Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development

The objectives of these experiments were to characterize separation of frozen-thawed bovine spermatozoa on a Percoll gradient and then to compare sperm separation by either a swim-up or Percoll gradient procedure for the ability of spermatozoa to fertilize oocytes in vitro. The Percoll gradient was a 45 and 90% discontinuous gradient. Initial experiments found that centrifugation of semen on the Percoll gradient for 15 min at 700 g was sufficient to obtain optimal recovery of motile spermatozoa. Most of the nonmotile spermatozoa were recovered at the interface of the 45 and 90% Percoll layers, while the motile spermatozoa were primarily in the sperm pellet at the bottom of the gradient. When frozen-thawed semen from each of 7 bulls was separated by swimup, a mean +/- SEM of 9% +/- 1 of the motile spermatozoa were recovered after the procedure. In contrast, more spermatozoa were recovered after Percoll gradient separation (P < 0.05), with 40% +/- 4 of the motile spermatozoa recovered. The effect of separation procedure on in vitro fertilization found swim-up separated spermatozoa penetrated a mean +/- SEM of 74% +/- 5 of the oocytes, while fewer oocytes were penetrated by Percoll separated spermatozoa at 52% +/- 8 (P < 0.05). There was no effect of the separation procedure on the rates of polyspermy as measured by sperm/penetrated ova, with a mean +/- SEM of 1.25 +/-.09 for swim-up separated spermatozoa and 1.14 +/-.07 for Percoll separated spermatozoa (P>0.05). A carry over of Percoll into the fertilization medium with the Percoll separated spermatozoa was found not the cause for the decreased penetration of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decreased penetration of oocytes by Percoll separated spermatozoa could be overcome by increasing the sperm concentration during fertilization from 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized by either swim-up or Percoll separated spermatozoa was compared for the semen from 2 bulls, a difference in cleavage rate was found in favor of swim-up separated spermatozoa (P < 0.05), but there was no effect of separation procedure on development (Day 7) to the morula + blastocyst or blastocyst stage (P>0.05). The disadvantages of the Percoll procedure could easily be overcome and the procedure was faster and yielded a six-fold greater recovery of motile spermatozoa than the swim-up method.     

Isolation and viability of presumptive spermatids collected from bull testes by Percoll density gradient

The objective of the present study was to develop a procedure for isolating pure populations of round spermatid(s) (RS) by Percoll density gradient from bull testes. Bull testes were de-capsulated and testicular tissues were dissociated enzymatically to recover RS. After being filtered through a 20 microm nylon mesh, the cells were centrifuged at 650 x g for 25 min through the discontinuous Percoll density gradients (20, 35, 40, 45 and 90% Percoll solution). Isolated cells were analyzed by microscopic observation for survivability and apoptosis. In Experiment 1, both microscopic observation and DNA analysis by flow cytometry showed that approximately 40% of cells collected from 35% Percoll gradient were presumptive RS, whereas in 40% Percoll gradient, mostly primary spermatocytes were observed. Experiment 2 compared the effect of 35% Percoll density isolation on the incidence of apoptosis and necrosis in fresh and frozen-thawed cells to those of untreated cells. The percentage (mean+/-S.E.M.) of necrosis in cells collected from 35% Percoll gradient was less (P<0.05) than in untreated and frozen-thawed cells from 35% Percoll gradient (11.7+/-3.1% compared with 26.3+/-2.0% and 53.5+/-1.3%, respectively), but the rate of apoptosis did not differ (1.2+/-0.49% compared with 2.5+/-0.8% and 0.9+/-0.04%, respectively). The proportional data (mean+/-S.E.M.) of live cells in Percoll treated group were greater (P<0.05) than in untreated and frozen-thawed cells from the 35% Percoll gradient (86.7+/-3.26% compared with 70.8+/-2.73% and 41.9+/-1.69%, respectively). Experiment 3 compared the development rates of embryos injected with RS isolated from fresh and frozen-thawed cells collected with the 35% Percoll gradient to those of untreated cells, and parthenotes as control. There were no significant (P>0.05) differences in the rates of cleavage and blastocyst development between untreated fresh cells and fresh cells collected from the 35% Percoll gradient (75.4 and 10.5% compared with 82.4 and 12.8%). However, there were lesser (P<0.05) cleavage and blastocyst rates in frozen-thawed cells from the 35% Percoll gradient (51.6 and 6.3%) and parthenotes (60.7 and 4.1%) were observed. These results suggest that isolation of presumptive RS by 35% Percoll density gradient is effective in eliminating apoptotic and early necrotic cells. However, the use of RS in improving the developmental potential of embryos merits further studies.     

Integrated morphophysiological assessment of two methods for sperm selection in bovine embryo production in vitro

Extensive work was done regarding the ability of Swim up and Percoll gradient to select functional sperm for in vitro embryo production (IVP) systems. The aim of this work was to compare Swim up and Percoll as methods of sperm selection by ultrastructural, biochemical and functional studies. Frozen-thawed semen from two bulls (Experiments 1 and 2, respectively) were treated using Swim up or Percoll discontinuous gradients. Motility, sperm membrane ultrastructure, sperm proteins, in vitro embryo production (insemination doses, cleavage, embryo yield and quality) and embryo sex ratio were scored and compared. Electron transmission microscopy of outer sperm membranes showed higher (P<0.05) percentage of sperm with lost acrosomes in Percoll treated samples compared to Swim up. A differential protein pattern was also detected. When in vitro embryo production was performed, Percoll gradient produced higher (P<0.05) number of fertilizing doses (7.6 versus 5.9, Bull 1; 13.5 versus 7.8, Bull 2) and higher sperm motility (90% versus 76.6%, Bull 1; 81.7% versus 68.3%, Bull 2) than Swim up. The percentage of cleavage (Day 3) was similar in both treatment groups, whereas embryo production rate (Day 7) was higher (39.4% versus 30.2%, Bull 1; 38% versus 32.4%, Bull 2; P<0.05) when Percoll gradient was used. The percentage of hatched embryos (Day 11) and sex ratio did not differ. Total cell counting and embryo differential staining (inner cell mass and trophoblast cells) of Day 7 embryos showed that Percoll treated sperm produced better quality embryos compared to Swim up. We concluded that Percoll had a better performance selecting sperm and an enhanced capacity for embryo production when compared with the Swim up procedure; this could be attributed to a better acrosome exocytosis, associated to the absence of certain membrane proteins.     

Improved purification of Piscirickettsia salmonis using Percoll gradients

Viable preparations of intact Piscirickettsia salmonis, purified from host cell material, are necessary for studying characteristics associated with this bacterium. However, purification of the organism is difficult due to its obligate intracellular nature. A simple and effective method for isolating whole P. salmonis, which is quick and easy to perform, but still maintains the viability and antigenicity of the bacterium is described. P. salmonis was purified by differential pelleting and density gradient centrifugation using 30%, 40%, or 50% (v/v) Percoll gradients. Following fractionation, a band with a density of 1.056-1.080 was found to be composed entirely of rickettsiae, confirmed by fluorescent antibody technique (IFAT). Purity of the P. salmonis from different stages of the purification process was assessed by light and transmission electron microscopy, and the viability of yields determined from a plaque assay and a tissue culture infective dose (TCID(50) ml(-1)). P. salmonis recovered from the 30% Percoll gradient appeared to retain their intracellular structure better than P. salmonis obtained from the 40% and 50% Percoll gradients, and appeared to have a greater viability. Differences were seen between P. salmonis-infected CHSE-214 cells and purified P. salmonis when compared by SDS-PAGE and Western blotting, and less host cell contamination was present in preparations obtained from the 30% Percoll gradient. Finally ten different P. salmonis isolates obtained from three different geographical locations and four different fish species, were purified using the 30% Percoll gradient. When the morphology of these was compared by transmission electron microscopy (TEM), they appeared similar in size and appearance, although isolate R980769 was highly pleomorphic and isolate R-29 was larger than the other isolates examined.     

Fractionation of Different Life Cycle Stages of Microsporidia Nosema grylli from Crickets Gryllus bimaculatus by Centrifugation in Percoll Density Gradient for Biochemical Research

ABSTRACT. A new method of fractionation and purification of different life cycle stages of microsporidia Nosema grylli , parasitizing the fat body of cricket Gryllus bimaculatus , by centrifugation in Percoll density gradient is elaborated. The whole procedure can be summarized as: 1) infected fat body preparation, 2) homogenization in buffer and filtration through cotton wad and filter paper, 3) first centrifuging, resulting in the separation of the pellet into three layers containing different life cycle stages, 4) second centrifuging of the chosen layer in Percoll density gradient, 5) washing out the Percoll from the fraction under study. After centrifugation in Percoll density gradient, meronts and early sporonts form a band in the area corresponding to density 1.016 g/ml. Mature spores form the pellet at the bottom of centrifuge tube, while immature spores are distributed throughout the layer of 1.016 g/ml up to the bottom of the centrifuge tube, according to their buoyant densities. The offered technique is simple, it takes about one hour and may become a routine procedure for biochemical studies on microsporidia.     

Isolation of highly purified rat liver lysosomal membranes using two Percoll gradients

Normal rat liver lysosomal membranes in the form of membrane vesicles have been purified using Percoll density gradient centrifugation. Lysosomes (density = 1.111) were purified approximately 63 +/- 12-fold (mean +/- standard deviation, n = 5) using a gradient of Percoll made isotonic with sucrose and buffered to pH 7.0. These lysosomes were then exposed to 10 mM methionine methyl ester, pH 7.0, the uptake of which resulted in swelling and breakage of the lysosomes with subsequent vesicle formation. These vesicles (density = 1.056) were further separated from residual mitochondrial and plasma membrane enzyme activities using a second Percoll density gradient. Marker enzyme analysis and electron microscopy indicated that the lysosomal membranes were essentially free of both beta-hexosaminidase, a soluble lysosomal enzyme, and contaminating organelles. The specific activity of lysosomal ATPase in the lysosomal membranes was fourfold greater than in the intact lysosomes.     

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.     

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.     

Motility, acrosome integrity, membrane integrity and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from frozen-thawed buffalo semen

Frozen-thawed semen of five buffalo bulls was used to compare efficacy of swim-up and Percoll gradient methods for separating viable spermatozoa. Sperm separated by the two methods were also tested to differentiate buffalo bulls on the basis of in vitro fertilization (IVF) rates. Recovery of motile sperm (%), increase in membrane integrity (%) and acrosome integrity (%) were compared after two sperm separation methods in experiment I, and in vitro fertilization rate (cleavage rate and cleavage index) was compared in experiment II. Swim-up separated sperm showed a higher motility (P<0.05), while percent recovery of motile sperm was higher with Percoll separation (P<0.05). Membrane integrity (%) of sperm separated with swim-up was significantly higher (P<0.05) as compared to sperm separated with Percoll gradient. Swim-up separated sperm gave a higher cleavage rate and cleavage index (P<0.001). Sperm separated by swim-up showed significant difference among the bulls in cleavage rate and cleavage index (P<0.05), while the Percoll gradient method did not. It has been concluded that separation of sperm from frozen-thawed buffalo semen by swim-up method can be more expedient for IVF in buffalo.     

The isolation of intact adrenal chromaffin granules using isotonic Percoll density gradients

An improved method for the isolation of intact adrenal chromaffin granules under isotonic conditions, using a Percoll density gradient, is presented. After dissection, homogenization, and differential centrifugation, the crude granule homogenate was layered onto a gradient medium previously centrifuged at 20,200g × 5 min, consisting of 30% ($\text{v}\text{v}$) Percoll, 0.27 m sucrose, and 10 mm Tris-maleate, pH 7.0. After centrifugation for 40 min at 8650g in a standard preparative centrifuge, the chromaffin granules were found to band in the lowest fraction, where 45% of the catecholamines and 60% of the ATP could be recovered. With respect to other published methods, the percentage of lysosomal and mitochondrial contamination compared favorably. In addition, granules isolated by the Percoll gradient method were found to have at least 42 and 14% higher ATP and catecholamines, respectively, per milligram of protein. It is suggested that this method offers the advantages of ease of preparation, purity, and cost efficiency when compared with previously published techniques.     

Assessment of sperm viability in boar,rabbit and rooster: a modification of the fluorometric ethidium bromide exclusion procedure

The ethidium bromide (EtBr) exclusion procedure, a fluorometric method for measuring sperm cell viability, was studied to optimize the use of this technique on boar, rabbit and rooster semen. Diluted semen was used for boars and roosters. Diluted rabbit semen did not allow for reliable fluorescence readings; the interference of granules characteristic of rabbit seminal plasma was suggested as its cause. Therefore, rabbit semen was washed on several Percoll and Optiprep density gradients, with the aim of removing the granules from the sperm suspension. The complete absence of granules was not obtained, however, the best result was provided by the 35/70% Percoll density gradient. Most spermatozoa formed a loose pellet with low contamination. Although the washing procedure resulted in a selective action, Percoll washed semen was used to assess the EtBr procedure. The fluorescence intensities of stained fresh and stained digitonin-permeabilized samples were corrected, respectively, for the nonspecific fluorescence measures of fresh and digitonin-permeabilized samples both unstained. The contribution of the dye was subtracted from the corrected values, then the ratio between the corrected values of fresh and permeabilized cells provided the proportion of damaged cells in the sample. The working cell concentration range giving a constant proportion of damaged cells was set using diluted semen for boars and roosters (8-32 x 10(6) cell/ml) and Percoll washed semen for rabbits (4-16 x 10(6) cell/ml). The reliability of the fluorometric method was compared with the traditional nigrosin-eosin (NE) staining technique. The intactness of sperm samples containing known proportions of fresh and killed cells was measured in defined working cell ranges. For boars and roosters the values determined by fluorometry agreed closely with those determined using the NE method.     

Purification and properties of cytoplasmic granules from cytotoxic rat LGL tumors

To evaluate the role of NK cell granules in the lytic activity of NK cells, cytoplasmic granules of rat NK tumors were purified by centrifugation of the cell homogenates in a Percoll gradient. Analysis of such gradients showed a band of light-scattering material near the bottom of the tube; assay of gradient fractions for lytic activity against SRBC showed a potent lytic activity giving a sharp peak in this region. Complete lysis of SRBC was achieved with less than 1 microgram/ml protein of the most active fractions. Examination in the electron microscope showed that a pool of fractions containing lytic activity consisted of pure cytoplasmic granules showing similar morphology to those found in the LGL tumors. The lytic band was associated with a peak in the activity of four different lysosomal enzymes. Analysis of Percoll gradient fractions showed that marker enzymes for mitochondria, plasma membrane, and cytosol were well separated from this activity peak. Analysis of the Percoll gradient fractions by SDS gel electrophoresis showed that this granule fraction was free of contamination of proteins from other parts of the gradient. The granules contained major protein bands of 62, 58, 30, 29, and 28 kilodaltons. In addition to protein, the purified granule fractions contain hexose and uronic acid, but no nucleic acids or phospholipids were detected in chemical assays. Major amounts of chymotryptic, tryptic, and elastase activities were not present, nor were peroxidase or lysozyme activities detectable in substantial amounts. These data show that NK tumor cell cytoplasmic granules contain a potent lytic activity and have biochemical properties that distinguish them from granules present in granulocytes and mast cells.     

Isolation and purification of intact peroxisomes from green leaf tissue

Intact peroxisomes were prepared from green leaves of a number of C₃ species, both monocots and dicots. A protoplast extract from which chloroplasts have been removed by a 1-minute 10,000g centrifugation was applied to a step gradient of 5, 15, 30, and 45% Percoll containing 0.5 molar sucrose, 0.1% BSA, and 25 millimolar Hepes-KOH (pH 7.5). After centrifugation, a peroxisomal fraction with low contamination by chloroplastic and mitochondrial markers was recovered from the 30/45% Percoll interface. This fraction was passed through a Sepharose 2B column to remove the Percoll which resulted in a peroxisomal preparation exhibiting high intactness (estimated from enzyme latency) and stability.     

Separation of canine epididymal spermatozoa by Percoll gradient centrifugation

The objective was to characterize the separation of canine epididymal spermatozoa on a Percoll gradient. Epididymal spermatozoa were overlaid on a 45 and 90% discontinuous Percoll gradient and centrifuged at 700 x g for 20 min. The Percoll column was separated into six fractions (top to bottom, A-F) after centrifugation. Fractions A-C contained few spermatozoa. Spermatozoa with bent or folded tails and a large amount of granular debris were observed in Fraction B. Fraction D contained many nonmotile spermatozoa, erythrocytes and round epithelial cells. Spermatozoa in Fraction E had significantly lower motility than those in the initial layer. Spermatozoa in Fraction F had motility similar to those before separation. Fraction F contained 40.6% of the motile spermatozoa layered and 67.5% of all motile spermatozoa recovered. There was no significant difference between Fraction F and the initial layer in sperm membrane integrity. In the sperm-oocyte penetration assay, spermatozoa from Fraction F had a significantly higher penetration rate into the immature homologous oocytes than those from Fraction E. Although the recovery rate of the motile spermatozoa was low, the canine epididymal spermatozoa with motility, membrane integrity and penetrating capability could be separated by two-layer discontinuous Percoll gradient centrifugation.     

Acetylcholinesterase in sea urchin spermatozoa

Flagella contain the bulk of spermatozoan acetylcholinesterase. Brief sonication of sea urchin sperm suspended in Tris-buffered (pH 8.0), Ca, Mg-free artificial sea water (F-ASW) containing 10 mM ethylene diaminetetracetic acid, (EDTA) doubled the specific activity over that of the intact spermatozoa. Lipids were removed from the solubilized supernatant of the tail membrane fraction by ether extraction. Hydrolysis of acetylthiocholine in the presence of dithiobisnitrobenzoic acid (DTNB) was monitored spectrophotometrically at 412 nm by the Ellman procedure. The enzyme was purified by affinity chromatography on a Sepharose cyanogen bromide gel to which the cholinesterase inhibitor trimethyl (para-aminophenyl) ammonium chloride was coupled. The enzyme was eluted from the column with a discontinous NaCl gradient (0.1–0.5 M). The active fraction recovered at 0.35 M NaCl contained 0.007% of the initial total sperm cell protein with a 500-fold increase in specific activity. Twenty-four hr centrifugation on a 5–20% sucrose density gradient at 50,000g in a Beckman L5-75 centrifuge yielded peaks at 14.7 S and 9.1 S. In the presence of 1% Triton X-100, three peaks appeared: 23.3 S, 13.7 S, and 9.1 S. These sedimentation coefficients resemble those of the electroplax acetylcholinesterase (AChE) forms A₈ and A₄. Eserine completely inhibited the activity of the purified enzyme, which exhibits a substrate optimum at 4 mM acetylcholine. The activity is depressed by 75% at 10 mM ACh and by 90% at 25 mM. The Km was 2.1 × 10^?4 M. In the sperm cell the enzyme that terminates the action of intracellularly synthesized ACh may be involved in controlling ionophoric channels that regulate transmembrane transport of calcium.     

Effects of centrifugation through three different discontinuous Percoll gradients on boar sperm function

In this study, different combinations of 2-step, discontinuous gradient centrifugation were used, consisting of three different combinations of isotonic Percoll (45/60, 60/75 and 45/90%) that allowed us to select different sperm subpopulations from fertile and normozoospermic boars. Our objective in this study is to evaluate the effects of centrifugation through three different discontinuous Percoll gradients on sperm function parameters (motility, viability, morphology, acrosome status, chromatin condensation, DNA fragmentation, ROS generation, tyrosine phosphorylation and intracellular calcium concentration) and the sperm penetrating capacity in an IVF system. All the Percoll treatments evaluated increased the percentage of spermatozoa with normal morphology, the proportion of un-damaged DNA, normal chromatin condensation, motion parameters measured by CASA and the percentage of capacitated spermatozoa with tyrosine phosphorylated proteins compared to control group. Finally, the in vitro oocyte penetrating capacity of boar spermatozoa was significantly affected by Percoll centrifugation. All the Percoll treatments increased the penetration rates and mean number of sperm per penetrated oocyte. Despite the efficiency of all three of the sperm treatments tested in selecting spermatozoa with improved sperm parameters and capacity to penetrate oocytes in vitro, the optimum performance of this system was demonstrated after preselecting spermatozoa by centrifugation on a discontinuous 45/90 Percoll gradient. The P45/90 treatment leads to obtain a higher percentage of spermatozoa which develop properly the capacitation process as it was shown measuring tyrosine phosphorylation and intracellular calcium concentration.     

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO(2) in air) for 1h in the following treatments either 500 microL of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1h either FM or FM with alpha L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 x 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha L-PGDS: (1) 26.4+/-3.0; (2) 25.6+/-3.0; (3) 59.7+/-3.0; (4) 56.4+/-3.0; and (5) 57.1+/-3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P<0.05) compared with the control: (1) 89.2+/-2.0%; (2) 87.5+/-2.0%; (3) 19.4+/-2.0%; (4) 27.2+/-3.1%; and (5) 14.1+/-3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization.     

Physiological state of bull sperm affects fucose- and mannose-binding properties

In cattle, sperm are stored in a reservoir in the caudal isthmus of the oviduct until the time of ovulation approaches. Bull sperm are trapped in the reservoir by binding to fucosylated molecules on the oviductal epithelium. Capacitated sperm lose binding affinity for the epithelium; therefore this study was undertaken to determine whether this occurs because capacitated bull sperm lose binding affinity for fucose. BSA conjugated to alpha-L-fucopyranosylphenyl isothiocyanate and fluorescein isothiocyanate (fuc-BSA-FITC) was used in conjunction with flow cytometry to monitor the capacity of bull sperm to bind fucose. Dead sperm were identified using ethidium homodimer and were excluded from analysis. BSA-FITC conjugated with mannose (man-BSA-FITC) and BSA-FITC were used as controls. When examined by epifluorescence microscopy, motile bull sperm that exhibited labeling by any of the probes were fluorescent over the acrosomal region of the plasma membrane. By flow cytometry, labeling of live sperm was greatest for sperm that had been washed in TALP medium and probed with fuc-BSA-FITC (mean +/- SD:167 +/- 6.0 relative fluorescence units, collected in logarithmic mode). Labeling by fuc-BSA-FITC was lower in unwashed sperm (60 +/- 2.7) and in washed sperm with seminal plasma added back (56 +/- 8.0). Labeling was also reduced by centrifuging washed sperm through a Percoll step gradient (103 +/- 6.3) and by capacitating washed sperm in medium containing 10 microg/ml heparin (50 +/- 4.4). BSA-FITC labeling was barely detectable in all treatments. Man-BSA-FITC produced little labeling of washed sperm (22 +/- 0.6), as expected; however, intense labeling appeared over the acrosomal region of sperm incubated under capacitating conditions (128 +/- 21.6). It was concluded that removal of seminal plasma exposes fucose-binding sites, which are then lost or modified during capacitation, thereby allowing the release of sperm from the reservoir. At that time, mannose-binding sites are revealed or activated, which might serve to bind sperm to the zona pellucida.     

Nuclear buoyant density determination and the purification and characterization of wild-type neurospora nuclei using percoll density gradients

A procedure has been developed using Percoll density gradients for the isolation and purification of nuclei from germinated conidia of wild-type Neurospora crassa St. Lawrence strain 74A. Crude nuclei were purified isopycnically in gradients of Percoll, which is silica coated with polyvinylpyrrolidone. A DNA:RNA:protein ratio of 1:3.5:6.5 was found in purified nuclei. Cytoplasmic contamination was found to be negligible in the nuclear preparations, as determined by electron microscopy and by following a radioactively-labeled ribosome tag during the isolation procedure. A small amount of endogenous ribonuclease activity was detected in the crude nuclear preparations, but not in suspensions of nuclei purified in the Percoll gradients. Ribosomal RNA was extracted from the nuclei in good yields, and electrophoretic analysis indicated the presence of precursor rRNA molecules, as well as the mature 17S and 25S rRNA species. Using the Percoll gradient system, the buoyant density of purified Neurospora nuclei was determined to be 1.08 grams per milliliter based on refractive index measurements.     

Intraplastidic Localization of the Enzymes That Convert delta-Aminolevulinic Acid to Protoporphyrin IX in Etiolated Cucumber Cotyledons

The intraplastidic localization of the enzymes that catalyze the conversion of δ-aminolevulinic acid (ALA) to protoporphyrin IX (Proto) is a controversial issue. While some researchers assign a stromal location for these enzymes, others favor a membranebound one. Etiochloroplasts were isolated from etiolated cucumber cotyledons (Cucumis sativus, L.) by differential centrifugation and were purified further by Percoll density gradient centrifugation. Purified plastids were highly intact, and contamination by other subcellular organelles was reduced five- to ninefold in comparison to crude plastid preparations. Most of the ALA to Proto conversion activity was found in the plastids. On a unit protein basis, the ALA to Proto conversion activity of isolated mitochondria was about 2% that of the purified plastids, and could be accounted for by contamination of the mitochondrial preparation by plastids. Lysis of the purified plastids by osmotic shock followed by high speed centrifugation, yielded two subplastidic fractions: a soluble clear stromal fraction and a pelleted yellowish one. The stromal fraction contained about 11% of the plastidic ALA to Proto conversion activity while the membrane fraction contained the remaining 89%. The stromal ALA to Proto conversion activity was in the range of stroma contamination by subplastidic membrane material. Complete solubilization of the ALA to Proto activity was achieved by high speed shearing and cavitation, in the absence of detergents. Solubilization of the ALA to Proto conversion activity was accompanied by release of about 30% of the membrane-bound protochlorophyllide. It is proposed that the enzymes that convert ALA to Proto are loosely associated with the plastid membranes and may be solubilized without the use of detergents. It is not clear at this stage whether the enzymes are associated with the outer or inner plastid membranes and whether they form a multienzyme complex or not.