Isolation of motile spermatozoa by density gradient centrifugation in Percoll®

A procedure using centrifugation in density gradients composed of Percoll was developed for isolation of spermatozoa from mammalian semen. To evaluate the technique, rabbit, human, or bovine semen was layered over continuous Percoll gradients ranging in density from 1.02 to 1.13 gm/ml and centrifuged at 1,500g for 45 min. After centrifugation, the seminal plasma remained above the gradient, whereas the spermatozoa and seminal particles were distributed within the gradient according to their buoyant densities. Unlike most washing techniques, no sperm pellet was formed; instead, the spermatozoa were concentrated into a compact band above the most dense layer of Percoll. The spermatozoa recovered from the gradient were easily resuspended by gentle techniques. Thus, the mechanical stress to the spermatozoa was minimized. Osmotic stress to the spermatozoa was also negligible as the Percoll gradients were isotonic throughout. Spermatozoa obtained by this technique possessed motility equivalent to that of spermatozoa in the unfractionated semen. Sperm suspensions recovered from the gradients contained less than 5% of the nonspermatozoal particles present in the original samples of unfractionated semen. Soluble seminal components were also efficiently removed from the spermatozoa. Thirty billion bovine spermatozoa could be fractionated on a single gradient without loss of effectiveness. Recovery of spermatozoa from these preparative separations averaged 80%. These results demonstrated that Percoll was a superior medium for efficient density gradient isolation of motile spermatozoa free of contamination by other seminal constituents.     

A Novel Technique for Isolation of Pure Sperm Heads from Disintegrated Mammalian Spermatozoa

Abstract

The phenomenon of differential charge distribution on sperm surface membrane has been utilised here in a low e.m.f. (electro motive force) capillary electrophoresis system to effect separation of sperm heads from disintegrated mixed spermatozoal subfractions. Washed caudal sperm of goat (Black Bengal variety) and ejaculated washed human sperm were fractionated by sonication into head, mid-piece and tail portions. Routine techniques of density gradient centrifugation on Percoll and/or sucrose were performed with sonicated spermatozoa for separation into their respective subfractions. The products obtained were not free of contamination in either case. Mixed sperm fractions when subjected to the afore mentioned modified capillary electrophoresis technique only the head pieces exhibited high affinity for migration towards the cathode terminal. With this method around 50% of the total sperm heads were separated and collected in absolutely pure form at the cathode side within 2 hrs. at 150 volts (V) and 1.5 milliampere (mA) current at 37.5°C. A 4 cm. long capillary tube with a bore size 1.2 mm. was used for this purpose.     

Separation of European flat oyster, Ostrea edulis, haemocytes by density gradient centrifugation and SDS-PAGE characterisation of separated haemocyte sub-populations

A two-step gradient centrifugation with Percoll and Ficoll successively as density medium was developed to separate European flat oyster, Ostrea edulis, haemocytes into three sub-populations representing granulocytes, large hyalinocytes and small hyalinocytes, respectively. After a Percoll gradient centrifugation, granulocytes and agranulocytes were separated and a pure fraction of granulocytes was obtained. The agranulocytes were further separated by centrifugation through a Ficoll gradient, and two haemocyte subpopulations representing large hyalinocytes and small hyalinocytes were obtained. No significant impact on the haemocyte viability was detected after separation with this two-step density gradient centrifugation. The three haemocyte sub-populations showed different protein patterns in SDS-PAGE.     

Comparative studies on the lysosomal association of monomeric 239Pu and 241Am in rat and Chinese hamster liver: analysis with sucrose, metrizamide, and Percoll density gradients of subcellular binding as dependent on time

The binding of 239Pu and 241Am in the livers of Chinese hamsters and rats was analyzed by centrifugation of a mitochondrial-lysosomal fraction in sucrose, metrizamide, and Percoll density gradients at intervals between 4 and 70 days after nuclide injection. The behavior of 239Pu and 241Am during the centrifugation experiments was very similar. In contrast to the results for rats, the median densities of the nuclide profiles from hamsters decrease with time in hyperosmolar sucrose gradients, as does the nuclide fraction liberated by addition of Triton X-100, and the nuclide profiles do not respond typically to Triton WR 1339 treatment of the animals. The results with nearly iso- osmolar metrizamide gradients, together with those for Percoll, agree with the assumption that there is an initial lysosomal association of the transuranium elements. It was concluded from the results that the major fraction of 239Pu and 241Am remained bound to typical lysosomes in rat liver, whereas those in hamster liver may be transformed into telolysosomes . Possibly, a vesicular biliary transport system for certain heavy metals, for which evidence exists in rat liver, does not occur in Chinese hamster liver.     

Membrane Populations of Bovine Choroid Plexus: Separation by Density Gradient Centrifugation in Modified Colloidal Silica

Abstract: The choroid plexus is intimately involved in the production and regulation of the cerebrospinal fluid. Populations of surface membranes from this epithelial tissue were separated by density gradient centrifugation by use of modified colloidal silica (Percoll). A fraction of heavy microsomes (P₃) containing plasma membranes was prepared by differential centrifugation. Membranes in fraction P₃ were mixed with a given concentration of Percoll and density gradients generated during centrifugation. When fraction P₃ was mixed with 20% (v/v) Percoll and centrifuged at 20,000 r.p.m. for 1 h in a 50.2 Ti fixed-angle rotor, membranes containing alkaline phosphatase (AP) were found at a density of 1.037 g/cm³ while those containing NaK ATPase were found at 1.047 g/cm³. With more shallow density gradients using 12% and 14% Percoll, a broad shoulder of AP activity became manifest at densities greater than 1.060 g/cm³ suggesting multiple populations of membranes containing AP. Membranes containing AP could also be separated from membranes containing γ-glutamyl transpeptidase (γ-GTP); this separation was most pronounced in 12% Percoll. The activity of γ-GTP could not be separated from activity of NaK ATPase. Total protein was distributed broadly throughout the gradients. Studies have been undertaken to compare the behavior of choroidal membranes in Percoll gradients with that of renal membranes because the biochemical anatomy of the kidney has been extensively studied. In contrast to choroidal membranes, renal membranes with NaK ATPase activity were found to have densities lower than those membranes with AP. Thus, the distribution of membrane-bound enzymes from kidney in a Percoll gradient was exactly the opposite of that observed for these same enzymes from choroid plexus. In addition, unlike the γ-GTP activity of choroid plexus, γ-GTP from kidney could be separated from the activities of both alkaline phosphatase and NaK ATPase. These marked differences in membrane populations between choroid plexus and kidney as defined by Percoll density gradient centrifugation analyses are presumably reflective of differences in the functions of the two epithelial tissues.     

Is Urografin density gradient centrifugation suitable to separate nonculturable cells from <Emphasis Type="Italic">Escherichia coli</Emphasis> populations?

The ability of Urografin or Percoll density gradient centrifugations to separate nonculturable subpopulations from heterogeneous Escherichia coli populations was analysed. Bacterial counts (total, active and culturable cells) and flow cytometric analyses were carried out in all recovered bands. After Urografin centrifugation, and despite the different origin of E. coli populations, a common pattern was obtained. High-density bands were formed mainly by nonculturable cells. However, the increase in cell density would not be common to all nonculturable cells, since part of this subpopulations banded in low-density zones, mixed with culturable cells. Bands obtained after Percoll centrifugation were heterogeneous and culturable and nonculturable cells were recovered along the gradient. Thus, fractionation in Urografin cannot be only attributed to changes in buoyant densities during the transition from culturable to nonculturable state. Urografin density gradients allow us to obtain enriched fractions in nonculturable subpopulations from a heterogeneous population, but working conditions should be carefully chosen to avoid Urografin toxicity.     

Evaluation of separating X- and Y-sperms by percoll density gradient centrifugation using sperms of transgenic mice carrying a transgene on Y-chromosome

Using sperms of the transgenic mice carrying a human A gamma/beta-globin gene on Y-chromosome, we attempted to separate X- and Y-bearing sperms by the Percoll density gradient centrifugation. The ratio of X- and Y-sperms was determined by DNA dot blot hybridization procedure with sperm DNA. Sperm suspension collected from cauda epididymidis was loaded on the gradient composed of 7 Percoll concentrations (35-84%) and was centrifuged at 300 x g for 10, 15 or 20 minutes, respectively, at room temperature. After centrifugation, sperms were collected from each gradient fraction and washed with 0.85% saline solution. DNA was extracted from sperms, dotted and fixed on nitrocellulose filter, and was hybridized with the 32P-labeled DNA probe derived from the beta-globin gene. Each DNA spot was cut out, immersed in the liquid scintillator and was counted for radioactivity. There was no difference among the radioactivities in the DNA spots, indicating that the ratio of X- and Y-sperms was the same in all the gradient fractions of three different centrifugal conditions. The results suggests to be difficult to separate X- and Y-sperms by Percoll density gradient centrifugation, at least, using sperms from cauda epididymidis of mouse.     

Percoll-gradient separation of Leydig cells from postnatal rat testes

The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other. Similarly, intertubular cell preparations from 21- and 25-day-old rats resolved into two peaks of hCG binding; adult-like Leydig cells were observed in the first peak, but fetal Leydig cells were rarely observed in the second of these peaks. These results demonstrate the separation of two Leydig cell populations from intertubular cells obtained from animals aged up to 15 days. Thereafter the pattern of the hCG binding profile is similar but is not due to the presence of the same cell types. Therefore these results emphasize the necessity for morphological identification of cell types to permit the correct interpretation of the corresponding biochemical data.     

Wet and dry bacterial spore densities determined by buoyant sedimentation.

The wet densities of various types of dormant bacterial spores and reference particles were determined by centrifugal buoyant sedimentation in density gradient solutions of three commercial media of high chemical density. With Metrizamide or Renografin, the wet density values for the spores and permeable Sephadex beads were higher than those obtained by a reference direct mass method, and some spore populations were separated into several density bands. With Percoll, all of the wet density values were about the same as those obtained by the direct mass method, and only single density bands resulted. The differences were due to the partial permeation of Metrizamide and Renografin, but not Percoll, into the spores and the permeable Sephadex beads. Consequently, the wet density of the entire spore was accurately represented only by the values obtained with the Percoll gradient and the direct mass method. The dry densities of the spores and particles were determined by gravity buoyant sedimentation in a gradient of two organic solvents, one of high and the other of low chemical density. All of the dry density values obtained by this method were about the same as those obtained by the direct mass method.     

Wet and dry bacterial spore densities determined by buoyant sedimentation

The wet densities of various types of dormant bacterial spores and reference particles were determined by centrifugal buoyant sedimentation in density gradient solutions of three commercial media of high chemical density. With Metrizamide or Renografin, the wet density values for the spores and permeable Sephadex beads were higher than those obtained by a reference direct mass method, and some spore populations were separated into several density bands. With Percoll, all of the wet density values were about the same as those obtained by the direct mass method, and only single density bands resulted. The differences were due to the partial permeation of Metrizamide and Renografin, but not Percoll, into the spores and the permeable Sephadex beads. Consequently, the wet density of the entire spore was accurately represented only by the values obtained with the Percoll gradient and the direct mass method. The dry densities of the spores and particles were determined by gravity buoyant sedimentation in a gradient of two organic solvents, one of high and the other of low chemical density. All of the dry density values obtained by this method were about the same as those obtained by the direct mass method.     

Percoll and Ficoll self-generated density gradients by low-speed osmocentrifugation

Percoll and Ficoll self-generated density gradients can be obtained by low-speed centrifugation of their solutions within dialysis cells. Useful Percoll density gradients can be obtained after 10-30 min centrifugation at 220-2010g, within dialysis cells. Ficoll density gradients, which are more difficult to self-generate, can be obtained by the same technique. Red cell band formation in a Percoll density gradient can be done in a single step by using dialysis cells as the centrifugation solution container.     

Functional and physical characteristics of rat Leydig cell populations isolated by metrizamide and Percoll gradient centrifugation

The physical and functional properties of Leydig cell populations obtained by centrifugation of testicular cells in two different density gradient media, Percoll and Metrizamide, were compared. Percoll-gradient centrifugation yielded two Leydig cell bands (Peak I and Peak II) that were comparable, as to their density and testosterone-producing capacity, to the respective Leydig cell bands, Population I and Population II, isolated in a Metrizamide gradient. The denser Leydig cell band (II) had a greater capacity for testosterone production than the less dense band (I), regardless of the type of gradient used for its isolation. Metrizamide gradient centrifugation separated the majority of germ cells from the "light" (Population I) Leydig cells, whereas in the Percoll gradient, germ cells comigrated with Peak I Leydig cells. Leydig cell separation by Percoll gradients was highly dependent on the presence of Ca2+ and Mg2+ in the medium, while these cations had no effect on the separation of Leydig cells by Metrizamide. In conclusion, Metrizamide gradient centrifugation yielded two Leydig cell populations of similar functional and physical properties to the respective populations isolated in Percoll gradients.     

Separation of Non-filamentous Micro-organisms from Soil by Density Gradient Centrifugation in Percoll

Soil suspensions were homogenized, and desorbed non-filamentous micro-organisms were concentrated in a minimum volume of buffer by low speed centrifugation. The cells were separated from inanimate material by flotation at the interface between the buffer and a silica sol/polyvinyl pyrrolidone density gradient medium (Percoll). Cell suspensions were removed from the interface and fractionated according to density by high speed centrifugation on discriminating density gradients in Percoll.
Preliminary experiments indicated that most non-filamentous soil micro-organisms had densities in the range 1.081–1.123 g%sol;ml while Rhizobium isolated from crushed root nodules on Percoll was split into two bands of densities 1.081–1.110 and 1.041–1.073 g/ml. The lighter cells were the more pleomorphic.
The efficiency of extraction of cells from soil was governed by the extent of their desorption from inanimate particles. As rigorous desorption procedures damage cells, extraction efficiencies were low; 10–20% of cells counted microscopically in soil were recovered from density gradients. Electron microscopy of soil micro-organisms isolated by this method showed an unusual range of surface ornamentations on cell-like structures of bacterial dimensions.     

Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.     

Sex ratio of early embryos fertilized in vitro with spermatozoa separated by percoll

Early bovine embryos were obtained by in vitro fertilization and sexing carried out by chromosome analysis. Separation of bovine X- and Y-bearing spermatozoa was performed using Percoll density gradient centrifugation and the enrichment of X-sperm proportion was investigated. Through treatment with vinblastin sulfate and podophyllotoxin, 880 (48.6%) of 1812 embryos at two- to seven-cell stages at 48 to 53 h after sperm-egg incubation produced metaphase spreads, and 399 (45.3%) of these were successfully sexed; the sexable rate reaching 53.4% for four-cell embryos. Sexing rates for embryos from the original sperm of two bulls were 69.6% (32/46) in Bull A and 54.2% (58/107) in Bull B. Embryos fertilized in vitro with sperm sedimented at the bottom of sperm centrifuged under conditions (I) 50 to 85% of Percoll, 15 °C; (II) 30 to 80%, 10 °C; (III) 30 to 80% 20 °C; (IV) 30 to 90%, 20 °C, gave rise to male sex ratios of (I) 58.3% in Bull A and 53.5% in Bull B, (II) 65.9% in Bull A, (III) 49.3% in Bull B and (IV)_66.7% in Bull B. In conclusion, Percoll density gradient centrifugation under these four conditions was unsuccessful in separating X- and Y-bearing bull spermatozoa.     

The heterogeneous distribution of acid hydrolases within a homogeneous population of cultured mammalian cells

1. Chinese-hamster ovary fibroblasts were cultured to provide a homogeneous cell population. Homogenates obtained from these cells were fractionated by centrifugation techniques and the resulting fractions were analysed for protein and for enzymes representative of certain subcellular particles. 2. Unlike those in rat liver homogenates, the mitochondrial and lysosomal populations proved impossible to separate by differential centrifugation owing to the similarity of their sedimentation properties. Their resolution was possible by using isopycnic centrifugation in a continuous sucrose density gradient. 3. The mitochondrial population equilibrated at a density of 1.17g.cm(-3) as in rat liver homogenates. However, the lysosomal population equilibrated at a lower rather than a higher density position than the mitochondria and the probable reasons for this are discussed. 4. The lysosomal population subdivided into two groups characterized by differences in acid hydrolase content and equilibrium densities. The fraction with a density of 1.15g.cm(-3) contained the majority of arylsulphatases A and B, of cathepsin and of beta-acetylglucosaminidase activities, whereas that with a density of 1.09g.cm(-3) contained the majority of the acid phosphatase and acid ribonuclease activities. The probable division of the lysosomal population of a single cell into a number of distinguishable subgroups is suggested.     

Variation in the polysome assembly and incorporation of [3H]-uridine in the cells of pine buds during the cold season

The ribosome assemblies isolated from buds of Scots pine ( Pinus sylvestris L.) containing microsporangiate strobili varied both quantitatively and qualitatively in samples collected from October to April. The seasonal fluctuation in the amount of ribosonnes was more evident in the cytosolic fraction than in the smaller membrane-bound fraction. The profiles obtained after sucrose density gradient centrifugation were of two types. One type was commonly obtained from samples collected late in the autumn and early in the spring, and this type was characterized by a relatively high peak for the large subunits, a low or negligible peak for the dimers, and an even or ascending series of peaks for the polymers. The other type was obtained from samples collected during the winter, and was characterized by small peaks for both subunits, a moderate to large peak for the dimers and a descending series of peaks for the polymers. However, the scanning electron microscope investigations indicated that the winter-time samples did not lack polysomes and clusters of ribosomes. They did not become visible in the polysome profiles because they pelleted too tightly at the bottom of the centrifuge tubes to be removed with gradient fractionation. The au-toradiographic analyses suggested that the cells were capable of synthesizing mRNA throughout the winter, whereas rRNA synthesis was arrested. On the basis of the above results, we postulate that the synthesis of the enzyme proteins needed for the maintenance of winter-time metabolism takes place in the cytosolic ribosome fraction. The possible existence of winter-time polysome stores is also pointed out.     

Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro

Various times of in vitro maturation of oocytes, and three methods of separating spermatozoa from frozen-thawed semen (Percoll density-gradient centrifugation, swim-up, and glass-wool filtration), were compared for their effects on goat embryo production in vitro. Cumulus-oocyte-complexes (COCs) from abattoir ovaries were matured in M199 supplemented with 10% fetal calf serum and hormones. In Experiment 1, COCs were fixed at 4 h intervals from 0 to 27 h of culture to assess oocyte nuclear maturation. A higher proportion cultured for 27 h than for 24 h were in Metaphase II (27/37, 73% vs. 18/33, 55%, P < 0.05). In Experiment 2, the effects of separation methods on total numbers and numbers of membrane-intact spermatozoa, and the acrosome reaction were compared. Total numbers after Percoll density-gradient centrifugation were approximately 4 times higher than after swim-up and approximately 2 times higher than after glass-wool filtration (P < 0.001). Progression of the acrosome reaction was not affected differentially. In Experiments 3 and 4, after 27 h of culture the COCs were inseminated with sperm isolated by the three methods. In Experiment 3, presumptive zygotes were examined for pronucleus (PN) formation at 6, 12, 18 and 24 h post-insemination. At 12 h, male PN formation rate from Percoll-treated spermatozoa was higher than from sperm subjected to swim-up and glass-wool treatments (20/37, 54% vs. 6/37, 16% and 6/38, 16%, respectively; P < 0.001). In Experiment 4, embryos were compared for cleavage at 48 h and development into blastocysts, hatching rates and cell number at 192 h. The rates of cleavage and blastocyst formation in the Percoll-treated group were higher (P < 0.05) than in the swim-up and glass-wool groups (62% and 18% vs. 50% and 11%, and 45% and 8%, respectively). Similarly, the mean cell number in the Percoll group was higher (P < 0.05) than in the swim-up and glass-wool groups (167 +/- 5 vs. 149 +/- 4 and 126 +/- 4, respectively). We conclude that Percoll density-gradient centrifugation is superior to the other two methods for separating goat spermatozoa from frozen-thawed semen in preparation for IVF.     

Localization of the membrane-associated CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary cells with an altered membrane composition

The subcellular localization of the membrane-associated CTP:phosphocholine cytidylyltransferase was determined in Chinese hamster ovary cells in which the phospholipid composition had been altered by growth in the presence of N-methylethanolamine or treatment with phospholipase C. Cell homogenates were fractionated on Percoll density gradients, and marker enzyme activities were used to determine the location of the cellular membrane fractions. The peak of cytidylyltransferase activity occurred in the gradient at a density intermediate to that of the peaks of endoplasmic reticulum and plasma membrane markers. The profile of cytidylyltransferase activity most closely resembled that of the Golgi membrane marker; however, upon sucrose gradient centrifugation, the profile of the Golgi apparatus was very different from that of cytidylyltransferase. Differential centrifugation suggested a nuclear membrane association of the enzyme. Cytidylyltransferase was associated with a membrane fraction that sedimented when subjected to very low speed centrifugation (65 x g, 5 min). From Percoll gradient fractions, nuclei were identified by microscopy, and they migrated with cytidylyltransferase activity. The data are consistent with a localization of cytidylyltransferase in the nuclear membrane.