Single UV excitation of Hoechst 33342 and propidium iodide for viability assessment of rhesus monkey spermatozoa using flow cytometry

Many fluorescent probes excited by visible light have been used to assess sperm quality by flow cytometry. Developing a viability evaluation method using UV excited stains would be useful for multiparameter analysis of sperm function. This investigation was conducted to determine the efficacy of Hoechst 33342 (H342) and propidium iodide (PI) dual staining for evaluating rhesus monkey sperm viability through use of flow cytometry and excited by a single UV laser. The results showed that the live cells stained only with H342 strongly correlated with expected sperm viability, and flow cytometric analyses were highly correlated with fluorescence microscopic observation. Using H342/PI/SYBR-14 triple staining method, it was found that the live/dead sperm distributions were completely concordant in both H342/PI and SYBR-14/PI assays. In addition, this dual staining was extended with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) to simultaneously analyze viability and acrosome integrity of sperm cryopreserved using two different extenders, TTE and TEST, and indicated that TTE offered better Preservation of plasma and acrosome integrity than TEST Therefore, the H342/PI dual staining provides an accurate technique for evaluating viability of rhesus monkey sperm and should be valuable for multiparameter flow cytometric analysis of sperm function.

Regulation of zygotic gene activation in the preimplantation mouse embryo: Global activation and repression of gene expression

Superimposed on the activation of the embryonic genome in the preimplantation mouse embryo is the formation of a transcriptionally repressive state during the two-cell stage. This repression appears mediated at the level of chromatin structure, because it is reversed by inducing histone hyperacetylation or inhibiting the second round of DNA replication. We report that of more than 200 amplicons analyzed by mRNA differential display, about 45% of them are repressed between the two-cell and four-cell stages. This repression is scored as either a decrease in amplicon expression that occurs between the two-cell and four-cell stages or on the ability of either trichostatin A tan inhibitor of histone deacetylases) or aphidicolin tan inhibitor of replicative DNA polymerases) to increase the level of amplicon expression. Results of this study also indicate that about 16% of the amplicons analyzed likely are novel genes whose sequence doesn't correspond to sequences in the current databases, whereas about 20% of the sequences expressed during this transition likely are repetitive sequences. Lastly, inducing histone hyperacetylation in the two-cell embryos inhibits cleavage to the four-cell stage. These results suggest that genome activation is global and relatively promiscuous and that a function of the transcriptionally repressive state is to dictate the appropriate profile of gene expression that is compatible with further development.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

Applicability of the MTT assay for measuring viability of cyanobacteria and algae, specifically for Microcystis aeruginosa (Chroococcales, Cyanobacteria)

Several assay methods were screened for viability assessment in cyanobacteria using Microcystis aeruginosa FACHB 905. Compared with fluorescent diacetate (FDA), Evan's Blue and autofluorescence, the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) assay, which was based on the ability of viable cells to reduce MTT to formazan, was found to be reliable and was selected for further study. MTT concentration, incubation time and temperature were optimized for M. aeruginosa. Improvements to the sensitivity and reproducibility of the MTT assay included performing it in the dark to reduce the effects of formazan light sensitivity when extracted in DMSO. Another improvement involved collecting viability data by cell by counting rather than colourimetrically, which was concluded from the fact that oxidoreductase activity, responsible for MTT reduction, would elevate or decrease under stress conditions. Half-life of oxidoreductase in dead cell was calculated to be 3 h. The MTT assay was also found to be applicable to other cyanobacteria and diatoms, including field samples, but not for algae belonging to Chlorophyta, Euglenophyta, Pyrrophyta or Chrysophyta. Based on the above results, we proposed an optimized procedure for the MTT method on Microcystis strains. The use of this assay may be of importance to better understand the dynamics of bloom and the fate of Microcystis under natural or disturbed conditions.

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%). (C) 2002 Wiley-Liss, Inc.

The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach, Misguruns mizolepis Gunthe

Microcystin-LR, a specific and potent hepatotoxin, was tested for its effects oil loach embryo-larval and juvenile development, The results of this study showed that loach embryos were more sensitive when exposed to microcystin-LR at a later than at an earlier stage of development, Juveniles were far less sensitive to MC-LR than were embryos and larvae. Mortality and developmental abnormality were proven to be dose-dependent and to be stage-specific sensitive. Among the abnormal changes noted were: pericardial edema and tubular heart, bradycardia, homeostasis, poor yolk resumption. small head, curved body and tail, and abnormal hatching, Liver and heart were the main targets of microcystin-LR toxicity. Ultrastructural analysis documented a complex set of sublethal effects of microcystin-LR on loach hepatocytes, chiefly including morphological alteration in nuclear and RER of loach liver cells. fit addition, microcystin-LR was lethal to loach juvenile in the subacute (7 days) exposure (LC50) = 593.3 mug/l). (C) 2002 Elsevier Science Ltd. All rights reserved.

Effects of subretinal implant materials on the viability, apoptosis and barrier function of cultured RPE cells

Background: Subretinal microphotodiode array (MPDA) is a type of visual prosthesis used for the implantation in the subretinal space of patients with progressive photoreceptor cell loss. The present study aimed to evaluate the effect of materials for MPDA on the viability, apoptosis and barrier function of cultured pig retinal pigment epithelium (RPE) cells.Methods: Primary culture of pig RPE cells was performed and 24 pig eyes were used to start RPE culture. The third passage of the cultures was plated on different materials for MPDA and MPDAs. The tetrazolium dye-reduction assay (MTT) was used to determine RPE cell viability. Flow cytometry was measured to indicate the apoptosis rates of RPE cells on different materials. RPE cells were also cultured on microporous filters, and the transepithelial resistance and permeability of the experimental molecule were measured to determine the barrier function.Results: The data from all the methods indicated no significant difference between the materials groups and the control group, and the materials tested showed good biocompatibility.Conclusions: The materials for MPDA used in the present study had no direct toxicity to the RPE cells and did not release harmful soluble factors that affected the barrier function of RPE in vitro.

Organic-inorganic hybrid material for the cells immobilization: Long-term viability mechanism and application in BOD sensors

In this paper, organic-inorganic hybrid material, which is composed of silica and the grafting copolymer of poly (vinyl alcohol) and 4-vinylpyridine (PVA-g-P(4-VP)), was employed to immobilize Trichosporon cutaneum strain 2.570 cells. Cells entrapped into the hybrid material were found to keep a long-term viability. The mechanism of such a long-term viability was investigated by using confocal laser scanning microscopy (CLSM). Our studies revealed that arthroconidia produced in the extracellular material might play an important role in keeping the long-term viability of the immobilized microorganism. After the arthroconidia were activated, an electrochemical biochemical oxygen demand (BOD) sensor based on cell/hybrid material-modified supporting membrane was constructed for verifying the proposed mechanism.

beta-catenin in early development of the lancelet embryo indicates specific determination of embryonic polarity

The lancelet (amphioxus) embryo develops from a miolecithal egg and starts gastrulation when it is approximately 400 cells in size, in a fashion similar to that of some non-chordate deuterostomes. Throughout this type of gastrulation, the embryo develops characteristics such as the notochord and hollow nerve cord that commonly appear in chordates. beta-Catenin is an important factor in initiating body patterning. The behavior and developmental pattern of this protein in early lancelet development was examined in this study. Cytoplasmic beta-catenin was localized to the animal pole after fertilization and then was incorporated asymmetrically into the blastomeres during the first cleavage. Asymmetric distribution was observed at least until the 32-cell stage. The first nuclear localization was at the 64-cell stage, and involved all of the cells. At the initial gastrula stage, however, concentrated beta-catenin was found on the dorsal side. LiCl treatment affected the asymmetric pattern of beta-catenin during the first cleavage. LiCl also changed distribution of nuclear beta-catenin at the initial gastrula stage: distribution extended to cells on the animal side. Apparently associated with this change, expression domains of goosecoid, lhx3 and otx also changed to a radially symmetric pattern centered at the animal pole. However, LiCl-treated embryos were able to establish embryonic polarity. The present study suggests that in the lancelet embryo, polarity determination is independent of dorsal morphogenesis.

Early expressed genes showing a dichotomous developing pattern in the lancelet embryo

Lancelets (amphioxus), although showing the most similar anatomical features to vertebrates, never develop a vertebrate-like head but rather several structures specific to this animal. The lancelet anatomical specificity seems to be traceable to early developmental stages, such as the vertebrate dorsal and anterior-posterior determinations. The BMP and Wnt proteins play important roles in establishing the early basis of the dorsal structures and the head in vertebrates. The early behavior of BMP and Wnt may be also related to the specific body structures of lancelets. The expression patterns of a dpp-related gene, Bbbmp2/4, and two wnt-related genes, Bbwnt7 and Bbwnt8, have been studied in comparison with those of brachyury and Hnf-3 beta class genes The temporal expression patterns of these genes are similar to those of vertebrates; Bbbmp2/4 and Bbwnt8 are first expressed in the invaginating primitive gut and the equatorial region. respectively, at the initial gastrula stage. However, spatial expression pattern of Bbbmp2/4 differs significantly from the vertebrate cognates. It is expressed in the mid-dorsal inner layer of gastrulae and widely in the anterior region, in which vertebrates block BMP signaling, The present study suggests that the lancelet embryo may have two distinct developmental domains from the gastrula stage, the domains of which coincide later with the lateral diverticular and the somitocoelomic regions. The embryonic origin of the anterior-specific structures in lancelets corresponds to the anterior domain where Bbbmp2/4 is continuously expressed.

The encysted dormant embryo proteome of Artemia sinica

The possibility of the brine shrimp Artemia to produce dormant embryo (cysts) in diapause is a key feature in its life history. In the present study, we obtained a proteomic reference map for the diapause embryo of Artemia sinica using two-dimensional gel electrophoresis with a pH range of 4-7 and a molecular weight range of 10-100 kDa. Approximately 233 proteins were detected, and 60 of them were analyzed by capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Of these, 39 spots representing 33 unique proteins were identified, which are categorized into functional groups, including cell defense, cell structure, metabolism, protein synthesis, proteolysis, and other processes. This reference map will contribute toward understanding the state of the diapause embryo and lay the basis and serve as a useful tool for further profound studies in the proteomics of Artemia at different developmental stages and physiological conditions.

Comparative growth and viability of hybrids between two populations of Chinese shrimp (Fennropenaeus chinensis)

Fenneropenaeus chinensis is confined to the Yellow Sea and Bohai Sea in China and the West Coast of the Korean Peninsula. Intra- and intercross populations were produced between Rushany (YP) and Korean (KN) populations. Seven traits were recorded. The heterosis of hybrids was computed and comparison between treatments was performed by ANOVA. At the fourth month after post-larvae, six indexes of growth trait and viability showed a range of heterosis, ranging from 0.514% to 14.950%. At the fifth month after post-larvae, six indexes of growth trait and viability ranged from -9.000% to 19.090%. The negative heterosis was observed in CL, HST and viability. The heterosis of KN female xYP male tended generally to increase as the age of the Chinese shrimp increased while the heterosis of YP female xKN male tended to decrease. The results indicated that the viability of reciprocal hybrids were not significantly different (P > 0.05) from their parents during the experiment. The result of ANOVA indicated that the F1 hybrids were significantly different (P < 0.05) in WST and TW at the fourth month. The multiple comparisons of LSD test indicated that the hybrids of YP female xKN male were significantly different (P < 0.05) from their parents in TW. The hybrids of YP female xKN male were significantly different (P < 0.05) from the other three combinations in WST. At the fifth months, the F1 hybrids had significant difference (P < 0.05) in body weight while other traits showed no significant differences (P > 0.05) from their parents. The multiple comparisons of LSD test indicated that the hybrids of KN female xYP male were significantly different (P < 0.05) from the KN parents in TW. The results indicate that in experimental conditions, the F-1 hybrids created from two populations of Chinese shrimp showed a certain level of heterosis for growth performance and viability. The crossing scheme may improve growth performance and viability in Chinese shrimp, but the improvement may be limited because effective crossbreeding requires the maintenance of pure, preferably inbred, lines and possibly involves specialized sire and dam lines. Therefore, the exploitation of heterosis through single crossbreeding in Chinese shrimp is of limited utility in practical commercial shrimp aquaculture in spite of the potential of significant heterosis. The crossbreeding of different populations can be applied in the establishment of base populations.

Genetic improvement on Chinese shrimp (Fenneropenaeus chinensis): growth and viability performance in F1 hybrids of different populations

Fenneropenaeus chinensis distributed in the Yellow Sea and Bohai Sea of China and the west coast of the Korean Peninsula. Different geographical populations represent potentially different genetic resources. To learn further the characteristics of different geographical population, crosses among two wild and three farmed populations were produced. The two wild populations were from the Yellow Sea and Bohai Sea (WYP), and the west coast of the Korean Peninsula and coast (WKN). The three farmed populations included the offspring of first generation of wild shrimp from coast in Korea (FKN), the Huang Hai (the Yellow Sea in Chinese) No.1 (HH1), and JK98. The phenotypes growth and survival rates of these populations were compared to confirm the feasibility for crossbreeding. The body length (BL), carapace length (CL), carapace width (CW), height of the second and third abdominal segment (HST), width of the second and third abdominal segment (WST), length of the first abdominal segment (LF), length of the last abdominal segment (LL), live body weight (BW), and survival rate were measured. Different combinations were statistically performed with ANOVA and Duncan's Multiple Range Test. The results show that the survival rate of JK98(a (TM) Euro)xWKN(a (TM),) was the highest, followed by WYP(a (TM) Euro)xWKN(a (TM),), FKN(a (TM) Euro)xWYP(a (TM),), FKN(a (TM) Euro)xHH1(a (TM),) and WYP(a (TM) Euro)xFKN(a (TM),); the body weight of FKN(a (TM) Euro)sxHH1(a (TM),) was the highest, followed by FKN(a (TM) Euro)xWYP(a (TM),), WYP(a (TM) Euro)xWKN(a (TM),), WYP(a (TM) Euro)xFKN(a (TM),) and JK98(a (TM) Euro)xWKN(a (TM),); the total length had the same ranking as the body weight. All growth traits in hybrids JK98(a (TM) Euro)xWKN(a (TM),) were the lowest among all combinations. F1 hybrids had significant difference (P < 0.05) in BL, CL, HST, LL, and BW; and insignificant difference (P > 0.05) in other growth traits and survival rate. The results of Duncan's Multiple Range Test are that BL and CL of JK98(a (TM) Euro)xWKN(a (TM),) were significantly different from the other combinations; HST different from the combination of FKN(a (TM) Euro)xWYP(a (TM),), FKN(a (TM) Euro)xHH1(a (TM),) and WYP(a (TM) Euro)xWKN(a (TM),); and BW different from FKN(a (TM) Euro)xWYP(a (TM),) and FKN(a (TM) Euro)xHH1(a (TM),). As a whole, the results indicate that the FKN(a (TM) Euro)xHH1(a (TM),) was the best combination in all growth traits. Therefore, hybridization can introduce the variation to base populations. The systematic selection program based on additive genetic performance may be more effective than crossbreeding.