Progesterone does not potentiate the acrosome reaction in human spermatozoa: flow cytometric analysis using CD46 antibody

The study was designed in order to investigate the action ofprogesterone on the spontaneous and ionophore-induced humanspermatozoa acrosome reaction in vitro. The principle of theassay system is flow cytometric analysis of CD46 antibody bindingto the inner acrosomal membrane. The technique is a simple andobjective method of analysis, allowing fluorescent analysisof a large segment (5000 spermatozoa) of the spermatozoa populationunder investigation, with concomitant isolation of the livefraction of the spermatozoa population. Four concentrationsof progesterone (1, 25, 50, and 100µg/ml) were examinedfor their effects on spermatozoa capacitated for 4 and 24 h.In addition, motility parameters were examined by the CellSoft2000 automated semen analyser system. Analysis of variance revealedthat progesterone had no effect on either the spontaneous acrosomereaction or the ionophore-induced acrosome reaction at both4 h and 24 h of spermatozoa capacitation times. Further, noeffects on sperm motility parameters or on spermatozoa viabilitycould be attributed to progesterone. We therefore conclude thatprogesterone had no objectively measurable effects on eitherthe sperm acrosome reaction or sperm motility parameters, asmeasured in normal sperm populations.     

High resolution flow cytometric analysis of electronic nuclear volume and DNA content in normal and abnormal human tissue

Background: The NPE Analyzer^® flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit. Methods: Calibrated beads and biological standards (lymphocytes, trout erythrocytes [TRBC], calf thymocytes, and tumor cells) were analyzed for ENV versus DNA content. Parallel data (forward scatter versus DNA) from a conventional flow cytometer were obtained. Results: ENV linearity studies yielded an R value of 0.999. TRBC had a coefficient of variation (CV) of 1.18 ± 0.13. DNA indexes as low as 1.02 were detectable. DNA content of lymphocytes from 42 females was 1.9% greater than that for 60 males, with a noninstrumental variability in total DNA content of 0.5%. The ENV/DNA ratio was constant in 15 normal human tissue samples, but differed in the four animal species tested. The ENV/DNA ratio for a hypodiploid breast carcinoma was 2.3 times greater than that for normal breast tissue. Conclusions: The high-resolution ENV versus DNA analyses are highly reliable, sensitive, and can be used for the detection of near-diploid tumor cells that are difficult to identify with conventional cytometers. ENV/DNA ratio may be a useful parameter for detection of aneuploid populations.     

Fertilization and early embryology: Female age does not affect the capacity of human zona pellucida to bind spermatozoa

The purpose of this study was to evaluate the effect of femaleage on the capacity of the zona pellucida to bind spermatozoa.A total of 1008 unfertilized oocytes obtained from 210 women(aged 21–43 years) participating in the in-vitro fertilizationprogramme were tested using a hemizona assay. Spermatozoa takenfrom a cryopreserved pool of fertile donor specimens servedas a control in the hemizona assay, and were used to assessthe ability of the zona pellucida to bind spermatozoa. The mean± SD number of spermatozoa attached to the hemizona was107 ± 42. The binding capacity of different oocytes fromthe same cohort varied substantially (coefficient of variation= 28%). Age was not found to be correlated with the number ofspermatozoa bound to the zona pellucida (r = -0.02; P > 0.1).It was concluded that female age has no role in the abilityof the human zona pellucida to bind spermatozoa. Key words:female age/spermatozoa/zona binding.     

Hydrosalpinx fluid does not adversely affect the normal development of human embryos and implantation in vitro

Several retrospectively designed studies have shown an associationbetween the presence of hydrosalpinx and impaired implantationand pregnancy rates among in-vitro fertilization (IVF) patients.In the present study we have evaluated the influence of hydrosalpinxfluid on normal human embryo development and implantation. Surplus,donated frozen embryos (n = 183) from IVF patients were usedto study the effects on blastocyst development of hydrosalpinxfluid at concentrations of 50 and 100% compared with controlsin S2 medium. The fluids were analysed for concentrations ofelectrolytes, osmolarity, protein content, endotoxin levels,bacterial or fungal contamination, pH and haemoglobin content.There was no difference in blastocyst development in culturesunder mineral oil when control cultures (15/42 = 36%) were comparedwith cultures in 50% hydrosalpinx fluid (32/96 = 33%). The onlybiochemical parameter which correlated with capacity for blastocystdevelopment was pH in hydrosalpinx fluid/medium (50/50%) afterequilibration in 5% CO₂ in air. When embryos were cultured in100% hydrosalpinx fluid the blastocyst development was 14% (5/36)in comparison to control 33% (3/9). The original experimentwas repeated in an open culture system without the protectionof mineral oil but still in the presence of 50% hydrosalpinxfluid. The rate of blastocyst development was within the samerange in the open system. In three separate experiments, thecapability of expanded blastocyst to implant on multilayer artificialendometrium was tested. In these experiments, 1/3, 4/5 and 9/9blastocysts implanted. The present study demonstrates that hydrosalpinxfluid does not generally exert any major negative effects onin-vitro development of human embryos or on the implantationprocess in vitro.     

Aging does not affect calcium response to CCL2 and LPS in human monocytes

Monocytes play important roles in anti-microbial and anti-viral responses and chronic inflammatory diseases. Monocytes' functions are altered by aging. We investigated age-changes in calcium (Ca²⁺) response to CCL2 and LPS in human monocytes. CCL2 and LPS induced a slow increase of the cytosolic Ca²⁺ level, with a maximum response at ～360 s and ～300 s, respectively, in monocytes of young and older adults. No difference was observed in the magnitude and in the Ca²⁺ kinetic with both stimuli. Furthermore, store-operated Ca²⁺ entry and plasma membrane expression of ORAI1 showed no difference between both groups. In summary, monocytes from older adults maintained the capacity to mobilize calcium as their counterparts in young adults suggesting that the mechanisms underlying the dysfunctions in monocytes in aging might not involve alterations in Ca²⁺ flow through the plasma membrane.     

Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay

The sperm chromatin structure assay (SCSA) is a flow cytometric (FCM) technique which exploits the metachromatic properties of Acridine Orange to monitor the susceptibility of sperm chromatin DNA to in-situ acid denaturation. SCSA was used to study the chromatin structure variations of human spermatozoa in semen, both before and after swim-up and after cryopreservation. Semen samples were provided by 19 healthy normozoospermic subjects attending pre-marriage checks. Each sample was divided into three aliquots: the first aliquot was evaluated without further treatment, the second underwent swim-up, and the third was stored according to standard cryopreservation techniques in liquid nitrogen at -196 degrees C. Samples were also analysed by light and fluorescence microscopy (after Acridine Orange staining to evaluate the number of green fluorescent sperm heads), and by computer-assisted semen analysis. The results showed that post-rise spermatozoa represent a subpopulation characterized by a general improvement of the morphological (reduction of the percentage of abnormal forms and heads, increase of the green head sperm percentage) and kinetic parameters. This subpopulation also exhibited improved chromatin structure properties, confirming that these cells have the best structural and functional characteristics, indicative of optimal fertilizing ability. On the other hand, overall sperm quality deteriorates after cryopreservation. When thawed spermatozoa underwent an additional swim-up round, a general improvement of nuclear maturity was seen in the post-rise spermatozoa.     

Improved sensitivity and resolution in the flow cytometric DNA analysis of human solid tumor specimens. Use of in vitro fine-needle aspiration and uniform staining reagents.

Twenty-five unselected fresh colon or breast tumors were studied to identify specific components of sample preparation, sample staining, and flow cytometer operation affecting the sensitivity of DNA stemline analysis. Solid tumors were disaggregated using both a published method for mechanical/enzymatic whole cell (M/EWC) isolation and a fine-needle aspiration (FNA) technique. Staining FNA samples with CycleTEST propidium iodide reagents demonstrated improved sensitivity in the recognition of near diploid and near tetraploid aneuploid populations: 9 of 20 resolvable aneuploid DNA stemlines identified in FNA suspensions were not detected or clearly resolved in M/EWC preparations. These results suggest that previously reported discordances between flow and static image cytometry in the recognition of near tetraploid DNA stemlines may be related to inherent limitations of M/EWC. The in vitro FNA technique, when compared to M/EWC, may yield increased sensitivity and precision in clinical DNA stemline analysis when using fresh, unfixed, solid tumor specimens.     

Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining.

The acrosome reaction is a necessary prerequisite for spermatozoa to acquire fertilizing ability. Several different moieties appear to promote the acrosome reaction through different pathways, including solubilized zona pellucidae, recombinant zona protein ZP3, follicular fluid, calcium ionophores, and mannosylated bovine serum albumin (BSA). Although many investigators have presented evidence that progesterone also promotes the acrosome reaction through the mediation of a non-genomic cell membrane receptor, this concept has been challenged. Other workers have suggested that progesterone does not promote an acrosome reaction in human spermatozoa, as judged by the detection of CD46, a complement regulatory protein present on the inner acrosome membrane, through flow cytometric analysis of large numbers of spermatozoa. Prior investigations were criticized by the limited numbers of spermatozoa enumerated visually, the use of non-specific staining techniques, and the failure to eliminate dead spermatozoa during the scoring of the acrosome reaction. We have repeated these experiments, using both a supravital dye to eliminate dead spermatozoa from flow cytometric analysis, and anti-CD46 monoclonal antibody to score acrosome-reacted spermatozoa. Care was taken to validate the adequacy of capacitation conditions, which were proven by the ability of spermatozoa to acrosome react in response to mannosylated BSA and to penetrate zona-free hamster eggs. Confocal microscopy was used to confirm that CD46 immunostaining was limited to the acrosomal region of the spermatozoon head. Our results indicate that progesterone does promote an acrosome reaction within capacitated spermatozoa.     

Pimecrolimus does not affect the differentiation,maturation and function of human monocyte-derived dendritic cells,in contrast to corticosteroids

Clinically, corticosteroids (CS) are among the first line drugs in the therapy of autoimmune and allergic diseases and potently inhibit the activation of immune cells. However, due to their pleiotropic mode of action, the prolonged use of CS is generally associated with a range of undesirable side‐effects. In this study, we compared the activity of pimecrolimus, a novel immunomodulatory drug for the treatment of inflammatory skin disorders, and the CS dexamethasone (Dex) and beta‐methasone‐valerate (β‐MSV) in different in vitro assays addressing the cytokine‐induced differentiation and maturation of monocyte‐derived dendritic cells (M‐DC), the susceptibility of M‐DC to drug‐induced apoptosis and the potency of differentiated M‐DC to induce primary T cell activation. In contrast to pimecrolimus, Dex and β‐MSV strongly induced apoptosis of M‐DC precursors if added at the start of the DC differentiation culture. Flow cytometric analysis of surviving cells on day 6 of culture showed that the expression of several DC‐specific antigens such as CD1a, CD40 and CD80 was inhibited by 50% to 80% at concentrations between 1 nm and 10 nm of either Dex or β‐MSV. Furthermore, the presence of CS during the final maturation of M‐DC inhibited the synthesis of IL‐12p70, the expression of critical DC costimulatory molecules, such as CD83 and CD86 and impaired their ability to activate primary CD4⁺ T cell proliferation. In contrast, pimecrolimus did not inhibit the LPS‐induced secretion of IL‐12, surface expression of costimulatory molecules or the maturation of M‐DC into potent stimulators of T cells. Taken together, these data indicate that pimecrolimus does not interfere with the differentiation and viability of dendritic cells and their precursors or with the function of mature M‐DC to prime naïve T lymphocytes, and thus may have a lower potential than CS to interfere with DC‐mediated immunosurveillance.     

CD71和Hoechst33258用于孕妇外周血中胎儿有核红细胞的分选

目的 比较ＣＤ７１单染和ＣＤ７１、Ｈｏｅｃｈｓｔ３３２５８（ＨＯ２５８）双染对单个核细胞的标记情况,并用于对孕妇外周血中有核红细胞（ｎｕｃｌｅａｔｅｄ ｒｅｄ ｂｌｏｏｄ ｃｅｌｌｓ,ＮＲＢＣｓ）的分选。方法 选用红细胞特异性抗体ＣＤ７１、核染料ＨＯ２５８对正常孕妇外周血、轻（或）中度妊娠高血压综合征（简称妊高症）患者外周血、初生婴儿脐血单个核细胞进行标记,并结合流式细胞术对有核红细胞进行分选。结     

Description of a flow cytometry approach based on SYBR-14 staining for the assessment of DNA content, cell cycle analysis, and sorting of living normal and neoplastic cells

RATIONALE: This study aimed to expand the utilization of a simplified flow cytometric approach that employing SYBR-14/PI staining into broader flow cytometry applications, including (i) measurement of the DNA content; (ii) performing cell cycle analysis on mammalian cells; and (iii) sorting of live SYBR-14-stained mammalian cells based on DNA content. MATERIAL AND METHODS: Cell lines of human origin were stained with SYBR-14 and propidium iodide (PI) and assessed by a dual-color flow cytometry. Finally, sorting of living SYBR-14-stained human cell lines was performed. RESULTS: Dual staining with SYBR-14 and PI of human cells followed by flow cytometry analysis demonstrates that in addition to quality assessment, this staining could be utilized to determinate the DNA content on mammal cells. In addition, it resolves the diploid, tetraploid, and aneuploid DNA content. Furthermore, the SYBR-14-stained mammal cells were efficiently sorted based on DNA content and live cells were obtained. All these features have not been previously described with the utilization of this staining approach. CONCLUSIONS: Results of this study demonstrate that this flow cytometric approach not only allows assessment of the viability of cells, but also the DNA content of mammal cells. In addition, this approach allows one to sort viable cells stained with SYBR-14. These findings open-up unexpected and unrestricted avenues for sorting of living mammal cells and provide significant advantages over the traditionally cumbersome sorting approaches for living cells, which demand very specialized and expensive UV light sources as well as sophisticated sorting procedures.     

Low dose exposure to sodium arsenite synergistically interacts with UV radiation to induce mutations and alter DNA repair in human cells

Inorganic arsenic is a known human carcinogen, yet its mechanism of action remains poorly understood. Epidemiological data suggest that arsenic exposure interacts with UV radiation exposure to increase the risk of skin cancer. Studies have suggested that arsenic is able to impair DNA repair enzymes and alter the repair of UV-induced DNA damage. Here we have tested the hypothesis that arsenite [As(III)] and UV interact synergistically to enhance mutagenesis. TK6 human lymphoblastoid cells that are functionally heterozygous at the thymidine kinase (TK) locus were pre-exposed to As(III) alone and in combination with UV. Our data suggest that As(III) is mutagenic only at high doses at the TK locus. As(III) enhanced UV mutagenesis in a more than additive fashion. To investigate the mechanism underlying this synergy we assessed the removal of UV-induced dimers in TK6 cells using the T4 endonuclease-incorporated Comet assay. Pre-treatment with As(III) specifically inhibited the repair of UV-induced pyrimidine dimer-related DNA damage. Taken together, these data suggest that pre-treatment of human cells with arsenic impairs the nucleotide excision repair pathway and leads to enhanced UV mutagenesis.     

Chronic exposure to valproic acid promotes insulin release, reduces KATP channel current and does not affect Ca2+ signaling in mouse islets

Hyperinsulinemia is one of the reported side effects of valproic acid (VPA), a medicine used to treat epilepsy. However, its underlying mechanism remains unknown. The present study was designed to investigate a direct effect of VPA on insulin secretion by using mouse pancreactic islets and β-cells. VPA had no acute effect on insulin secretion from islets, or on cytosolic Ca²⁺ ([Ca²⁺]_i) in single β-cells. However, following long-term exposure to VPA (48 h), both basal and glucose-stimulated insulin secretion were markedly elevated (5-fold), while the insulin gene expression level was unaltered. Following long-term exposure to VPA, β-cells showed a decrease in whole cell K_ATP channel current. However, the increase in [Ca²⁺]_i in response to the sulfonylurea drug, tolbutamide was attenuated. The present study shows that VPA has no acute effects, but long-term treatment results in enhancement of both basal and glucose-stimulated insulin secretion. This long-term effect may mediate the K_ATP channel, while VPA can also attenuate the effect of the K_ATP channel blocker tolbutamide.     

Chronic exposure to morphine does not induce dependence at the level of the calcium channel current in human SH-SY5Y cells.

mu-Opioid receptors mediate inhibition of the N-type calcium channel current in the human neuroblastoma cell line SH-SY5Y. We have previously shown that chronic exposure to morphine induces homologous tolerance to this effect. Here we show that chronic incubation with morphine (1 microM for three to seven days) does not, however, induce physical dependence at the level of the calcium channel current. Initial experiments were performed using the whole cell voltage-clamp technique. Chronically treated cells were bathed in superfusate which also contained morphine (1 microM). On washout of morphine the current amplitude increased by 12% and this was reversed by re-addition of morphine. Naloxone (1 microM) elicited a similar increase. However, this increase is most likely due to a reversal of the residual inhibitory effect of morphine on the calcium channel current rather than being a novel withdrawal response. Chronic exposure to morphine did not change the voltage-sensitivity of the calcium channel current or induce the appearance of a current sensitive to the L-type calcium channel agonists Bay K 8644 (3 microM) and S(+)-PN 202-791 (1 microM). In a further series of experiments the nystatin-perforated patch technique was employed in order to prevent washout of any L-type current in these cells. Under these conditions a Bay K 8644-sensitive, L-type current was unmasked following treatment with omega Conus Toxin GVIA. The peak current was depressed by omega Conus Toxin GVIA (1 microM) by approximately 90% both in control cells and cells chronically exposed to morphine. Now Bay K 8644 (3 microM) almost doubled the remaining current but the effect was equal in both groups of cells. It is concluded that chronic exposure to morphine does not induce physical dependence and a withdrawal syndrome in the human SH-SY5Y neuroblastoma cell line by changing either N-type or L-type calcium channel activity.     

Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells

Zeocin is a member of the bleomycin/phleomycin family of antibiotics, known to bind and cleave DNA. We established human SK-OV-3 cells that stably express the Zeocin resistance gene (Sh ble) using an ecdysone-inducible mammalian expression system. Surprisingly, our results demonstrated that Zeocin, added in the culture medium to maintain the expression of the ecdysone receptor, was responsible for the formation of DNA strand breaks in the recombinant cells. This suggests that the Zeocin is not completely detoxified and is still able to cleave DNA, despite the stable expression of the Sh ble gene in the recombinant clones. Our study indicates that one needs to be very cautious in the interpretation of data involving stable cell lines selected with Zeocin.     

Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells

Mutagenesis 20     

N-Methyl-N-nitrosourea-induced DNA damage detected by the comet assay in Vicia faba nuclei during all interphase stages is not restricted to chromatid aberration hot spots

The genotoxic effect of the monofunctional alkylating agent N:-methyl-N:-nitrosourea (MNU) on root-tip nuclei of the field bean, Vicia faba, has been tested by comparative application of three protocols of the comet assay. While the alkaline denaturation/alkaline electrophoresis (A/A) procedure proved to be most sensitive at low doses, the alkaline denaturation/neutral electrophoresis (A/N) procedure yielded an optimal dose-response curve within a wider dose range. With the neutral electrophoresis without alkaline denaturation (N/N) procedure only minimal response was found. MNU-mediated single-strand breaks occurred in nuclei of all interphase stages. Detection of tandemly repeated FOK:I elements on comets by fluorescence in situ hybridization showed an average involvement of these heterochromatin-specific sequences in MNU-mediated single-strand breaks. This, together with previous results, suggests that the pronounced clustering of chromosomal aberrations in heterochromatic regions after treatment with S phase-dependent mutagens is mainly due to an error-prone interference of recombinative repair and replication in damaged basic repeats of large tandem repeat arrays.     

Monocytes are primed to produce the Th1 type cytokine IL-12 in normal human pregnancy: an intracellular flow cytometric analysis of peripheral blood mononuclear cells

This paper considers both monocytes and peripheral blood lymphocytes as potential targets for maternal immunological modulation in pregnancy. Peripheral blood mononuclear cells (PBMCs) from non-pregnant and normal pregnant donors were stimulated in vitro, and cytokine production detected intracellularly by flow cytometry. It was found that monocyte production of TNF-alpha was unaltered in pregnancy, while production of IL-12 was significantly enhanced. In contrast, production of the Th1 type cytokine IFN-gamma was suppressed in the lymphocyte subsets: CD4+ T helper cells and CD56+ NK cells. Production of the Th2 type cytokine IL-4 in CD4+ cells was not significantly altered in pregnancy. These data suggest that the concept that pregnancy is a 'Th2 phenomenon' cannot be generalized to the function of all aspects of maternal cellular immunity as, paradoxically, circulating monocytes are 'primed' to produce the Th1 cytokine IL-12. Furthermore, these data support the hypothesis that components of maternal innate immunity are activated in normal pregnancy.