Mass Isolation and Preservation of Viable Sperm Cells in Brassica campestris var.purpurea

The two-step osmotic shock and grinding methods reported by Yang and Zhou (1989) were modified for isolation of viable sperm cells in large quantities from pollen grains of Brassica campestris var. purpurea. Factors affecting the yield and survival of isolated sperm cells have been investigated. These included physiological status of donor flowers, sucrose concentration used for pollen hydration, basic media, protectants and osmotica supplemented in the medium etc. As a result, two procedures have been developed. For osmotic shock method, pollen grains at the day of anthesis were hydrated in 25% sucrose solution for 30 min and, after centrifugation and removal of the supernatant, the pellet was shocked by a medium containing 12.5% sucrose, 0.1 g/L KNO3, 0.36 g/L CaCl2, 2H2O, 0.3% potassium dextran sulphate (PDS), 0.6% bovine serum albumin (BSA), and 0.3% polyvinylpyrrolidone (PVP). The viable sperm yield was 34%. After removal of pollen wall debris by filtration and centrifugation, the sperm cell-rich pellets were resuspended in a medium containing 20% sucrose, 5% sorbitol, 0.1 g/L KNOs, 0.36g/L CaCl2·2H2O, 0.6% BSA and 0.3% PDS, and preserved at 4℃ for two days. For grinding method, the pollen grains hydrated in 30% sucrose solution for 30 min. were resuspended in a medium containing 20% sucrose, 5% sorbitol, 0.1g/L QNO3, 0.36g/L CaCl2·2H2O, 0.3% PDS, 0.6% BSA, 0.3% PVP and 20 μg/ml fluorescein diacetate, then ground with a glass homogenizer to release the sperm cells. The viable sperm yield was up to 86%. Following filtration and centrifugation for removal of pollen wall debris, the sperm cells were stored at 4℃ in the same medium but without supplementation of PVP. Tested by fluorochromatic reaction, the sperm cells could survive up to one week with a gradual decline of viability. Cytological observations revealed that pairs of ellipsoidal sperm cells just released were linked together; one of the pair had a long tail-like extension which also show fluorochromasia. Soon after, the sperm cells separated and turned to be spherical. The present results open a prospect to use isolated viable sperm cells for further experimental manipulations.     

Two-Dimensional Electrophoretic Studies of the Proteins and Polypeptides in Mature Pollen Grains and the Male Germ Unit of Plumbago zeylanica

Three fractions (male germ unit [MGU]-rich, cytoplasmic-particulate and water-soluble proteins) were isolated from pollen of Plumbago zeylanica L. Proteins were extracted using a phenol procedure and polypeptide patterns were compared on one- and two-dimensional polyacrylamide gels. The MGU-rich fraction contains the sperm and vegetative nucleus of the pollen grain and yielded 427 spots >33 kilodaltons. The cyto-plasmic-particulate fraction contained 515 spots >33 kilodaltons. The third fraction consisted of water-soluble proteins and polypeptides from the pollen cytoplasm, in which 285 spots (>33 kilodaltons) were identified. Of 133 polypeptide spots suitable for comparison, 18 were unique to the MGU-rich fraction, 3 to the cytoplasmic-particulate fraction, 14 to the water-soluble fraction, 65 were common to two different fractions (and absent in one), and 33 were common to all three of the fractions examined.     

Structural and Biochemical Characterization of Isolated Trichocysts of the Ciliate Pseudomicrothorax dubius1

ABSTRACT Trichocysts of Pseudomicrothorax dubius were ejected by 15% ethanol in phosphate-buffered culture medium (CM) and purified on discontinuous sucrose gradients, in which they concentrated in the lower part of the 27% phase and in the 57% phase. These phases were washed by 15% ethanol in CM, or by CM alone, and pooled. Ejected trichocysts observed by Nomarski interference contrast microscopy and after negative-staining for electron microscopy show a shaft with periodic cross-bands and four opened-out arms, sometimes with electron-dense droplets at both ends of each arm. On SDS-PAGE, trichocysts show ?20 protein bands. The major bands are at 31 and 30 kD (G₁), 27 and 26.5 kD (G₂), 25 kD, 23 kD, and six bands at 15–20 kD (G₃). Minor bands are observed above 30 kD, among them ciliary components which contaminate the trichocyst fraction. The trichocyst banding pattern was reproducible with different ejection media; however, the 30 kD disappeared when the buffered ejection medium contained no added Ca²⁺ or contained EDTA. When the trichocyst extract is solubilized in sample buffer without 2-mercaptoethanol, the major trichocyst bands are those of G₁ and bands at 32.5–35 kD and 41 kD, which appear to be dimers of a few of the G₃ proteins. On two-dimensional gels of trichocysts, ?40 acidic protein spots are resolved with pI's of 4.6–6.6. On Western blots of two-dimensional gels, glycoproteins were revealed by Concavalin A-peroxidase labeling in three spots of G₃, in two spots at 23 kD, in all five spots of G₁, and in seven spots over 35 kD.     

Comparative cryopreservation of avian spermatozoa: benefits of non-permeating osmoprotectants and ATP on turkey and crane sperm cryosurvival

A comparative approach was used to evaluate the cryosurvival of turkey and crane sperm frozen in a dimethylacetamide (DMA) cryodiluent supplemented with osmoprotectants and ATP. A range (6-26%) of DMA concentrations was used alone or in combination with ATP (30, 60 or 118mM) or one of the following osmoprotectants: (1) sucrose (turkey, 8.0%; crane, 5.0%); (2) 5.0% sucrose and 5.0% trehalose; or (3) betaine hydrochloride (0.1, 0.2 or 0.4mM). The viability of thawed sperm was assessed using the nigrosin-eosin stain and sperm motility was determined using the hanging-drop technique. For semen frozen only with DMA, post-thaw sperm motility was greatest (P<0.05) for the 6.0%, 10.0% and 18% concentrations, regardless of species. Turkey sperm frozen with the sucrose/trehalose combination had greater (P<0.05) post-thaw motility for all DMA treatments compared to DMA alone. The lowest concentration of the osmoprotectant betaine hydrochloride substantially improved turkey sperm viability post-thaw in all treatments compared to DMA alone (P<0.05). The post-thaw motility of crane sperm was improved (P<0.05) with a combination of 18.0%, 24.0% or 26.0% DMA and 30mM ATP. Moreover, in the presence of osmoprotectants, crane sperm motility decreased as the osmoprotectant concentration increased. The lowest concentration of ATP also improved crane sperm viability post-thaw, especially for DMA concentrations 18% or greater. The combination of sucrose and trehalose improved (P<0.05) crane sperm viability only with 6% and 10% DMA. These data affirm that there are avian-specific differences in sperm survival after cryopreservation and suggest that post-thaw survival can be enhanced by including species-based osmoprotectant/ATP combinations in a cryodiluent where DMA is the cryoprotectant.     

Generation of a mouse sperm membrane fraction with zona receptor activity.

To identify mouse sperm components involved in primary sperm binding to zonae pellucidae, a mouse sperm plasma membrane-enriched fraction was generated using a vortex method. The crude membrane fraction recovered after vortexing was resolved into three bands and a pellet by centrifugation on a discontinuous sucrose gradient. Ultrastructural analysis indicated that Bands 2 and 3 were composed predominantly of membranes, although Band 3 was contaminated with mitochondria; Band 1 and the gradient pellet contained insufficient material and were unsuitable for ultrastructural analysis. To determine where plasma membranes migrate in the gradient, sperm were labeled vectorially with 125I; subsequently, membrane fractions were analyzed by SDS-PAGE and autoradiography. Band 2 was enriched threefold in radiolabel when compared with Band 3. Examination of intact and vortexed sperm stained with regionally distributed anti-mouse sperm monoclonal antibodies revealed that vortexing removed anterior head plasma membrane preferentially. Bioactivity, defined as the ability to inhibit primary sperm binding to the zona pellucida in a concentration-dependent manner, was contained in the crude membrane fraction and Band 2 exclusively, with inhibition of 53% and 44%, respectively, at the maximum concentration tested. Band 3 exhibited no significant bioactivity. We conclude from these results that a plasma membrane-enriched fraction, Band 2, isolated from mouse cauda epididymal sperm, exhibits zona pellucida receptor activity.     

Improved flow cytometric sorting of X‐ and Y‐chromosome bearing sperm: Substantial increase in yield of sexed semen

The yield of flow cytometric sorted X‐ and Y‐chromosome‐bearing sperm in a given time period is an important factor in the strategies used for fertilization and the production of sex‐preselected offspring. This yield is dependent on the efficiency with which the modified flow cytometer/cell sorter analyzes the DNA of spermatozoa. The efficiency is directly related to the number of sperm with the correct orientation during DNA analysis. Currently, the efficiency of flow cytometric sperm sorting is low since orientation of the sperm head to laser excitation is rate limiting. To overcome this problem, a new nozzle was designed to enhance sperm orientation and tested under flow cytometric sorting conditions. The degree of orientation improvement was determined with different sample rates using viable sperm and dead sperm of several different species. There was at minimum, a two‐fold increase in the proportion of oriented sperm when comparing the new nozzle with the currently used modified flow cytometer/cell sorter employing a beveled needle. More than 60% of intact bull sperm and boar sperm were correctly oriented compared with 25% to 30% using the beveled needle system. A unique characteristic of the novel nozzle was that the proportion of oriented sperm was independent of sample rate and of sperm motility. The accuracy of DNA measurement together with high purity sorting was tested using the novel nozzle. The novel nozzle was unique in that accuracy of measurement and sorting performance were not diminished. Using the new nozzle, samples of 88% purity of sorted X‐sperm and Y‐sperm were obtained for viable bull and boar sperm. The yield of flow cytometric sorted X‐ and Y‐chromosome‐bearing sperm using the novel nozzle was, on average, twice that obtained by using the beveled needle system in conjunction with a standard equipment nozzle for orientation. Mol. Reprod. Dev. 52:50–56, 1999. Published 1999 Wiley‐Liss, Inc.     

DNA integrity of Polyodon spathula cryopreserved sperm

Comet assay was used to detect DNA integrity of paddlefish (Polyodon spathula) sperm following cryopreservation. At the same time, sperm velocities prior to freezing and post‐thawing were also assessed by the computer‐assisted sperm analysis (CASA) system. Significant differences (P < 0.05) were detected in the degree of DNA damage in cryopreserved sperm using different extenders. According to osmolality of the extenders, DNA damages of Sb (20 mm Tris, 75 mm sucrose, 0.5 mm KCl, pH 8.5) sperm was the least, which showed that the percentage of tail DNA of Sb (17.87–35.28%) was lower than those of Sa (20 mm Tris, 50 mm sucrose, 0.5 mm KCl, pH 8.5) and Sc (20 mm Tris, 100 mm sucrose, 0.5 mm KCl, pH 8.5). Moreover, A and B class sperm cells provided most of the Sb sperm (>50%). However, in light of the concentration of methanol, DNA damages of M8 (8% methanol concentration) sperm were the least, including a lower percentage of the tail DNA (21.56–30.86%), and C and D class sperm cells (<30%), regardless of the osmolality of the extenders. In conclusion, when the dilution was 20 mm Tris, 75 mm sucrose, 0.5 mm KCl, pH 8.5 and the concentration of methanol was 8%, the extenders were the best for cryopreservation of paddlefish sperm. In addition, the results indicated that the extent of damage to sperm motility caused by freeze‐thawing (VCL, VSL) was correlated with DNA breakage (|r| > 0.8). This implied that cryopreservation could damage sperm DNA of paddlefish and affect the sperm velocities when the osmolality and the concentrations of the cryoprotectants of the extender were inappropriate.     

Characterization of the semen quality of postpuberal boars with spontaneous unilateral abdominal cryptorchidism on the right side.

In recent studies, we found that the ectopic testis from postpuberal boars with unilateral abdominal cryptorchidism does not produce sperm. Therefore, in these males, the seminal characteristics can be used as indicators of the activity of the scrotal testis and its epididymis and also the accessory glands. The semen quality (ejaculate volume, cell-rich fraction volume, sperm concentration, sperm vitality, sperm motility, sperm morphology and cephalic stability of spermatozoa) was evaluated in healthy postpuberal boars and in postpuberal boars with unilateral abdominal cryptorchidism on the right side. In comparison with the healthy boars, the unilateral abdominal cryptorchid boars showed a significant decrease of the ejaculate volume, sperm concentration and sperm motility. The low sperm concentration indicated that unilateral abdominal cryptorchidism severely impairs the sperm production of the scrotal testis. The decrease of ejaculate volume was attributed to an abnormal activity of the accessory glands. The alterations in sperm motility develop as a result of dysfunctions in the epididymal epithelium and/or the accessory glands. The sperm vitality, sperm morphology and cephalic stability of spermatozoa maintained normal values; therefore, at testicular level, despite the low sperm production, the germ cell differentiation is not disturbed. At epididymal level, the morphological maturation of spermatozoa is not altered.     

Some properties of acid and alkaline phosphates from boar sperm plasma membranes

Boar sperm plasma membranes were purified by differential and sucrose density equilibrium centrifugation and were found to yield a single band at a density of 1.14 g/cm3. Both alkaline and acid phosphatase activities were enriched in this fraction. The alkaline phosphatase activity was optimal in 100 mM tris (hydroxymethyl) methylamine (Tris)-NaHCO3 at pH 9.9 with 0.05% Triton X-100 and 1 mM MgCl2. This activity was inhibited by ethylenediaminetetraacetic acid (EDTA), cadmium, zinc or heating at 60 degrees C for 30 min. Also, L-homoarginine caused approximately 70% inhibition and L-phenylalanine or L-leucine caused about 10 to 20% inhibition. Acid phosphatase activity was optimal in 100 mM sodium acetate at pH 5.1 with 0.05% Triton. Sodium dodecyl sulfate, potassium fluoride (KF) or sulfhydryl reagents inhibited the activity, while EDTA or heating at 60 degrees C had no effect. These data for enzymes from boar sperm plasma membranes can be used for future work on the quantitation of the enzymes, distinguishing these two phosphatases from other phosphohydrolases, purification of the enzymes and for comparison to phosphatases in other tissues.     

Successful ultrarapid cryopreservation of wild Iberian ibex (Capra pyrenaica) spermatozoa

A method for cryopreserving wild ibex sperm at high cooling rates was developed. To design a freezing solution based on Tris, citric acid, and glucose (TCG), two preliminary experiments were performed using glycerol (GLY) and dimethyl sulfoxide (DMSO) at different concentrations (5%, 10%, 20%). The 10% GLY + 10% DMSO combination reduced (P < 0.05) frozen-thawed sperm motility, which reached a minimum when 20% GLY + 20% DMSO was used. In the second experiment, sperm tolerance to three sucrose concentrations was evaluated (100-mM sucrose, 300-mM sucrose, 500-mM sucrose). Frozen-thawed sperm motility and sperm viability decreased (P < 0.05) at concentrations above 300 mM. The ultrarapid cooling procedure finally used involved a TCG egg yolk (ey)–based extender with 100-mM sucrose, either alone or with 5% GLY with or without BSA. Two warming procedures (37 °C vs. 60 °C) were also evaluated. The TCG ey with 100-mM sucrose but without GLY/BSA returned the best sperm quality variables. Slow warming at 37 °C strongly affected (P < 0.05) sperm motility and viability in all groups. Sperm selection by density gradient centrifugation produced no motile sperm when slow warming was performed. In contrast, when fast warming was used, sperm selection increased (P < 0.05) percentage of motility, viability, and the percentage of sperms with intact acrosomes. Heterologous in vivo fertilization involving domestic goats was performed to evaluate the in vivo fertilization capacity of the ultrarapidly cooled cryopreserved sperm (in TCG-ey + 100 mM sucrose), with warming undertaken at 60 °C. Inseminations of domestic goats resulted in three pregnancies (3 of 16, 18.7% fertility). In conclusion, ibex spermatozoa are strongly sensitive to high concentrations of permeable cryoprotectants and sucrose. However, the combination of ultrarapid cooling, using TCG-ey + 100-mM sucrose, and fast warming at 60 °C, followed by sperm selection by density gradient centrifugation to collect the motile sperm, has a positive effect on sperm viability.     

The effects of extenders and sperm‐egg ratios on fertilizing ability of cryopreserved testicular sperm of northern pike (Esox lucius L.)

The effects of four different extenders and two sperm‐to‐egg ratios on fertilizing ability of cryopreserved testicular sperm of northern pike (Esox lucius L.) were tested in the present study. Testicular sperm was diluted with each of the four extenders (0.6 m sucrose + 15% DMSO, 0.3 m glucose + 15% DMSO, 0.6 m sucrose + 10% methanol and 0.3 m glucose + 10% methanol, all supplemented with 10% egg yolk and 1.7 g KCl L^?1) and frozen in 0.5‐ml straws at 2 cm above the surface of liquid nitrogen and thawed at 25°C for 30 s. Then 125 μl or 50 μl of frozen‐thawed testicular sperm was poured onto about 1250 eggs for fertilization. The results showed that both sperm‐to‐egg ratio and diluent had no significant influence on cryopreservation efficiency of testicular sperm, whereas cryoprotectant had a significant influence. The highest fertilization rate (92.2%) was obtained from testicular sperm cryopreserved in glucose‐based extender containing 10% methanol at a sperm‐to‐egg ratio of 1 × 10⁶ spermatozoa per egg. The results indicated that glucose‐based extender containing 10% methanol, 10% egg yolk and 1.7 g KCl L^?1 was a useful combination.     

Spot overlapping in two-dimensional maps: a serious problem ignored for much too long

In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the DeltapI and DeltaMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.     

Fate of the sea urchin egg receptor for sperm following fertilization

The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH- terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi-associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane-bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane-bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.     

Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane

Ram spermatozoa were obtained from different regions (caput, corpus, and cauda) of the epididymis and their plasma membrane was removed using a nitrogen cavitation treatment (750 psi, 10 min equilibration at 4 degrees C). Membrane was recovered after sucrose gradient centrifugation and identified using 125I-succinylated concanavalin A (125I-succConA) as a surface marker. Based on fluorescein isothiocyanate-succConA (FITC-succConA) labeling and electron microscopy, cavitation removed plasma membrane from the anterior sperm head in the area overlying the acrosome. Cholesterol was the major sterol in plasma membrane, with desmosterol present in sperm entering the epididymis (caput sperm) but negligible in sperm after epididymal transit (cauda sperm). Ethanolamine and choline phosphoglycerides represented 70-80% of membrane phospholipids, with the ethanolamine fraction decreasing relative to choline phosphoglycerides during epididymal transit. The molar ratio of cholesterol to phospholipid increased in the plasma membrane during maturation. The bulk phospholipid-bound fatty acids consisted primarily of palmitoyl acyl groups (16:0) in caput sperm and docosahexaenoyl acyl groups (22:6) in cauda sperm. The choline phosphoglyceride fraction was purified and analyzed. It consisted of a mixture of ether acyl glycero-3-phosphocholine and diacyl phosphoglyceride, with the dominant acyl residue, at all stages of epididymal maturation, being 22:6 throughout epididymal transit. The significance of these findings relative to acquisition of fertilization capacity by sperm during epididymal maturation is discussed.     

牛精子蛋白质组学技术平台的建立及冻融前后精子差异蛋白初步分析

本研究通过探索不同的精子蛋白制备方法、水化液成分和优化2D电泳程序以建立牛精子蛋白质组学研究技术平台,同时以牛鲜冻精为实验材料通过差异凝胶电泳寻找冻融前后精子蛋白的改变。结果表明：使用改进的热TRIzol法裂解精子细胞制备蛋白,结合优化的2D电泳技术可建立稳定的牛精子蛋白质组学研究技术平台。差异凝胶电泳揭示牛精子在冻融后有质和量的改变：冻融后缺失的蛋白点有20个,表达下调的有2个,表达上调的有10个。作为一项阶段性的实验成果,本研究建立的2D平台和所发现的冻融引起的差异表达蛋白质点为揭示冷冻损伤机理和性控精液的差异蛋白质组学研究奠定了较好的基础。     

Vanadium ion inhibition of alkaline phosphatase-catalyzed phosphate ester hydrolysis.

The intracellular location of guanylate cyclase was examined in sperm from two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus, and from the tube worm Chaetopterus variopedatus. Cells suspended in a medium isotonic with sea water were passed repeatedly through a 23-gauge hypodermic needle to break flagella from heads. This preparation was then fractionated by two methods, one based on centrifugation over a 25% sucrose medium and the other involving repeated differential centrifugation, to resolve flagella from heads. Guanylate cyclase specific activity was increased 3.5–4.5-fold in the flagellar fraction relative to the starting sperm homogenate. Relatively little activity was present in the head fraction where specific activity was $\text{1}\text{10}\text{–}\text{1}\text{100}$ that of the flagella. Plasma membranes were separated from axonemal microtubules by dialyzing flagella against low ionic strength buffer, followed by centrifugation over a 40% sucrose medium. Although the overall recovery of guanylate cyclase was low, the specific activity in the plasma membrane fraction was increased two- to threefold over the dialyzed flagella, and over 90% of the recovered activity resided in this fraction. Thus the flagellar plasma membrane is a site rich in guanylate cyclase. It could not be determined, however, whether this is the only intracellular locale of the enzyme.     

Effect of cooling and freezing,the two first steps of a freezing protocol,on the fertilizing ability of the rabbit sperm

The effect of different phases of a freezing protocol on the fertilising ability of rabbit spermatozoa was evaluated. An extender containing 1.75 M DMSO and 0.05 M sucrose (final concentration) was used to freeze rabbit sperm. In the first experiment, the results obtained with fresh and cooled (5 degrees C for 45 min) sperm were compared; no differences were observed between fresh and cooled semen for any of the parameters studied: fertility rate (78% vs. 91% for fresh and cooled sperm, respectively), and normal embryos two days after insemination (6.8 vs. 8.5 normal embryos for fresh and cooled sperm). In the second experiment, the results obtained with fresh semen and sperm which had passed the first two steps of a freezing protocol (5 degrees C for 45 min and -30 degrees C for 30 min, and thawed at 50 degrees C for 15 s) were compared; the differences between them were obtained for fertility rate (94% vs. 61% for fresh and frozen sperm, respectively) and normal embryos two days after insemination (7.8 vs. 3.8 fresh and frozen sperm). These observations indicated that the differences in the results obtained with fresh and cryopreserved sperm were produced during the second step of the freezing protocol, and that apparently no toxic effect of DMSO was produced.     

Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.     

Isolation and Purification of Viable Sperm Cells from Stored Bicellular Pollen of Lilium davidii Duch.

A successful mass isolation of viable sperm cells from stored bicellular pollen of Lilium davidii Duch. was reported. When fresh pollen was cultured in BKS 15 medium, 87 % germinated in which the generative cells of the fresh pollen underwent mitosis and formed sperm cells within 28 hours. For pollen stored at -20℃ and -70℃ for 6 months, only less than 20 % germinated; but the germination percentage rose to 80 % after they have been hydrated and gradually warmed. Pollen grains of L. davidii which have been stored at -70 ℃ for 6 months, after being thawed were firstly germinated in a 15% sucrose medium for 28 hours, and then osmotically shocked with 10 % sucrose Solution. The solution was later adjusted to a final sucrose concentration of 15%. After density gradient centrifugation, 4 mL suspension of purified sperm cells with a density of 6 × 106 cells/mL were obtained at the interphase of 5%—15% percoll, with a 12% yield of viable sperm cells. The purified sperm cells had a diameter of 13—15 μm and reacted positive to fluorochrome, indicating that they have intact plasma membrane.     

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 μm s⁻¹ swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 °C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 °C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 × 10⁶/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 °C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.