes after thawing. The highest cleavage (76.2 percent) and blastocysts formation rates (63.6 percent) were obtained when the egg yolk concentration was adjusted to 30 percent. To optimize the equilibration time, the spermatozoa were equilibrated in the optimized medium for 0, 10, 30, 50, 70, 90 min at 40 degree C before plunging into liquid nitrogen. After thawing, the highest cleavage rate (87.4 percent) of IVF embryos was observed when equilibrated for 30 min. In experiment 2, the cleavage and blastocyst rates in Group 1 (81.2 percent, 65.4 percent) and Group 3 (72.5 percent, 45.0 percent) were higher (P less then 0.05) than those in Group 2 (22.2 percent and 13.9 percent), respectively. When 2-cell embryos obtained in Group 1 and 3 were transferred, 32.1 percent and 22.7 percent of embryos in the pregnant receipts developed to term, respectively. In conclusion, the optimized protocol is highly efficient for the cryopreservation of KM mice spermatozoa; the ZIP technique is very useful for improvement of the fertilization efficiency using the cryopreserved gametes and normal offspring can be produced efficiently.     

Actin localisation and the effect of cytochalasin D on the osmotic tolerance of cauda epididymidal kangaroo spermatozoa

This study examined the hypothesis that filamentous actin associated with the complex cytoskeleton of the kangaroo sperm head and tail may be contributing to lack of plasma membrane plasticity and a consequent loss of membrane integrity during cryopreservation. In the first study, the distribution of G and F actin within Eastern Grey Kangaroo (EGK, Macropus giganteus) cauda epididymidal spermatozoa was successfully detected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam), respectively. G-actin staining was most intense in the acrosome but was also observed with less intensity over the nucleus and mid-piece. F-actin was located in the sperm nucleus but was not discernable in the acrosome or sperm tail. To investigate whether cytochalasin D (a known F-actin depolymerising agent) was capable of improving the osmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypo-osmotic media (61 and 104 mOsm) containing a range of cytochalasin D concentrations (0-200 microM). Cytochalasin D had no beneficial effect on plasma membrane integrity of sperm incubated in hypo-osmotic media. However, when EGK cauda epididymidal sperm were incubated in isosmotic media, there was a progressive loss of sperm motility with increasing cytochalasin D concentration. The results of this study indicated that the F-actin distribution in cauda epididymidal spermatozoa of the EGK was surprisingly different from that of the Tammar Wallaby (M. eugenii) and that cytochalasin-D does not appear to improve the tolerance of EGK cauda epididymidal sperm to osmotically induced injury.     

Head dimensions of cryopreserved red deer spermatozoa are affected by thawing procedure

The objective of this study was to evaluate the effects of the thawing procedure on red deer spermatozoa distribution in morphologically distinct subpopulations after freezing and thawing. For this purpose, epididymal spermatozoa were thawed using two different thawing protocols (I = 37 degree celsius for 20 s vs. II = 70 degree celsius for 5 s). The spermatozoa, from 10 Iberian deer stags, were diluted at room temperature in a Triladyl-20 percent egg yolk medium and frozen in nitrogen vapor. Standard sperm freezability was judged by microscopic assessments of sperm motility. The thawing procedure had an effect on sperm motility percentage (P = 0.05), with the best overall recovery rates found with the use of protocol I (76.8 + or - 1.8 vs. 70.6 + or - 1.8). Moreover, the morphometric dimensions for a minimum of 200 sperm heads were analyzed from each sample by means of the Sperm-Class Analysez (SCA), and the mean measurements recorded. Deer sperm heads were significantly (P = 0.01) smaller when spermatozoa were thawed using protocol II than when using procedure I (area = 30.02 square micrometers vs. 30.32 square micrometers; width = 4.47 micrometers vs. 4.51 micrometers; length = 8.05 micrometers vs. 8.11 micrometers), but not for all stags. All sperm head measurements were placed in a statistical database and a multivariate cluster analysis performed. Mean measurements for all parameters of the major clusters for the two different thawing procedures were compared by ANOVA. The mean values for length, width, area, perimeter, shape factor and width/length in the major cluster of sperm head dimensions for thawing protocol I were significantly different from those for protocol II (P = 0.001). In addition, differences were found within all stags for whole morphometric parameters (P = 0.001), with the smallest overall sperm head dimensions found with the use of protocol II. Additionally, the rapid thawing protocol produced a dramatic loss of heterogeneity. Finally, our results showed that the greater the loss of heterogeneity, the greater the degree of sperm cryoinjury.     

Cholesterol addition and removal in pacific oyster oocytes does not improve cryopreservation success

The optimal cholesterol content in cells could provide the benefit of lowering or eliminating the lipid phase transition temperature, while maintaining membrane fluidity and strength; thus, making cells less sensitive to chilling injury and more amenable to cryopreservation. Such effects were shown in some gametes and embryos of certain mammalian species, however, some other cell types, benefited from cholesterol removal. The experiments developed in this study aimed to determine the effect of incubating Pacific oyster (Crassostrea gigas) oocytes in cholesterol-addition or removal solutions prior to cryopreservation on their post-thaw fertilization ability. The results showed a positive association of cholesterol with the oocytes when assessed by fluorescent microscopy. However, this uptake was not reflected by an increase in cholesterol as determined by colorimetric analysis or in the post-thaw fertilization rate of treated oocytes. It is presumed either that oyster oocytes already contain a substantial amount of cholesterol or other lipids in their plasma membranes and do not benefit from any additional cholesterol or there is no lipid phase transition temperature in oyster oocytes.     

Analysis of ploidy and the patterns of amplified fragment length polymorphism and methylation sensitive amplified polymorphism in strawberry plants recovered from cryopreservation

Shoot-tips of 10 strawberry genotypes were successfully cryopreserved using a modified encapsulation-dehydration method. All genotypes survived cryopreservation with high survival and regeneration rates. Eight Joho single-bud sibling lines were established as a model system for genetic analysis. Although cytological examination found chromosomal variation in both non-cryopreserved and cryopreserved samples, the ploidy constitution remained relatively stable after cryopreservation. DNA samples digested with MseI and PstI were used for amplified fragmentation length polymorphism (AFLP) assay. In 16 primer combinations, only one, namely, PCCA-MCAG, detected one site where band pattern changed after cryopreservation, which might be contributed to the change in DNA methylation status at PstI recognition site. Methylation sensitive amplified polymorphism (MSAP) assay was carried out for further investigation on the influence of cryopreservation on DNA methylation status. It was found that cryopreservation induced a significant change in DNA methylation status.     

Acrosome reaction in Octopus tankahkeei induced by calcium ionophore A23187 and a possible role of the acrosomal screw

The octopod sperm is unique especially in two aspects: the screw-shaped acrosome and its inner layered substructure (striation). The present study aims to investigate morphological changes of Octopus tankahkeei spermatozoa during the acrosome reaction (AR) and to pursue functions of the internal substructures revealed by inducing AR with the calcium ionophore A23187. Gradual changes of the spermatozoa were traced using fluorescence and electron microscopy. The AR process included the bulging, vesiculation, and dehiscence of the plasma membrane around the acrosome and the nucleus, as well as the vesiculation of the mitochondrial sheath. Membrane vesiculation outside the nucleus has never been reported in the order Octopoda. The rigid screw and the inner striation of the acrosome remained intact surmounting the nucleus, suggesting that these two structures have potential functions during fertilization. In addition, the detachment of the sperm head and the tail was commonly observed in this study, both in intact and acrosome-reacted sperm. Fluorescence microscopy revealed that the detached mitochondrial sheath usually gave weaker and more dispersive signals than the joint ones. This phenomenon implied that the intense energy release might promote the detachment of the mitochondrial sheath.     

Cryopreservation of human sperm using rapid cooling rates

The effect of cryopreservation on human spermatozoa has been investigated during the past few decades. The majority of current cryopreservation protocols are carried-out using low cooling rates. However, theoretical calculations have shown that for human spermatozoa an optimal cooling rate is about 7,000 C/min. In our work we have studied the effect of cryopreservation with high cooling rates, variation of osmolarity of the cryoprotectant medium and the glycerol content. The results of experiments have demonstrated that within the range of high cooling rates, after thawing the dependency of sperm survival on the cooling rate has a maximum recovery at 2,500-3,300 C/min in moderately hyperosmolar medium, containing 4-5% glycerol. When decreasing the cooling rate down to 1,750-2,500 C/min there was a statistically significant reduction in sperm motility. Using the cooling rate of 8,000-11,000 C /min only a small percentage of spermatozoa retained their motility.     

Characterization of porcine oviductal epithelial cells, cumulus cells and granulosa cells-conditioned media and their ability to induce acrosome reaction on frozen-thawed bovine spermatozoa

The mammalian female reproductive tract cells have been wildely used in in vitro fertilization. We studied the secretory proteins of porcine oviductal epithelial cells (POEC), and cumulus cells (CC) co-cultured with granulosa cells (GC) in conditioned media (CM), and their ability in inducing acrosome reaction (AR) on frozen-thawed bovine spermatozoa. POEC and CC+GC were cultured in M 199 medium for 48, 96 and 144h prior to investigation of protein secretion. The results from SDS-PAGE showed secretory proteins sizes of about 17, 22 and >220kDa in both CM from POEC and CC+GC. To test the ability of CM from POEC and CC+GC in inducing AR, the CM was frozen for 1-3 months before incubating with frozen-thawed bovine spermatozoa. Percentages of the spermatozoa with AR were determined under an inverted microscope and it was found that the tested group 1 (incubated with fresh and frozen CM from POEC for 1-3 months) were 78.44+/-7.25, 75.78+/-4.41, 65.22+/-5.59 and 50.56+/-6.25, respectively. The tested group 2 (incubated with fresh and frozen CM from CC+GC for 1-3 months) had the percentages of the spermatozoa with AR at 88.67+/-4.03, 82.22+/-3.46, 71.00+/-3.16, and 58.56+/-4.69, respectively. Statistical analysis of all group percentages indicated that they were significantly different (P<0.05). Transmission electron microscope studies demonstrated that there were morphological changes on the sperm heads which resulted in the leakage of acrosin and subsequent AR. We concluded that the CM from both POEC and CC+GC efficiently increased in vitro AR on frozen-thawed bovine spermatozoa. Identification and functions of these secretory proteins are currently under investigation which may lead us to a better understanding of other beneficial effects in the utilization of both CM conditions.     

Characteristics of fresh gander semen and its susceptibility to cryopreservation in six generations derived from geese inseminated with frozen-thawed semen

The paper summarises seven years experiments designed to determine the effect of continuous insemination with frozen-thawed semen on fresh semen quality and sperm susceptibility to freezing stress in succeeding generations. During course of experiments, semen was collected from 10-12 White Koluda ganders at the age of 8-9 months, then subjected to freezing and used after thawing for insemination of 10 geese in order to obtain the subsequent generation of males. Semen was diluted 1 to 0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with 6 percent (v/v) of dimethyl-formamide (DMF), frozen to temp. -140 degrees C at a rate 60 degree C per min and then transferred into liquid nitrogen container. Semen samples were thawed prior to insemination in a 60 degree C water-bath. It is difficult to conclude whether freezing stress affected the fresh semen quality, since average volume of SQF (index comprising ejaculate volume, sperm concentration and percentage of live normal cells) varied between generations from 19.3 to 56.2. Continuous goose reproduction by insemination with frozen-thawed semen resulted in significant increase (P less than 0.01) in spermatozoa resistance to cryoinjury in every subsequent generation. In the relation to adequate fresh semen the percentage of live morphologically intact spermatozoa which withstood freezing procedure increased from 27.2 in first generation to 74.4 in sixth generation.     

The effect of cryopreservation process on morphology and fertilising ability of Japanese quail (Coturnix japonica) spermatozoa

Changes in the morphology and fertilising ability of Japanese quail spermatozoa were studied after semen dilution, equilibration and freezing-thawing process in order to determine the optimal diluent, cryoprotectant and the freezing-thawing method. Subsequent stages of quail semen cryopreservation caused significant decline in spermatozoa morphology and their ability to fertilise the ovum. Semen dilution with Lake's extender alone reduced the number of morphologically normal spermatozoa and decreased their fertilising ability. Dimethylacetamide (DMA) was the least detrimental but equilibration of quail spermatozoa with this cryoprotectant caused further decline in the number of morphologically normal cells. However, despite these changes, after artificial insemination with semen equilibrated with DMA 25.8 percent of fertile eggs were obtained. Further loss in the number of normal spermatozoa was observed following the freezing-thawing process. Of the two investigated freezing-thawing methods, the rapid rate (60 C/min) appeared less detrimental to spermatozoa morphology and their ability to fertilize the ovum than the "slow" rate. Also the number of sperm holes appearing in the inner perivitelline layer and the number of spermatozoa trapped in the outer perivitelline layer of the ovum was higher after the rapid than the 'slow' freezing-thawing procedure. Nevertheless, both rates did not yield any fertile eggs.     

Effects of cryopreservation on developmental competency, cytological and molecular stability of citrus callus

Cell suspensions of twelve citrus genotypes were successfully cryopreserved by vitrification. All genotypes survived cryopreservation with >90% viability and the surviving cells of some genotypes regenerated somatic embryos better than the controls. Single-cell sibling lines of cultivar Newhall were used for cytological and molecular examination. It was found that the ploidy constitution remained genetically stable and that no DNA sequence variation was detected by randomly amplified polymorphic DNA (RAPD) assay after cryopreservation. In addition, the methylation sensitive amplified polymorphism (MSAP) assay indicated that cryopreservation caused a significant change in DNA methylation status.     

Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast

Sperm organization in the oysters Crassostrea gigas, Crassostrea nippona, Crassostrea cf. rivularis and Saccostrea cf. mordax inhabiting Asian Pacific coast was studied. The spermatozoa of all studied species had a number of common morphological characters such as a cup-like acrosome with heterogeneous matrix on its top, an axial rod in the subacrosomal space, a barrel-shaped nucleus, four mitochondria in the midpiece, pericentriolar complexes, and a 9+2-organized flagellum. The spermatozoa of C. cf. rivularis differed from the other species by having cytoplasm processes in the midpiece region. Such structures have never been described in the Ostreidae. Additionally, each species could be identified by the shape and size of sperm compartments (acrosome, nucleus, anterior nuclear fossa). The most significant interspecific difference was found in the size of an anterior nuclear fossa. The smallest anterior nuclear fossa was found in C. cf. rivularis (about 0.24 μm in length reaching about 22% of the nuclear length) while the biggest in C. gigas from the Sea of Japan (about 0.53 μm in length reaching about 46% of the nuclear length). The spermatozoa of C. gigas collected from the Sea of Japan and Taiwan Strait differed significantly in almost all the studied parameters. Since sperm morphology has been successfully used for species differentiation, this suggests the existence of two species rather than two populations. The data obtained indicate the species-specific difference in the sperm ultrastructure within the Ostreidae, which may be identified both ultrastructurally and morphometrically.     

Studies on the toxicity of dimethyl sulfoxide, ethylene glycol, methanol and glycerol to loach (Misgurnus fossilis) sperm and the effect on subsequent embryo development

The process of sperm cryopreservation consists of several steps: equilibration of sperm in cryoprotectant medium, freezing of sperm to subzero temperatures, low temperature storage and thawing of the sperm suspension. It has been shown that cryopreservation can cause some damage to the genetic material of cells although the mechanism and significance of these changes are still unknown. The aim of this work was to study the effect of cryoprotectant equilibration process on genetic damage of Loach (Misgurnus fossilis) sperm, using embryo survival as an indicator. Decrease in embryo survival after the 20th stage is generally believed to result from the failure in the genome function of embryos. In the first set of the experiments, Loach sperm were equilibrated in cryoprotectants Me2SO, ethylene glycol, methanol and glycerol (0.6, 1.2, 2.5 M) for 60 min at 10 degree C. The effect of cryoprotectant equilibration on sperm was evaluated based on the survival of embryos derived from cryoprotectant treated sperm. Embryo survival was evaluated at the following stages: 7th, 14th, 17th, 20th, 23rd, 26th, 31st, 34th, 35th, 36th and 37th. Cryoprotectants at concentrations greater than 1.2 M had significant effect on the survival of the embryos after the 20th stage. The effect of glycerol was the most significant with 64.8 +/- 2.4% of embryos survival compared to 77.0 +/- 2.4% for control. Me2SO treatment also effects embryo survival significantly. Possible mechanisms of the genetic instability of cryoprotectants are discussed.     

Relationship between the changes in cellular volume of fish spermatozoa and their cryoresistance

We have investigated the hypothesis that the spermatozoa of marine fish are more resistant than freshwater species to the dynamic changes in osmotic pressure that occur during the process of cryopreservation. We show that while the spermatozoa of marine fish can be successfully activated across a wide range of osmotic pressures (0-2000 mOsmol/l), those of the freshwater species only survive activation within a more restricted range (0-300 mOsm/l). After freeze-thawing, up to 30 percent of motile cells were found in silver carp samples, while up to 90 percent of motile cells were observed in samples from the haarder (Mugil soiuy B). Haarder spermatozoa showed no change of cell volume after dilution in activating or cryoprotective media, while the silver carp spermatozoa responded by swelling and eventual cell disruption. We propose that the differences in cryoresistance of silver carp (Hypophthalmichthys molitrix V.) and haarder spermatozoa may be determined by the ability to preserve cellular volume under non-isotonic conditions.     

The effect of saccharides on the post-thaw recovery of cane toad (Bufo marinus) spermatozoa

The effect of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose, trehalose) as diluents, in cryoprotective additives containing 15% (v/v) DMSO or glycerol as cryoprotectants, were investigated on the recovery of sperm motility after cryopreservation of cane toad (Bufo marinus) spermatoazoa at low (approximately 5 degrees C/min(-1)) and high cooling rates (approximately 35 degrees C/min(-1)). The results show that: 1. recovery of percentage motility was higher with slow cooling than with high cooling rates (37.0 +/- 2.5%, 15.3 +/- 1.6%, P<0.001, respectively), 2. disaccharides were more effective than monosaccharides in protecting spermatozoa with slow cooling (43.9 +/- 1.2%, 26.8 +/- 2.5%, P<0.02, respectively), 3. glycerol was more effective than DMSO with fast cooling (18.3 +/- 2.2%, 12.6 +/- 2.3%, P<0.02, respectively), 4. trehalose with glycerol was the most effective cryoprotective additive with fast cooling (31.0 +/- 3.2%, P<0.05), and 5. overall the recovery of degree (vigour) of motility (range, 1.9 - 3.2) was more resilient to cryopreservation than recovery of percentage motility (range, 8.9 - 51.5 %). Comparison of post-thaw percentage and vigour of sperm motility up to 24 minutes after activation showed disaccharides supported greater duration sperm motility than monosaccharides This result and the recovery of spermatozoa immediately after freeze-thaw, show the main effect of saccharides are as cryoprotectants and not as exogenous energy substrates.     

Ultrastructural changes associated with cryopreservation of potato (Solanum tuberosum l.) shoot tips

The ultrastructure of cells within shoot tips of S. tuberosum 'Désirée' was studied after different steps of the DMSO droplet cryopreservation method. After 2 h of DMSO treatment, cells contained numerous small vesicles, while at the same time mitochondria and chloroplasts had increased in size and vacuoles had assumed an irregular shape. After rapid cooling in liquid nitrogen, subsequent rewarming, and 1 h incubation there were no apparent changes in the ultrastructural organization of the cells, suggesting that they might be still intact. However, two days after rewarming, the meristematic dome area and part of the epidermis showed signs of extensive damage. Rupture of plasmalemma, plasmolysis and destruction of cell organelles as well as strong heterochromatisation of nuclei were observed. Survival and regeneration of cells were found mainly in leaf primordial regions. Here cells were very active, containing many mitochondria and intact or regenerating chloroplasts. Alternating temperature preculture of donor plants before shoot tip isolation improved the cryopreservation results (plant regeneration 46.5 percent) as compared to constantly warm precultured shoot tips (plant regeneration 20.0 percent), which showed slightly stronger damage after rewarming from liquid nitrogen.     

Effect of the extender supplement Equex-STM on cryopreserved semen in the Assaf sheep

This study was designed to evaluate the effect of adding the detergent Equex-STM to the extender used to dilute semen for cryopreservation on several indicators of sperm preservation. Two consecutive ejaculates per day were obtained from 5 Assaf sheep on two days out of every week over three alternate months. The freezing protocol involved diluting the semen in Fiser's extender, to which 0.7 percent Equex-STM was added or omitted before cryopreserving the semen in straws by exposure to nitrogen vapor. Equex-STM supplementation gave rise to significantly (p=0.05) improved sperm quality variables after different periods of freezing (0 hours, 1 week and 1 month). The variables examined were: individual motility, viability, acrosome integrity, plasma membrane integrity (HOS test) and morphological anomalies. This improvement was independent of the ram and month of testing. In a second experiment in which we incubated the semen (0 and 6 hours) at 37 degree C after thawing, Equex-STM also showed a beneficial effect on sperm quality.     

Cryopreservation of adult bovine testicular tissue for spermatogonia enrichment

To develop a procedure for cryopreservation of adult bovine testis tissue, the effects of dimethyl sulphoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), and their concentrations (v/v), as well as different thawing temperatures, on the cell viability of bovine testis tissue after freezing/thawing were examined. The highest testicular cell viabilities came from the media containing DMSO (85.3 ± 1.2 percent), PG (82 ± 1.0 percent) and EG (83.4 ± 1.0 percent) at 10 percent concentration respectively. Using 10 percent DMSO gave significantly higher spermatogonia percentage (61.1 ± 1.2 percent, P < 0.001) than processing with 10 percent PG (54.3 ± 0.6 percent) or 10 percent EG (55 ± 1.8 percent) after differential plating. Thawing in water bath of 37 or 97-100 degree C also provided significantly higher viabilities (85.1 ± 1.0, 85 ± 1.0 percent, P < 0.01, respectively) and spermatogonia percentages (56.6 ± 2.0, 56.6 ± 2.6 percent, P < 0.01, respectively) than that thawing at 4C (23.4 ± 0.8 percent for total viability, 8.97 ± 1.0 percent for spermatogonia percentage). Collectively, 10 percent DMSO and thawing in 37-100 degree C water baths were appropriate for the cryopreservation of bovine testicular tissue and subsequent spermatogonia enrichment.