- Keywords
- FOLANDROL,
- MeSH
- Centrifugation, Density Gradient methods MeSH
- Fertilization in Vitro methods MeSH
- Sperm Injections, Intracytoplasmic methods MeSH
- Humans MeSH
- Magnetics MeSH
- Infertility, Male complications MeSH
- Oligospermia drug therapy MeSH
- Dietary Supplements MeSH
- Cell Separation methods MeSH
- Spermatozoa cytology MeSH
- Check Tag
- Humans MeSH
- Male MeSH
In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations.
- MeSH
- Centrifugation, Density Gradient methods MeSH
- Cryopreservation methods MeSH
- Sperm Motility physiology MeSH
- Silicon Dioxide chemistry MeSH
- Povidone chemistry MeSH
- Proteomics MeSH
- Fishes physiology MeSH
- Spermatozoa physiology MeSH
- Semen Preservation methods MeSH
- Cell Survival physiology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Research investigating the dynamics of male gametophyte (MG) development has proven to be challenging for the plant science community. Here we describe our protocol for separating Arabidopsis MG developmental stages, which is based on the centrifugation of pollen through a discontinuous Percoll concentration gradient. This Percoll gradient can be formed using a pipette, and it does not require a gradient maker. The purity of the isolated developing spores is as high as 70%, and in most separations it is well above 80%. Using this protocol, we can separate four different stages of pollen development-uninucleate microspore (UNM), bicellular pollen (BCP), tricellular immature pollen (TCP) and mature pollen grain (MPG). The duration of the separation procedure, excluding the cutting of flower inflorescences, is 6 h. This is reduced to 4 h when using a vacuum cleaning method to remove the MPGs before the Percoll density separation.
More than one 80S monosome can translate an mRNA molecule at a time producing polysomes. The most widely used method to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes is a high-velocity centrifugation of whole cell extracts in linear sucrose gradients. This polysome profile analysis technique has been routinely used to monitor translational fitness of cells under a variety of physiological conditions, to investigate functions of initiation factors involved in translation, to reveal defects in ribosome biogenesis, to determine roles of 5' UTR structures on mRNA translatability, and more recently for examination of miRNA-mediated translational repression (see an application of this protocol on Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).
Velocity separation of translation complexes in linear sucrose gradients is the ultimate method for both analysis of the overall fitness of protein synthesis as well as for detailed investigation of physiological roles played by individual factors of the translational machinery. Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and Western blotting, genome-wide microarray analysis or qRT-PCR. This chapter provides a basic overview of the polysome profile analysis technique and the RNA isolation procedure from sucrose gradients. We also discuss possible experimental pitfalls of data normalization, describe main alternatives of the basic protocol and outline a novel application of denaturing RNA electrophoresis in several steps of polysome profile analysis.
- MeSH
- Centrifugation, Density Gradient methods MeSH
- Electrophoresis methods MeSH
- Data Interpretation, Statistical MeSH
- Yeasts MeSH
- Polyribosomes chemistry MeSH
- Protein Biosynthesis genetics MeSH
- Gene Expression Regulation genetics MeSH
- RNA isolation & purification MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
PURPOSE: To detect and isolate cells with stem cell (SC) characteristics in the limbus of the mouse. METHODS: Limbal tissues from BALB/c mice were trypsin-dissociated and separated on the gradient Percoll (Fluka, Buchs, Switzerland). Several fractions were isolated and characterized by real-time PCR for the presence of limbal SC markers and differentiation markers of corneal epithelial cells by flow cytometry for the determination of the side-population (SP) phenotype and growth properties in vitro. RESULTS: Cells retained in the lightest fraction (40% Percoll) and in the densest fraction (80% Percoll) of the gradient were both enriched for populations with a high expression of the SC markers ABCG2 and Lgr5 and also expressing the SP phenotype. However, the lightest fraction (representing approximately 12% of total limbal cells) contained cells with the strongest spontaneous proliferative capacity and expressed the corneal epithelial differentiation marker K12. In contrast the densest fraction (<7% of original cells) was K12 negative and contained small nonspontaneously proliferating cells, which instead were positive for p63. Unexpectedly, cells from this fraction had the highest proliferative activity when cultured on a 3T3 feeder cell monolayer. CONCLUSIONS: These findings demonstrate the presence of two distinct populations of corneal epithelial cells with limbal SC characteristics, based on differential expression of the keratin-specific marker K12 and transcription factor p63, and suggest a difference in developmental stage of the two populations, with the K12(-)p63(+) population being closer to the primitive limbal SC
- MeSH
- ATP-Binding Cassette Transporters analysis genetics MeSH
- Cell Division MeSH
- 3T3 Cells MeSH
- Centrifugation, Density Gradient methods MeSH
- Fibroblasts cytology MeSH
- Financing, Organized MeSH
- Stem Cells cytology physiology MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Silicon Dioxide MeSH
- Polymerase Chain Reaction MeSH
- Povidone MeSH
- Flow Cytometry MeSH
- Epithelium, Corneal cytology MeSH
- Cell Separation methods MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
