Freezing of stallion epididymal sperm

Inseminations with frozen-thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 degrees C for 24h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio, EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen-thawed epididymal sperm showing that Botu-Crio was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp+Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.     

Proceedings: Freezing injury to streptococcal L-phase variant

A comparative study of resistance to freezing in Streptococcus haemolyticus S.8 and its L-phase variant was made by viable counts of postthaw survival and also by morphological observation of freezing patterns with an electron microscope. It was recognized that L-phase cells are more susceptible to freezing than their original strains.     

Freezing injury to erythrocytes. II. Morphological alterations of cell membranes.

Morphological alterations of human red blood cell membranes were examined with the cells containing different concentrations of glycerol being subjected to rapid rates of cooling, approximately 10⁴ and 10⁵ °C/min, and subsequent rewarming. Small membrane defects, similar to holes, were observed in specimens frozen with and without 10% glycerol. Various degrees of roughness were found on the surface of the cells at all freezing rates tested. The membrane alterations were reduced with increasing glycerol concentration, although roughness also appeared on the surface of the cells in 30% glycerol suspensions, frozen rapidly, and rewarmed to ?80 or ?60 °C. The cell membrane surface texture correlated with the growth of intra- and extracellular ice particles. There was also a positive correlation between these alterations and post-thaw hemolysis. It is concluded, therefore, that morphological alterations appearing on the erythrocyte membranes may be a manifestation of freezing damage.     

The relationship between cell injury and osmotic volume reduction: III. Freezing injury and frost resistance in winter wheat

In order to distinguish between several possible mechanisms of frost hardening in winter wheat (Triticum aestivum L.) cells from two hardy and two tender cultivars were plasmolyzed in CaCl₂ solution at room temperature and cell volumes estimated by microscopic examination. Analyses of Boyle-van't Hoff plots of these data reveal that all cells from cultivars progressively increase their intracellular solute concentration up to 20 days hardening. This increase, which we had predicted from published calorimetric data to be the sole mechanism of hardening explained less than half of the increase in hardening seen in the most hardy cultivar, Kharkov. Hardening also increased the osmotically inactive volume.At CaCl₂ concentrations greater than 5%, plasmolyzed protoplasts departed further from the Boyle-van't Hoff prediction, remaining larger than expected until some higher concentration of CaCl₂, where protoplast volume again sharply decreased. In all cultivars except hardened Kharkov, the concentration of CaCl₂ producing this abrupt volume decrease had a freezing point corresponding to the killing temperature. If this concentration was exceeded during plasmolysis, then the protoplasts burst during deplasmolysis at some volume less than their original volume.We interpret these data to mean that, in addition to the often described hardening mechanism of increased cell solute and water binding, winter wheat shows a third mechanism, a mechanical resistance to protoplast shrinkage which produces volumes larger than those predicted during osmotic stress. The resisting element appears to be the plasma membrane itself. Shrinkage brings the membrane under compressive stress, developing tangential pressure within it. Cell injury occurs when the cell membrane area has been reduced to the point at which irreversible loss of membrane material is inevitable. Cell death occurs during deplasmolysis when the protoplast bursts because its membrane contains insufficient material to subtend the area of the cell wall.Of the cultivars tested, hardened Kharkov was unique in avoiding injury. Hardened Kharkov was injured only after the volume inflection had been greatly exceeded. Refractile droplets of lipid appeared in the cytoplasm of hardened Kharkov protoplasts during plasmolysis but disappeared during deplasmolysis suggesting that hardy Kharkov was able reversibly to store membrane lipids in cytoplasmic vesicles and return them to the membrane on deplasmolysis.     

Oxidative stress: the special case of diabetes

The implication of oxidative stress (OS) in diabetes is a major concern for the development of therapeutics aimed at improving the metabolic and/or vascular dysfunctions of this burdening disease. Ample evidence is available suggesting that OS is present in essentially all tissues and can even be observed in prediabetic states. This raises the question of the origin of OS and suggests that, although hyperglycemia is largely linked with free radical production, its role may mainly be the aggravation of a preexisting state. Indeed other factors are also causally linked to OS, such as hormones and lipids. The main debate is about the pertinence of antioxidant therapy since the large scale clinical trials performed recently have essentially failed to show any significant improvement in metabolic or vascular disturbances of diabetic patients. However this conclusion must be tempered by the fact that they have mainly been using vitamin E +/-C; indeed many arguments suggest that either the choice or the application modalities of these substances may have been inadequate. Potential reasons for the actual failure of antioxidant therapy in diabetes are discussed; the indisputable involvement of OS in this disease still leaves hope for alternative therapeutic approaches.     

Freezing response of white bass (Morone chrysops) sperm cells

The water transport response during freezing of sperm cells of Morone chrysops (white bass, WB) was obtained using a shape-independent differential scanning calorimeter (DSC) technique. Sperm cell suspensions were frozen at a cooling rate of 20 degrees C/min in two different media: (1) without cryoprotective agents (CPAs), or (2) with 5% (v/v) dimethyl sulfoxide (Me2SO). For calculations, the sperm cell was modeled as a cylinder of length 24.8 microm and diameter of 0.305 microm, while the osmotically inactive cell volume (Vb) was assumed to be 0.6 Vo, where Vo was the isotonic or the initial cell volume. By fitting a model of water transport to the experimentally determined water transport data, the best fit membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined, and ranged from Lpg = 0.51-1.7 x 10(-15) m3/Ns (0.003-0.01 microm/min-atm), and ELp = 83.6-131.3 kJ/mol (20.0-31.4 kcal/mol). The parameters obtained in this study suggest that the optimal rate of cooling for M. chrysops sperm cells is approximately 22 degrees C/min, a value that compares closely with experimentally determined optimal rates of cooling (approximately 16 degrees C/min).     

Freezing injury in cold-acclimated and unhardened spinach leaves

Spinach plants (Spinacia oleracea L.) were frost-hardened by cold-acclimation to 1° C or kept in an unhardy state at 20°/14° C in phytotrons. Detached leaves were exposed to temperatures below 0°C. Rates of photosynthetic CO₂ uptake by the leaves, recorded after frost treatment, served as a measure of freezing injury. Thylakoid membranes were isolated from frost-injured leaves and their photosynthetic activities tested. Ice formation occurred at about-4° to-5° C, both in unhardened and cold-acclimated leaves. After thawing, unhardened leaves appeared severely damaged when they had been exposed to-5° to-8° C. Acclimated leaves were damaged by freezing at temperatures between-10° to-14° C. The pattern of freezing damage was complex and appeared to be identical in hardened and unhardened leaves: 1. Inactivation of photosynthesis and respiration of the leaves occurred almost simultaneously. 2. When the leaves were partly damaged, the rates of photosynthetic electron transport and noncyclic photophosphorylation and the extent of light-induced H⁺ uptake by the isolated thylakoids were lowered at about the same degree. The dark decay of the proton gradient was, however, not stimulated, indicating that the permeability of the membrane to-ward protons and metal cations had not increased. 3. As shown by partial reactions of the electron transport system, freezing of leaves predominantly inhibited the oxygen evolution, but photosystem II and photosystem I-dependent electron transport were also impaired. 4. Damage of the chloroplast envelope was indicated by a decline in the percentage of intact chloroplasts found in preparations from injured leaves. The results are discussed in relation to earlier studies on freezing damage of thylakoid membranes occurring in vitro.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenol indophenol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES 2(N-morpholino) ethane sulfonic acid     

Lens crystallins and oxidation: the special case of gammaS

Among lens crystallins, gamma-crystallins are particularly sensitive to oxidation, because of their high amount of Cys and Met residues. They have the reputation to induce, upon ageing, lens structural modifications leading to opacities. A combination of small angle X-ray scattering and chromatography was used to study the oxidation of gamma-crystallins. At pH 7.0, all the gamma-crystallins under study were checked to have the same structure in solution. Under gentle oxidation conditions at pH 8.0, human gammaS (hgammaS) and bovine gammaS (bgammaS) formed disulfide-linked dimers, whereas the other bgamma-crystallins did not. Cys20 was shown to be responsible for dimer formation since the C20S mutant only formed monomers. The hgammaS dimers were stable for weeks and did not form higher oligomers. In contrast, monomeric gammaS-crystallins freshly prepared at pH 8.0, and submitted to more drastic oxidation by X-ray induced free radicals, were rapidly transformed into higher oligomers. So, only extensive oxidation causing partial unfolding could be detrimental to the lens and linked to cataract formation. The gammaS-crystallins lack the temperature-induced opacification observed with the other gamma-crystallins and known as cold cataract. The oxidation-induced associative behaviour and cold cataract are therefore demonstrated to be uncoupled.     

Freezing dog semen in presence of the antioxidant butylated hydroxytoluene improves postthaw sperm membrane integrity

In an attempt to evaluate the protective effect of a lipid-soluble antioxidant (butylated hydroxytoluene; BHT), semen from four dogs (Canis familiaris) was frozen in two different extenders (Uppsala or INRA-96 plus glycerol) with or without 1 mM BHT. Sperm membrane integrity using flow cytometry and motility using a computerized system were evaluated in each experimental group. The Uppsala extender was superior in all aspects of sperm function. The percentage of sperm membranes was significantly higher in semen samples frozen in presence of BHT. Our results suggest that the Uppsala extender can be improved with the addition of BHT.     

Mitotic catastrophe: a special case of apoptosis

Mitotic catastrophe is a poorly defined type of cell death linked to the abnormal activation of cyclin B/Cdk1. Here we propose that a conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe, provided that cell cycle checkpoints are inhibited, in particular the DNA structure checkpoints and the spindle assembly checkpoint. Two subtypes of mitotic catastrophe can be distinguished. First, mitotic catastrophe can kill the cell during or close to the metaphase, in a p53-independent fashion, as this occurs in Chk2-inhibited heterokarya generated by fusion. Second, mitotic catastrophe can occur after failed mitosis, during the activation of the polyploidy checkpoint, in a partially p53-dependent fashion. In these conditions, cells die as a result of caspase activation and mitochondrial membrane permeabilization that constitute hallmarks of apoptosis. Prevention of caspase activation and/or mitochondrial damage avoids mitotic catastrophe, indicating that this form of cell death indeed constitutes a special case of apoptosis. Importantly, the suppression of mitotic catastrophe can favor asymmetric division and the generation of aneuploid cells. This delineates a molecular pathway through which failure to arrest the cell cycle and inhibition of apoptosis can favor the occurrence of cytogenetic abnormalities which are likely to participate in oncogenesis.     

The tailored sperm cell

Sperm are ubiquitous and yet unique. Genes involved in sexual reproduction are more divergent than most genes expressed in non-reproductive tissues. It has been argued that sperm have been altered during evolution more than any somatic cell. Profound variations are found at the level of morphology, motility, search strategy for the egg, and the underlying signalling mechanisms. Sperm evolutionary adaptation may have arisen from sperm competition (sperm from rival males compete within the female’s body to fertilize eggs), cryptic female choice (the female’s ability to choose among different stored sperm), social cues tuning sperm quality or from the site of fertilization (internal vs. external fertilization), to name a few. Unquestionably, sperm represent an invaluable source for the exploration of biological diversity at the level of signalling, motility, and evolution. Despite the richness in sperm variations, only a few model systems for signalling and motility have been studied in detail. Using fast kinetic techniques, electrophysiological recordings, and optogenetics, the molecular players and the sequence of signalling events of sperm from a few marine invertebrates, mammals, and fish are being elucidated. Furthermore, recent technological advances allow studying sperm motility with unprecedented precision; these studies provide new insights into flagellar motility and navigation in three dimensions (3D). The scope of this review is to highlight variations in motile sperm across species, and discuss the great promise that 3D imaging techniques offer into unravelling sperm mysteries.     

Distinguishing medical practice and research: the special case of IVF

The debate over the acceptability of human embryo research takes place within the context of a broader regulatory framework for experimentation with human subjects. Using the Australian situation for illustration, Gaze and Dawson examine the concepts of therapeutic, nontherapeutic, and destructive experimentation in medical practice. They explore the problems of applying these concepts to human embryo research by asking whether the embryo or the woman undergoing in vitro fertilization (IVF) treatment is the experimental subject. They argue that when the embryo is treated as the sole subject, the woman is lost sight of, and that research that is therapeutic for the embryo may be nontherapeutic for her. Regulation of embryo research should take into account the relationship between research and the woman's treatment, the dependence of embryos upon women for gestation, and the empirical, uncertain nature of biomedical knowledge.     

Tolerance to cadmium in plants: the special case of hyperaccumulators

On sols highly polluted by trace metallic elements the majority of plant species are excluders, limiting the entry and the root to shoot translocation of trace metals. However a rare class of plants called hyperaccumulators possess remarkable adaptation because those plants combine extremely high tolerance degrees and foliar accumulation of trace elements. Hyperaccumulators have recently gained considerable interest, because of their potential use in phytoremediation, phytomining and biofortification. On a more fundamental point of view hyperaccumulators of trace metals are case studies to understand metal homeostasis and detoxification mechanisms. Hyperaccumulation of trace metals usually depends on the enhancement of at least four processes, which are the absorption from the soil, the loading in the xylem in the roots and the unloading from the xylem in the leaves and the detoxification in the shoot. Cadmium is one of the most toxic trace metallic elements for living organisms and its accumulation in the environment is recognized as a worldwide concern. To date, only nine species have been recognized as Cd hyperaccumulators that is to say able to tolerate and accumulate more than 0.01 % Cd in shoot dry biomass. Among these species, four belong to the Brassicaceae family with Arabidopsis halleri and Noccaea caerulescens being considered as models. An update of our knowledge on the evolution of hyperaccumulators will be presented here.     

Freezing stresses and hydration of isolated cell walls

The hydration of the cell walls of the giant alga Chara australis was measured as a function of temperature using quantitative deuterium nuclear magnetic resonance (NMR) of samples hydrated with D2O. At temperatures 23-5K below freezing, the hydration ratio (the ratio of mass of unfrozen water in microscopic phases in the cell wall to the dry mass) increases slowly with increasing temperature from about 0.2 to 0.4. It then rises rapidly with temperature in the few Kelvin below the freezing temperature. The linewidth of the NMR signal varies approximately linearly with the reciprocal of the hydration ratio, and with the freezing point depression or water potential. These empirical relations may be useful in estimating cell wall water contents in heterogeneous samples.     

Roles for the extracellular matrix in plant development and pollination: a special case of cell movement in plants.

Pattern formation in plants is now thought to be primarily dependent on positional information during development. We discuss the prevalent theories on how position is deciphered by cells in an organism and highlight the recent advances implicating molecules of the cell wall or extracellular matrix (ECM) in this process. We compare the functions of the ECM in plants and animals and describe the various cell and substrate adhesion molecules of the animal ECM which play a role in morphogenesis and cell movement. We propose that analogous molecules may occur in plants and provide evidence for the presence of a substrate adhesion molecule like vitronectin in plants and algae. We provide a model for how substrate adhesion molecules may be involved in a special case of cell movement in plants, pollination.     

Arabinogalactan proteins at the cell surface ofBrassica sperm andLilium sperm and generative cells

Antibodies to arabinogalactan proteins were tested for binding to sperm cells ofBrassica campestris and to generative cells and sperm ofLilium longiflorum. Two monoclonal antibodies, JIM8 and JIM13, bound toBrassica sperm in pollen grains and pollen tubes and to isolated sperm. Sperm pairs retained within the vegetative cell inner plasma membrane fluoresced more brightly than single sperm, indicating that the vegetative cell inner plasma membrane that surrounds sperm pairs also contains arabinogalactan proteins. Isolated sperm pairs exhibited a uniform fluorescence while single sperm had patches of fluorescence. InLilium, isolated generative cells and single sperm cells bound antibodies in a patchy pattern. Antibodies to arabinogalactan proteins may be useful in describing the overall shape of sperm cells and for identifying sperm among other cell types.