Surface heterogeneity of bovine sperm revealed by aqueous two-phase partition

Ejaculated, bovine sperm have been subjected to multiple partition in aqueous two-phase systems. This partition, carried out in a countercurrent fashion, reveals heterogeneity of the sperm population with respect to surface properties. The sperm, when partitioned in phase systems that detect non-change associated surface properties (change-insensitive) are largely distributed as two distinct populations. In charge-sensitive phase systems (which principally detect cell surface molecules carrying charge) the sperm do not show any obvious surface heterogeneity. Considerable heterogeneity is revealed in affinity-ligand phase systems containing palmitic acid coupled to one of the phase components-poly(ethylene glycol). There is a difference in surface heterogeneity between sperm which have been washed in buffer or left unwashed, direct from the ejaculate. This is indicative of weak adsorption of proteins to the sperm surface in seminal fluid. These results show that bovine ejaculated sperm is a heterogeneous cell population having unequal distributions of a number of different surface molecules.     

Surface heterogeneity of rat sperm during maturation

Rat sperm isolated from the caput and caudal epididymis and the vas deferens were subjected to multiple partition in aqueous two-phase systems. The technique was used to reveal heterogeneity of a sperm population with respect to particular surface properties. Sperm from all three regions gave broad distributions indicative of heterogeneous cell populations. Greatest heterogeneity was observed for cauda sperm with caput and was sperm producing similar distributions.Following multiple partition sperm from different regions of the distribution profiles were immunostained with three antibodies known to recognise maturation antigens. The results show that some antigens are acquired during epididymal transit whilst others are present throughout.The partition (surface heterogeneity) seen cannot therefore be explained solely by the distribution of the antigens recognised by 2D6, 6B2 and 3D5.     

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems.

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging. The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6 degrees C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maxium occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.     

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging.The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6°C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to sialic acid-associated cell surface charges. The pattern of change in relation to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maximum occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.     

High level of male-biased Scandinavian admixture in Greenlandic Inuit shown by Y-chromosomal analysis

We have used binary markers and microsatellites on the Y chromosome to analyse diversity in a sample of Greenlandic Inuit males. This sample contains Y chromosomes typical of those found in European populations. Because the Y chromosome has a unique and robust phylogeny of a time depth that precedes the split between European and Native American populations, it is possible to assign chromosomes in an admixed population to either continental source. On this basis, 58+/-6% of these Y chromosomes have been assigned to a European origin. The high proportion of European Y chromosomes contrasts with a complete absence of European mitochondrial DNA and indicates strongly male-biased European admixture into Inuit. Comparison of the European component of Inuit Y chromosomes with European population data suggests that they have their origins in Scandinavia. There are two potential source populations: Norse settlers from Iceland, who may have been assimilated 500 years ago, and the Danish-Norwegian colonists of the eighteenth century. Insufficient differentiation between modern Icelandic and Danish Y chromosomes means that a choice between these cannot be made on the basis of diversity analysis. However, the extreme sex bias in the admixture makes the later event more likely as the source.     

The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems

It is of great interest and importance how different amino acid residues contribute to and affect the properties of a protein surface. Partitioning in aqueous two-phase systems has the potential to be used as a rapid and simple method for studying the surface properties of proteins. The influence on partitioning of the surface exposed amino acid residues of eight structurally determined monomeric proteins has been studied. The proteins were characterized in terms of surface exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP), and partitioned in two EO30PO70-dextran aqueous two-phase systems, only differing in polymer concentrations (system I: 6.8% EO30PO70, 7.1% dextran; system II: 9% EO30PO70, 9% dextran). We show for the first time that the partitioning behaviour of different monomeric proteins can be described by the differences in surface exposed amino acid residues. The contribution to the partition coefficient of the residues was found to be best characterized by peptide partitioning in the aqueous two-phase system. Compared to hydrophobicity scales available in the literature, each amino acid contribution is characterized by the slope given by the graph of log K against peptide chain length, for peptides of different length containing only one kind of residue. It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface. Surface hydrophobicity calculations realized for systems I and II gave respectively correlation coefficients of 0.961 and 0.949 for the linear relation between log K and calculated hydrophobicity values. To study the effect on the partition coefficient of different amino acids, they were grouped into classes according to common characteristics: the presence of an aromatic group, a long aliphatic chain or the presence of charge. Using these groups it was possible to confirm that aromatic residues have the strongest effect on the partition coefficient, giving preference to the upper EO30PO70 phase of the system; on the other hand the presence of charged amino acids on the protein surface enhances the partition of the protein to the lower dextran phase. It is also important to note that the sensitivity of the EO30PO70-dextran system for the surface exposed residues was increased by increasing the polymer concentrations. The partition coefficient of a monomeric protein can thus be predicted from its surface exposed amino acid residues and the system can also be used to characterize protein surfaces of monomeric proteins in general.     

Alterations in membrane surface properties during cell differentiation as measured by partition in aqueous two-polymer phase systems: Rat intestinal epithelial cells

Membrane surface properties of rat intestinal epithelial cells (crypt base to villus tips) were studied by cell partition in a two-polymer aqueous phase system. A higher partition generally reflects higher cell surface charge (or charge-associated properties) which is not necessarily the same as the charge determined by cell electrophoresis since the latter reflects only the charge at the plane of shear while the former gauges it deeper into the membrane [10]. Cells were prepared by the method of Weiser [22] which sequentially yields cell fractions from villus tips to crypt base. The isolated cells were subjected to countercurrent distribution in a dextran-polyethylene glycol aqueous two-phase system. Countercurrent distribution on the first fractions obtained by Weiser's method have a peak to the left and a smaller peak to the right indicating a surface membrane heterogeneity of upper villus cells; last fractions have a peak only to the right. When all fractions are pooled before countercurrent distribution two well-separated peaks are obtained with the right peak sometimes showing additional heterogeneities. Experiments combining isotope labeling of cells with countercurrent distribution lead us to conclude that the membrane charge (or charge-associated properties) of crypt base cells increases during differentiation and that the charge of the villus cells to which they give rise then diminishes during maturation. The charge of the bulk of the upper villus cells is the lowest of any in the intestinal cell population. The basis for the alteration in charge has not been established but the phenomenon of changing membrane surface charge (or charge-associated properties) as a function of cell differentiation, maturation and aging appears to be a general phenomenon having been found and traced in different cell populations [14, 16, 17, 28].     

On the use of mild hydrophobic interaction chromatography for "method scouting" protein purification strategies in aqueous two-phase systems: a study using model proteins

The behavior of a series of pure proteins partitioned in aqueous two-phase systems is compared with their behavior during mild hydrophobic interaction chromatography (HIC). A simple theoretical rationale for this comparison is presented based upon solvophobic theory. Similarities were found in the behavior of the model proteins in the two forms of partition chromatography. This indicates that HIC may be employed as a rapid instrumental technique for the broad characterization of protein behavior, which may be of benefit in the development of liquid-liquid partitioning strategies. However, it has proved difficult to completely account for this behavior on the basis of the known physical and structural properties of the proteins used. The variety in the detailed partitioning behavior of this small sample of protein types suggests that partition in aqueous two-phase systems is uniquely sensitive to subtle differences in surface properties of complex macromolecules. (c) 1994 John Wiley & Sons, Inc.     

Sperm DNA and sex chromosome differences between two geographical populations of the creeping vole, Microtus oregoni

The relative DNA content of the "O" and Y chromosome-bearing sperm is presented for the creeping vole, Microtus oregoni. The animals had been trapped in Oregon and in Washington State. The two populations had very similar autosomal chromosome relationships but differed greatly in the size of their X chromosome (which is not carried by vole sperm) and in their Y chromosome. The greater size and banding differences of the Y chromosome of the Washington State vole compared to the Oregon vole paralleled the greater differences in sperm DNA between the Y-bearing sperm and the sperm carrying no sex chromosome (O). The actual DNA differences between O and Y sperm was 12.5% for the sperm from the Washington State voles and 9.1% for sperm from the Oregon voles. The difference in sperm DNA content (12.5%) for Washington State voles was far greater than the difference shown for other voles or other mammals.     

A single partitioning step in aqueous polymer two-phase systems reduces hypotonized rat erythrocyte heterogeneity

Rat carrier erythrocytes prepared by hypotonic dialysis (80 mOsm/kg) are a heterogeneous cell population that can be fractionated into two-well-defined cell subpopulations by a single partition step, in charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. One subpopulation (65% of total cells) has a decreased cell surface charge and is partitioned at the interface in a single step and then fractionated by counter-current distribution as a low-G subpopulation. The other subpopulation (35% of total cells) has charge surface properties more like those of the untreated control rat erythrocytes. These last cells are partitioned in the top phase in a single step and then fractionated by counter-current distribution as a high-G subpopulation. Partitioning is more effective in reducing cell heterogeneity in hypotonized rat erythrocyte populations than is density separation in Ficoll-paque which only separates a small less dense cell subpopulation (5% of total cells), with the most fragile cells, from a larger and more dense cell subpopulation (95% of total cells), with a mixture of fragile and normal cells. This simple cell separation procedure quickly reduces carrier erythrocyte heterogeneity in a single partitioning step so it can be used to prepare cells for in vivo studies.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.     

Short-term inhibition of the energy metabolism affects motility but not surface properties of sperm cells

Centrifugal countercurrent distribution (CCCD) in aqueous two-phase systems has been proven to be a useful method to study subtle surface properties of spermatozoa. The present work shows that a short-term inhibition of the energy metabolism of sperm cells effected by incubating bovine sperm cells with KCN or ouabain, did not account for changes in the cell surface properties, as assessed either by estimation of the cell viability or by CCCD analysis. However, the short-term inhibition of energy metabolism provoked a clear decrease of cell motility, suggesting that a drop of cellular ATP levels brings about a rapid decrease of motility followed by a very delayed effect on cell surface properties. The relevance of these results on the handling of sperm and on the understanding of the molecular events underlying asthenospermia is discussed.     

Detection of Differences in Surface-charge-associated Properties of Cells by Partition in Two-polymer Aqueous Phase Systems

WHEN aqueous solutions of two polymers are mixed in certain proportions they may form two-phase systems^1,2 which can be buffered and used to partition and separate cells, particles and macromolecules by countercurrent distribution (CCD). Partition generally depends on polymer composition and concentration, the ionic composition and the charge sign of the material being partitioned. Such systems have been used to separate erythrocytes from white cells and erythrocytes on the basis of age. Changes in the surface properties of cells resulting from enzyme treatment or storage have also been demonstrated by this means³. Higher cell partition often accompanies increasing electrophoretic mobility which suggests that surface charge may be an important factor in partitioning^4–6. An apparent exception to this is the increased partition of stored human erythrocytes as compared with fresh⁷, as opposed to the mean electrophoretic mobility of both cell populations which remain identical⁸.     

Membrane surface properties of lymphocytes of normal (DBA/2) and autoimmune (NZB/NZW)F1 mice: effects of L-canavanine and a proposed mechanism for diet-induced autoimmune disease

Partitioning cells in a dextran polyethylene glycol aqueous two-phase system (countercurrent distribution, CCD) is a sensitive method for learning about cell surface membrane properties and for subfractionating cell populations. In this study, we subjected lymphocytes from normal DBA/2 mice and autoimmune F1 New Zealand black/New Zealand white [NZB/NZW)F1) mice to countercurrent distribution and found that T cells partition to the right and B cells partition to the left of the CCD curve. We found no difference between the CCD patterns of normal and autoimmune mice. When the murine lymphocytes were exposed to a cationic dietary amino acid (L-canavanine) in vitro, L-canavanine selectively affected the CCD pattern of autoimmune B cells, reflecting an alteration in surface membrane properties. We separated these lymphocytes with altered surface membrane properties by CCD. Impaired B-cell immune responses associated with L-canavanine were isolated to this lymphocyte fraction. This study provides the first evidence that alterations in the charged surface membrane properties are associated with abnormal (auto) immune response.     

Two Tests of Y Chromosomal Variation in Male Fertility of Drosophila Melanogaster

Deficiency mapping with Y autosome translocations has shown that the Y chromosome of Drosophila melanogaster carries genes that are essential to male fertility. While the qualitative behavior of these lesions provides important insight into the physiological importance of the Y chromosome, quantitative variation in effects on male fertility among extant Y chromosomes in natural populations may have a significant effect on the evolution of the Y chromosome. Here a series of 36 Y chromosome replacement lines were tested in two ways designed to detect subtle variation in effects on male fertility and total male fitness. The first test involved crossing males from the 36 lines to an excess of females in an attempt to measure differences in male mating success (virility) and male fecundity. The second test challenged males bearing each of the 36 Y chromosomes to competition in populations with males bearing a standard, phenotypically marked (BsY) chromosome. These tests indicated that the Y chromosome lines did not differ significantly in either male fertility or total fitness, but that interactions with autosomes approached significance. A deterministic population genetic model was developed allowing Y autosome interaction in fertility, and it is shown that, consistent with the experimental observations, this model cannot protect Y-linked polymorphism.     

Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH.     

Hydrophobic affinity partition of spinach chloroplasts in aqueous two-phase systems

The surface properties of spinach chloroplasts, both of intact chloroplasts with surrounding envelope and broken chloroplasts consisting of the inner lamellar system, have been studied by partitioning them between two aqueous phases, especially using counter-current distribution technique. The two-phase system consists of poly(ethyleneglycol), dextran and water. The two polymers are enriched in opposite phases and by binding deoxycholate or palmitate to one of the polymers the affinity of chloroplasts for the corresponding phase is strongly enhanced. The partition of the two classes of chloroplasts, however, is not affected to the same degree and the affinity of the chloroplast envelope for deoxycholate and palmitate is stronger than that of the lamellar system. This has been correlated to the chemical composition of the two types of membranes. By studying the effect of salts on the partition it has been found that the lamellar system bears a larger number of negative charges as compared to the envelope of the intact chloroplast.     

A model for the prediction of partition coefficients in aqueous two-phase systems.

A mathematical model has been developed to predict partition coefficients for aqueous two-phase systems. The model is based on a previously-developed equation for partitioning which arises from an osmotic pressure viral expansion. The model suggests that the properties of importance are the concentration difference of one of the phase-forming components, such as a polymer, and the hydrophobicity of the solute relative to the hydrophobic-hydrophilic difference between the two phases. Several two-phase systems have been studied, with a particular emphasis on the poly(ethylene glycol)/magnesium sulfate system. Numerous solutes, including peptides, were used in this system and their partition coefficients show good agreement with the model.     

Phenotypic effects of the Y chromosome are variable and structured in hybrids among house mouse recombinant lines

Hybrid zones between divergent populations sieve genomes into blocks that introgress across the zone, and blocks that do not, depending on selection between interacting genes. Consistent with Haldane's rule, the Y chromosome has been considered counterselected and hence not to introgress across the European house mouse hybrid zone. However, recent studies detected massive invasion of M. m. musculus Y chromosomes into M. m. domesticus territory. To understand mechanisms facilitating Y spread, we created 31 recombinant lines from eight wild‐derived strains representing four localities within the two mouse subspecies. These lines were reciprocally crossed and resulting F1 hybrid males scored for five phenotypic traits associated with male fitness. Molecular analyses of 51 Y‐linked SNPs attributed ~50% of genetic variation to differences between the subspecies and 8% to differentiation within both taxa. A striking proportion, 21% (frequencies of sperm head abnormalities) and 42% (frequencies of sperm tail dissociations), of phenotypic variation was explained by geographic Y chromosome variants. Our crossing design allowed this explanatory power to be examined across a hierarchical scale from subspecific to local intrastrain effects. We found that divergence and variation were expressed diversely in different phenotypic traits and varied across the whole hierarchical scale. This finding adds another dimension of complexity to studies of Y introgression not only across the house mouse hybrid zone but potentially also in other contact zones.     

Countercurrent distribution of L line monolayer and suspension cell cultures in a 2-phase polymer system

The distribution of two sulines of L cells, differing in adhesive properties (monolayer and suspension cultures), have been investigated using polymer two-phase system dextran-500-polyenthylenglycol-600. By counter-current distribution it has been shown that most of the cells growing in monolayer have low coefficients of partition, unlike cells grown in suspension having a high coefficient of partition. It is supposed that differences revealed in properties of the cell surface may be associated with the ability of cells to form adhesive contacts.