哺乳动物精子质量的评价方法

精子的各种功能状态反应了精子的受精能力。检测精子质膜完整性的荧光探针有SYBR－14,SYTO－17,CFDA、CDMFDA、CAM、PI、Hoechst33258、Hoechst33342,其中以SYBR－14结合PI使用效果最好,检测线粒体活性的荧光探针有JC－1、MITO、Rh123,JC-1比MITO和Rh123更适用于检测精子线粒体功能,检测顶体状态的荧光探针有PNA－FITC、PSA－FITC、LYSO－G及CTC等。检测获能状态的荧光探针有CTC。此外,还可以通过检测精子与透明带的结合能力、精子穿入去透明带卵子的能力以及使卵子受精的能力和其后胚胎的发育能力等方面来评价精子的功能状态。     

Effects of sperm preparation and bull fertility on in vitro penetration of zona-free bovine oocytes

The objectives of this study were to develop and validate a zona-free bovine oocyte penetration assay for detecting relative differences in bovine sperm fertility and to determine the effect of different sperm preparation methods on oocyte penetration. Oocytes were incubated with heparin-capacitated spermatozoa which either were or were not induced to acrosome-react with lysophosphatidylcholine. Heparin-capacitated spermatozoa treated with lysophosphatidyl-choline penetrated more oocytes and had more penetrations per oocyte than spermatozoa capacitated in heparin but not induced to acrosome-react with lysophosphatidylcholine. Spermatozoa stained with Hoechst 33342, fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate, alone or in combination, penetrated similar numbers and percentages of zona-free bovine oocytes as the similar to non-stained spermatozoa. When spermatozoa from the same ejaculate were stained with either fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate and competed in penetrating the same oocytes, the number of penetrations generated by the 2 differently stained spermatozoa was similar. Spermatozoa from bulls of differing in vivo fertilities were labeled with different fluorescent dyes, and their relative abilities to penetrate the same oocytes were assessed. Comparisons between spermatozoa from high and low fertility bulls demonstrated that high fertility spermatozoa had a significant oocyte penetrating advantage over low fertility spermatozoa in 13 of 16 paired competitions. We concluded that the results of the competitive penetration of zona-free bovine oocytes by fluorochrome-labeled spermatozoa from bulls of different fertilities were indicative of their relative in vivo fertility.     

Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14

The objective of these experiments was to determine the efficacy of the new membrane permeant nucleic acid stain, SYBR-14, for assessing boar sperm viability and to determine it's effect on fertilization and early embryonic development using the pig as a model. We examined the staining patterns of SYBR-14 and another vital stain, Hoechst 33342, both in combination with the dead cell stain, propidium iodide (PI), to quantify the proportion of living and dead spermatozoa in ejaculated and epididymal semen. Flow cytometry analyses of semen from 4 boars revealed significant differences among boars for the proportion of SYBR-14-stained spermatozoa in both epididymal and ejaculated samples, but not for Hoechst 33342 or PI stained spermatozoa. Gilts were inseminated with unstained spermatozoa or spermatozoa stained with 2 levels of SYBR-14 or 2 levels of the reference stain, Hoechst 33342. Embryos recovered at 42 to 48 h postinsemination were morphologically evaluated, and only 4 to 8-cell embryos were continued in culture. Overall, fluorescent staining of boar spermatozoa with SYBR-14 or Hoechst 33342 neither affected their ability to fertilize oocytes, nor the developmental competence of the resultant embryos.     

Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3′-dihexyloxacarbocyanine iodide)

We have examined living and fixed gametes and early embryos of surf clams, sea urchins, and hamsters stained with the supravital dyes Hoechst 33342 for DNA and 3,3′-dihexyloxacarbocyanine iodide (DIOC₆) for mitochondria and endoplasmic reticulum. Hoechst staining (10 μM) was confined exclusively to egg and sperm chromatin and, in living marine specimens, did not interfere with sperm motility, fertilization, or nuclear activity during meiosis or early embryogenesis. Although Hoechst staining did not appear to affect the motility of hamster sperm, only zonae-free eggs inseminated. Because chromatin retained Hoechst 33342 stain during fertilization, the paternally and maternally derived chromosomes of living and fixed preparations fluoresced and their number, organization, and location within the zygote cytoplasm could be determined. Hence, polyspermy and other nuclear abnormalities were amenable to examination in these stained preparations. DIOC₆ staining (8.7 μM) was restricted primarily to the mitochondria of spermatozoa. Eggs stained with DIOC₆ (0.87 to 8.7 μM) were brightly fluorescent because of their size and the presence of large numbers of mitochondria and other DIOC₆-positive organelles. Sea urchin and surf clam sperm stained with DIOC₆ fertilized unstained eggs and the location of the incorporated sperm mitochondrion up to first cleavage was followed. Although hamster sperm stained with DIOC₆ were less motile than unstained sperm, they were capable of inseminating only zonae-free eggs. These observations demonstrate that staining with supravital fluorochromes provides a rapid and useful method to analyze macromolecular and organelle changes in a variety of living and fixed gametes and embryos.     

Viable embryos and normal calves after nuclear transfer into Hoechst stained enucleated demi-oocytes of cows.

Bovine oocytes were bisected, stained with Hoechst 33342 and observed under a fluorescent microscope to identify nucleated and enucleated demi-oocytes. Other oocytes were bisected but not stained, or bisected and only half of each oocyte stained, and viewed under a fluorescent microscope. The oocytes were then used for nuclear transfer by fusing them with embryonic blastomeres from a 5-6 day bovine embryo. The fusion rate and proportion developing into compact morulae or blastocysts was compared among different types of demi-oocytes. Expt 1 examined the effect of staining and indicated no effect on either fusion rate or embryonic development whether or not the oocytes were stained. In Expt 2, stained and unstained nucleated and enucleated oocytes were compared. As in the first experiment, there were no differences between stained and unstained demi-oocytes. There was no difference between fusion rates of nucleated and enucleated oocytes. However, there was a significant difference in embryonic development between nucleated (10.4%) and enucleated (22.6%) demi-oocytes (P less than 0.05). In a final experiment, stained and unstained enucleated oocytes were used for nuclear transfer and the resulting embryos transferred into recipient cows. There was no difference in pregnancy rates or in the number of normal calves born whether stained or unstained recipient oocytes were used. Results from these experiments indicate that Hoechst staining and fluorescent microscopy can be used to identify enucleated demi-oocytes, and that these can be used for nuclear transfer, and result in viable embryos and normal calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorometric evaluation of cryopreserved bovine spermatozoa extended in egg yolk and milk

A comparison of fluorogenically quantifiable parameters of cryopreserved, bovine spermatozoa that had been processed in homogenized milk and egg yolk citrate-based extenders was made using flow cytometry. Semen from four bulls was processed in egg yolk-citrate or homogenized milk extenders, packaged in straws and frozen at -196 degrees C. Samples were thawed at 37 degrees C, subdivided into three portions and stained after 0, 1.5 and 3 h of incubation at 37 degrees C. Spermatozoa were stained using a combination of carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) and analyzed by dual parameter flow cytometry. The sperm cells were quantified fluorometrically at each time interval for both green and red fluorescence. The proportion of spermatozoa retaining the fluorescent CFDA derivative was larger at each time interval for samples in egg yolk citrate than those in milk. Differences in the retention of spermatozoal viability were detected between identical samples of bovine spermatozoa extended in milk or egg yolk based media.     

Microplate screening of the differential effects of test agents on Hoechst 33342, rhodamine 123, and rhodamine 6G accumulation in breast cancer cells that overexpress P-glycoprotein

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.     

New assays for detection and localization of endogenous lipid peroxidation products in living boar sperm after BTS dilution or after freeze-thawing

Reactive oxygen species have been implicated in sperm aberrations causing multiple pathologies including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries for semen storage prior to artificial insemination but unusual in porcine breeding industries as semen dilution and storage at 17 degrees C is sufficient for artificial insemination within 2-3 days. However, longer semen storage requires cryopreservation of boar semen. Freeze/thawing procedures induce sperm damage and induce reactive oxygen species in mammalian sperm and boar sperm seems to be more vulnerable for this than bull sperm. We developed a new method to detect reactive oxygen species induced damage at the level of the sperm plasma membrane in bull sperm. Lipid peroxidation in freshly stored and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous lipid classes, phosphatidylcholine and cholesterol and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY(581/591). Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in the living sperm cells. Cytoplasmatic droplets in incompletely matured sperm cells were intensely peroxidized. Furthermore, lipid peroxidation was particularly strong in the mid-piece and tail of frozen/thawed spermatozoa and significantly less intense in the sperm head. Induction of peroxidation in fresh sperm cells with the lipid soluble reactive oxygen species tert-butylhydroperoxide gave an even more pronounced effect, demonstrating antioxidant activity in the head of fresh sperm cells. Furthermore, we were able to show using the flow cytometer that spontaneous peroxidation was not a result of cell death, as only a pronounced subpopulation of living cells showed peroxidation after freeze-thawing. Although the method was established on bovine sperm, we discuss the importance of these assays for detecting lipid peroxidation in boar sperm cells.     

Staining of living bacteria with rhodamine 123

Abstract It is possible to stain live bacteria with rhodamine 123 (R123). The stained fluorescent cells still keep the ability to replicate ( Staphylococcus aureus, Bordetella pertussis ) and to swim (e.g., Salmonella minnesota ). Dead cells or cells with a dissipated transmembrane potential showed markedly diminished fluorescence. Gram-negative strains were stained with different efficiency, presumably reflecting the different constitutions of the outer membrane.     

Rhodamine 123 as a cytoplasmic stain for mammalian zygotes

A mitochondrion-specific fluorescent dye, rhodamine 123, stains the cytoplasm of ova from mice, rabbits, sheep, cattle and pigs. Mouse zygotes stained with rhodamine 123 are often observed with areas of negative stain where the pronuclei are located. However, such areas of negative staining are not observed in zygotes from rabbits, sheep or cattle. In vitro viability of mouse zygotes stained with rhodamine 123 is high if observed with fluorescent microscopy for 5 min or less. Observation for more than 5 min results in a significant effect on embryo developmental potential in vitro.     

Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.     

Development of bovine embryos after in vitro fertilization of oocytes with flow cytometrically sorted, stained and unsorted sperm from different bulls

The objective of this study was to determine the effect of staining bovine sperm, with or without flow cytometry, on in vitro fertilization of bovine oocytes and blastocyst development. Bovine oocytes (n=4273) were fertilized with frozen–thawed sperm from three bulls that was: stained with Hoechst 33342 and sorted (into X- or Y-chromosome-bearing sperm) with flow cytometry; stained but not sorted; and not stained or sorted (Control). Oocytes, aspirated from slaughterhouse ovaries, were matured in TCM199 (supplemented with 10% fetal calf serum and 15 ng FSH, 1.0 μg LH, 1.0 μg E2/ml) for 22–24 h at 39 °C in 5% CO₂ in air with maximum humidity. Presumptive zygotes were removed from culture and placed in chemically defined medium (CDM-1) 6–7 h after insemination and cultured for 65–66 h. Embryos that had cleaved by 72 h post-insemination were cultured an additional 96 h in CDM-2 containing 0.12 IU insulin/ml. Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7–8 after insemination, respectively. There was no significant difference in blastocyst rate among the three types of sperm; however, cleavage rates with stained and sorted sperm (53.1%) and unsorted, stained sperm (59.9%) were lower (P<0.05) than Control sperm (69.7%). Furthermore, there were significant differences due to semen from different bulls in cleavage and blastocyst rates.     

Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.     

PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.     

Blastomere Content of Cultured/Frozen Bovine Demiembryos

The purpose of the study was to evaluate whether a period of co-culture with bovine oviduct epithelial cells (BOEC) could improve the tolerance of bisected bovine embryos to freezing and thawing. Day 6 embryos were bisected and the resulting demiembryos were stained with Hoechst 33342 and cell counts were made by counting intact blastomere nuclei. Of these, 11 were stained as freshly manufactured demiembryos, 25 after co-culture for 24 h with BOEC and 37 stained after 24 h co-culture and freezing and thawing. The staining revealed, that there was no significant difference in cell count of demiembryos that were stained immediately after bisection, compared to those, that were co-cultured for a 24 h period. Also, the co-cultured/frozen/thawed demiembryos had a significant decrease in cell numbers compared to the non-frozen demiembryos. We conclude, that a 24 h period of co-culture with BOEC does not result in appreciable cellular proliferation in demiembryos and therefore instead of improving the survival of frozen/thawed demiembryos by giving them opportunity to multiply their cell number and thus make them more resistant to cell damage, rather compromised the viability of cryopreserved demiembryos.     

The use of the tetrazolium salt XTT for the estimation of biological activity of activated sludge cultivated under steady-state and transient regimes

We have shown that the growth, starvation and population heterogeneity of Salmonella typhimurium and its isogenic nuoG and cydA mutants can be monitored by flow cytometry. Bacterial cells were analysed unstained, and after staining with rhodamine 123, propidium iodide and acridine orange. In unstained cultures it was possible to distinguish flagellated and non-flagellated cells. nuoG and cydA mutants were less stained with rhodamine confirming their defects in generating membrane potential. Increase in propidium iodide staining associated with reduced membrane integrity was seen between day 4 and 14 in all the strains. Acridine orange staining showed that there was retarded development in stationary phase in nuoG and cydA mutants. Furthermore, up to day 28, a small portion of cells showed high RNA and DNA levels. To determine whether these cells represent a sub-population better adapted for long term survival, we measured the growth of the population by both OD values and viable counts. Because the OD values increased throughout the whole study in both wild-type and mutant strains, while the viable counts gradually decreased, we propose that even in very old cultures there must be a population of cells undergoing replication.     

In situ detection and localization of lipid peroxidation in individual bovine sperm cells

Reactive oxygen species (ROS) have been implicated in many pathologies, including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries in order to perform artificial insemination. This freeze/thaw procedure is known to induce ROS in sperm samples. Lipid peroxidation in fresh and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous phospholipid class, phosphatidylcholine, and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY(581/591). Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in living sperm cells. Lipid peroxidation was particularly strong in the midpiece and tail of frozen/thawed spermatozoa and significantly less intense in the head. Induction of peroxidation in fresh sperm cells with the lipid soluble ROS tert-butylhydroperoxide gave an even more pronounced effect, demonstrating antioxidant activity in the head of fresh sperm cells. Furthermore, we were able to show that spontaneous peroxidation was not a result of cell death, as only a pronounced subpopulation of living cells showed peroxidation after freeze/thawing.     

Evaluation of sperm tail membrane integrity by light microscopy

During routine evaluation of trypan blue-Giemsa stained semen smears, sperm cells can be found with unstained heads and with stained tails. It was hypothesized that these cells were immotile and should not be considered as live. Sperm motility was determined in isoosmotic, and presumably isotonic trypan blue-stained wet preparations. Bull, ram and boar semen smears were stained with hypoosmotic trypan blue-Giemsa to compare the relationship between the percentage of stained sperm tails and the percentage of sperm tails remaining straight under hypoosmotic conditions. Actively moving spermatozoa with unstained heads, but with stained tails were never observed in wet preparations. The correlation coefficient found between the percentage of sperm with stained tails and the percentage with straight tails was 0.81, 0.94 and 0.85 for bull, ram and boar spermatozoa, respectively. Results of this study show that sperm cells with an intact head membrane, but a stained and presumably membrane-damaged tail are not motile. Therefore these cells should be included in the dead category rather than alive in the usual live-dead studies with vital stains.     

Characterizing the silica deposition vesicle of diatoms

Summary The present study on a centric diatom,Ditylum brightwellii, includes two parts: detection of sugars in the silica deposition vesicle (SDV) with lectins and labeling the developing siliceous cell wall in the SDV with rhodamine 123. Cells with developing valves are treated with SDS to remove all the cytoplasmic contents, then either stained with fluorescein labeled lectins or thin-sectioned and stained with colloidal gold labeled lectins. The results show that mannose is part of the organic matrix in the SDV. Rhodamine 123, a non-toxic fluorescent laser dye, enters the cell immediately and is trapped in the SDV probably by the high reducing potential of the SDV. Silica is co-deposited with rhodamine 123 in the SDV, and the resulting valves and girdle bands become fluorescent. Implications of this study for the mechanism of silicification are discussed.Abbreviation SDV Silica deposition vesicle     

Stimulation of P-glycoprotein-mediated drug transport by prazosin and progesterone. Evidence for a third drug-binding site.

P-glycoprotein is a plasma membrane protein of mammalian cells that confers multidrug resistance by acting as a broad-specificity, ATP-dependent efflux transporter of diverse lipophilic neutral or cationic compounds. Previously, we identified two positively cooperative drug-binding sites of P-glycoprotein involved in transport [Shapiro, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 130-137]. The H site is selective for Hoechst 33342 and colchicine. The R site is selective for rhodamine 123 and anthracyclines. Substrate binding to one site stimulates transport by the other. In this paper, we show that prazosin and progesterone stimulate the transport of both Hoechst 33342 and rhodamine 123. Rhodamine 123 and prazosin (or progesterone) in combination stimulate Hoechst 33342 transport in an additive manner. In contrast, Hoechst 33342 and either prazosin or progesterone interfere with each other, so that the stimulatory effect of the combination on rhodamine 123 transport is less than that of each individually. Non-P-glycoprotein-specific effects of prazosin on membrane fluidity and permeability were excluded. These results indicate the existence of a third drug-binding site on P-glycoprotein with a positive allosteric effect on drug transport by the H and R sites. This allosteric site appears to be one of the sites of photoaffinity labeling of P-glycoprotein by [125I]iodoarylazidoprazosin [Safa, A. R., Agresti, M., Bryk, D. & Tamai, I. (1994) Biochemistry 33, 256-265] and is likely not to be capable of drug transport.