176 Localization and Quantitative Expression of Phospholipase C Zeta in Equine Sperm Using Commercial Antibodies

2018 ◽  
Vol 30 (1) ◽  
pp. 228
Author(s):  
R. A. Gonzalez-Castro ◽  
J. K. Graham ◽  
E. M. Carnevale
Keyword(s):  
Flow Cytometry ◽  
Phospholipase C ◽  
Fluorescence Intensity ◽  
Molecular Probes ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Flow Cytometric ◽  
Live Sperm ◽  
Commercial Antibodies ◽  
Flow Cytometric Evaluation

Fertilization failure in vivo and in vitro (intracytoplasmic sperm injection, ICSI) can be caused by the inability of sperm to elicit intracellular calcium oscillations and to induce oocyte activation. Phospholipase C zeta (PLCz) is sperm-associated protein that can induce oocyte activation. Male infertility has been associated with PLCz deficiency in various species, although this has not been studied in the stallion. We hypothesised that the location and amount of PLCz on sperm varies among stallions. The aim of this study was to validate commercial antibodies (Ab) to detect PLCz on stallion sperm, and then to use these Ab to quantify the amount of PLCz, using flow cytometry, with the long-term goal of correlating PLCz on sperm with stallion fertility. Frozen-thawed sperm were analysed (20 stallions in 3 replicates) using 2 commercial Ab (anti-mouse PLCz M163 and anti-human PLCz H50, Santa Cruz Biotechnology, TX, USA). Western blot and immunofluorescence microscopy were used to validate Ab binding. For microscopy, sperm DNA was counterstained with 1 µg mL−1 Hoechst 33258. For flow cytometry, samples were incubated with Live Dead Fixable Far Red Stain Kit (Molecular Probes, Eugene, OR, USA), fixed, permeabilized, incubated overnight with primary Ab, and labelled with conjugated secondary Ab (anti-rabbit IgG Alexa Fluor 488, Molecular Probes). Green and far red mean fluorescence intensity (MFI) were measured for 20,000 cells per sample. Results are presented as mean ± SEM. Wilcoxon test, Spearman rank correlation, and linear regression were performed for analyses. Immunoblot analyses for both commercial Ab identified an immunoreactive band of ~70 kDa in sperm heads, tails, and whole sperm; β-tubulin was used as loading control and for normalization. Microscopy revealed PLCz in the acrosomal and post-acrosomal regions, connecting piece, midpiece, and tail. Post-acrosomal localization was the pattern most frequently observed (55%), followed by acrosomal plus post-acrosomal regions (25%). The PLCz labelling was observed on >85% of midpiece and tail regions, independent of Ab used. Flow cytometric evaluation revealed that percentage of live sperm was 47 ± 2%. Similar fluorescence intensity was exhibited for both Ab (M163 and H50) with a wide range of values among stallions [M163, mean 30.7 ± 1.9 × 103 (range, 8.8-82.2 × 103); H50: 25.5 ± 3.2 × 103 (7.3-55.0 × 103)]. The percentage of live sperm within a sample was not associated with Ab MFI. However, when samples were gated for live/dead cells, live sperm exhibited higher (P < 0.001) MFI than dead sperm for M163 (42.6 ± 6.0 v. 30.6 ± 3.9 × 103) and H50 (38.4 ± 4.7 v. 25.6 ± 3.7 × 103). There was a strong and positive correlation between M163 and H50 MFI for total sperm and live sperm (total: r = 0.81, P < 0.001; live: r = 0.71; P < 0.001). In conclusion, 2 anti-PLCz commercial antibodies detected equine PLCz, and the PLCz was localised on the sperm as described. Flow cytometric evaluation showed that stallions have different quantities of PLCz on their sperm, and this may provide a mean to determine if PLCz on stallion sperm is associated with fertility.

Flow cytometric evaluation of bladder cancer: recommendations of the NCI flow cytometry network for bladder cancer

1992 ◽  
Vol 10 (1) ◽  
pp. 63-67 ◽  
Author(s):  
R. L. Aamodt ◽  
J. S. Coon ◽  
A. Deitch ◽  
R. W. deVere White ◽  
L. G. Koss ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Bladder Cancer ◽  
Flow Cytometric ◽  
Flow Cytometric Evaluation

scholarly journals CD161 Is Expressed in a Subset of T-Cell Prolymphocytic Leukemia Cases and Is Useful for Disease Follow-up

2019 ◽  
Vol 152 (4) ◽  
pp. 471-478
Author(s):  
Scott R Gilles ◽  
Sophia L Yohe ◽  
Michael A Linden ◽  
Michelle Dolan ◽  
Betsy Hirsch ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Nk Cells ◽  
Retrospective Review ◽  
T Cell Subsets ◽  
Prolymphocytic Leukemia ◽  
Flow Cytometry Data ◽  
Flow Cytometric ◽  
Flow Cytometric Evaluation

AbstractObjectivesCD161 (NKRP1) is a lectin-like receptor present on NK cells and rare T-cell subsets. We have observed CD161 expression in some cases of T-cell prolymphocytic leukemia (T-PLL) and found it to be useful in follow-up and detection of disease after treatment.MethodsRetrospective review of T-PLL cases with complete flow cytometry data including CD161.ResultsWe identified 10 cases of T-PLL with flow cytometric evaluation of CD161 available. Six of these cases were positive for CD161 expression. All CD161-positive cases were positive for CD8 with variable CD4 expression, whereas all CD161-negative cases were negative for CD8. In a case with two neoplastic subsets positive and negative for CD8, only the former expressed CD161.ConclusionsThese novel results suggest that CD161 is often aberrantly expressed in a defined subset of T-PLL positive for CD8. We are showing the utility of this immunophenotype in diagnosis and follow-up.

scholarly journals A flow cytometric assay of neutrophil degranulation.

1983 ◽  
Vol 31 (6) ◽  
pp. 737-744 ◽  
Author(s):  
W R Abrams ◽  
L W Diamond ◽  
A B Kane
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Ionophore A23187 ◽  
Flow Cytometric Assay ◽  
Elastase Activity ◽  
Flow Cytometric ◽  
Specific Granules ◽  
Neutrophil Degranulation ◽  
High Fluorescence Intensity ◽  
High Fluorescence

A quantitative assay of neutrophil degranulation was developed using flow cytometry. Dog neutrophils were purified to greater than 95% purity and viability by isopyknic density centrifugation in an isosmotic medium. These cells concentrated the fluorochrome acridine orange (AO) in their azurophilic granules, but not in specific granules. Also contained in the azurophilic granules are elastase, myeloperoxidase, and approximately 50% of the lysozyme activity. The fluorochrome was released concomitantly with elastase activity, as shown by flow cytometry, fluorescence microscopy, and biochemical assay in response to the ionophore A23187. By flow cytometry, unstimulated cells are distributed in a single broad peak of high fluorescence intensity. With increasing concentrations of A23187 (0.48-4.80 microM), a greater proportion of the cells shifted to a single peak of low fluorescence intensity. Few cells with intermediate fluorescence were observed. These analyses revealed that the neutrophils degranulated in a quantal, all-or-none response.

276 ACTIVATION OF ROUND SPERMATID-INJECTED OOCYTES USING PHOSPHOLIPASE C ZETA IN PIGS

2013 ◽  
Vol 25 (1) ◽  
pp. 286
Author(s):  
J. Ito ◽  
E. Yuhara ◽  
A. Nakamura ◽  
N. Kashiwazaki
Keyword(s):  
Phospholipase C ◽  
Mammalian Species ◽  
Treated Group ◽  
Round Spermatid ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Domestic Species ◽  
Electrical Pulses ◽  
Group 2 ◽  
Pronuclear Formation

In several mammalian species, the generation of offspring by round spermatid injection has been reported. However, in domestic species, including pigs, no one has reported success to date. One of the reasons is that round spermatid-injected oocytes require artificial stimuli for oocyte activation, but the developmental ability of the oocytes is low in pigs, suggesting that a more optimal activation protocol is needed. During fertilization, a sperm-derived factor induces repetitive increases in intracellular calcium, known as calcium oscillations. It is now acknowledged that phospholipase C zeta (PLCζ) has an essential role in inducing calcium oscillations, not only in mammals, but also in several other vertebrates. Therefore, if PLCζ is used as a stimulus for oocyte activation, the efficiency of oocyte activation can be improved. Recently, we found that equine PLCζ (ePLCζ) has higher activity than those of other mammalian species to be studied. In the present study, we examined whether injection of ePLCζ complementary RNA (cRNA) improves the activation of round spermatid-injected oocytes in pigs. First, we examined whether ePLCζ is expressed in round spermatids. Porcine round spermatids were isolated from adult testes, and immunostaining using anti-PLCζ antibody was carried out. The PLCζ was localised at the head and tail in mature sperm, and a part of the round spermatid was also stained. Next, we evaluated the developmental ability of round spermatid-injected oocytes activated by different protocols (electrical pulses v. injection of ePLCζ cRNA). The cytoplasts were then injected with round spermatids. One hour later, the oocytes were divided into two groups. In group 1, the oocytes were activated by a direct current pulse (150 V mm–1 and 60 µs). In group 2, the oocytes were injected with ePLCζ cRNA as follows: the reagent (0.1 µg µL–1) was diluted in injection buffer [100 mM KCl and 10 mM HEPES (pH = 7.0)], loaded into glass micropipettes by aspiration, and delivered to the ooplasm by pneumatic pressure (Narishige, Tokyo, Japan). Each oocyte received 3 to 10 pL (1 to 3% of the total volume of the oocyte). After the stimulations, oocytes were cultured in PZM-5 under 38.5°C in a humidified incubator (95% air, 5% CO2). In the ePLCζ-injected group, rates of pronuclear formation (n = 22/32, 68.8%) and blastocysts (n = 2/43, 4.7%) were higher than those in the electrical pulse-treated group (n = 9/41, 22%; and n = 0/51, 0%, respectively; P < 0.05). In conclusion, our data suggest that injection of PLCζ is effective for activation of round spermatid-injected oocytes in pigs.

326 MICROINJECTIONS OF SMALL INTERFERING RNA AND COMPLEMENTARY RNA TO ELUCIDATE THE INVOLVEMENT OF ENDOGENOUS PHOSPHOLIPASE C ISOFORMS IN BOVINE OOCYTE ACTIVATION DURING FERTILIZATION

2010 ◽  
Vol 22 (1) ◽  
pp. 319
Author(s):  
B. R. Sessions ◽  
A. H. Bayles ◽  
J. Collier ◽  
K. Perry ◽  
L. S. Whitaker ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Small Interfering Rna ◽  
Calcium Oscillations ◽  
Activation Process ◽  
Mrna Levels ◽  
Post Injection ◽  
Oocyte Activation ◽  
Bovine Oocyte ◽  
Interfering Rna ◽  
Bovine Oocytes

Phospholipase C (PLC) isoforms stimulate the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), with IP3 regulating the release of calcium (Ca2+) from the endoplasmic reticulum. This release of calcium is essential for oocyte activation, and a sperm-specific PLC isoform, PLCγ;, has been proposed as the primary agent that initiates the activation process. However, the oocyte contains many endogenous PLC isoforms (PLC β, γ, and δ) that could also be involved in regulating or initiating these calcium oscillations downstream of other initiating events. In order to better elucidate the involvement of endogenous PLC isoforms as well as the specific role of the sperm-specific form, small interfering RNA (siRNA) directed against the specific bovine PLC isoforms (PLCζ;, PLCγ1, PLCγ2, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3) were microinjected into bovine oocytes, and the subsequent effects on PLC mRNA levels and bovine fertilization were evaluated. Real-time PCR (qPCR) was used to quantify the levels of PLC message present in bovine oocytes at the time of injection (15 h post-maturation) and 6, 10, and 14 h post-injection. The qPCR results indicated a near-complete knockdown of mRNA levels in bovine oocytes 10 h post-injection for the isotypes PLCγ1, PLCγ2, PLCδ3, PLCδ4, PLCβ1, PLCβ3, but only partial knockdown of PLCS 1 mRNA. Oocytes microinjected with PLC siRNA were also fertilized and cultured in vitro according to our standard laboratory procedures (Reed et al. 1996 Theriogenology 45, 439-449). The oocytes microinjected with PLCζ;, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3 siRNA resulted in cleavage rates similar to the negative control siRNA, non-injected, and sham-injected treatment groups, whereas bovine oocytes microinjected with PLCγ1 and PLCγ2 siRNA had significantly lower cleavage rates compared with the controls. Additionally, complementary cRNA for each specific PLC isoform was microinjected into bovine oocytes to ascertain each isoform’s ability to induce parthenogenetic activation. Development was observed in oocytes microinjected with a variety of cRNAs, and the activating effects of the cRNA were negligible if the oocytes were microinjected with the corresponding siRNA before microinjection with cRNA. Interestingly, siRNA specific for PLCζ; failed to reduce cleavage when treated bovine oocytes were fertilized. These data illustrate the potential involvement of multiple endogenous PLC isoforms and not just the sperm-specific PLCζ; isoform in bovine oocyte activation during fertilization.

180 Stallion sperm phospholipase C zeta affects cleavage rates after intracytoplasmic injection in bovine oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 214 ◽  
Author(s):  
F. Amoroso-Sanches ◽  
R. Gonzalez-Castro ◽  
J. Stokes ◽  
E. Carnevale
Keyword(s):  
Phospholipase C ◽  
Polar Body ◽  
Cumulus Cells ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Oocyte Activation ◽  
Normal Morphology ◽  
Flow Cytometric ◽  
Zygote Development ◽  
Chi Squared

Phospholipase C zeta (PLCz) is a sperm protein linked to oocyte activation and zygote development in diverse species. Human intracytoplasmic sperm injection (ICSI) success is poor when sperm PLCz is reduced or mutated. We hypothesised that the expression of PLCz in stallion sperm corresponds with cleavage rates after ICSI. For this study, we selected sperm from 4 of 21 stallions for which frozen-thawed sperm were evaluated using flow cytometry to assess mean fluorescence intensity (MFI) and percentage of sperm positively labelled with PLCz. Before flow cytometric assessment, Western blotting and immunofluorescence were performed to validate antibody binding and to identify PLCz as a 71-kDa protein in stallion sperm, located in the acrosomal and postacrosomal region, and the tail (Gonzalez-Castro et al. 2017 Reprod. Fertil. Dev. 30, 228). Frozen sperm from 4 stallions were selected based on MFI and percentage of PLCz-labelled sperm per total sperm population, respectively (High, 87% with 10,384 MFI and 84% with 10,784 MFI; Low, 56% with 4,789 MFI and 59% with 5,360 MFI). The samples were selected so that other fertility indicators, such as normal morphology (&gt; 70%), DNA integrity (&lt;8%, flow cytometric evaluation using sperm chromatin structure assay), and viability (SYBR14+/propidium iodide-, flow cytometric assessment) were similar for High and Low. Bovine ovaries were transported at 25°C before collection of oocytes from 2- to 8-mm follicles. Oocyte maturation and embryo culture were done as previously described using a bovine system with chemically defined media (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585-596). Oocytes were matured for 22 to 24h at 38.5°C in 5% CO2 and air before removal of cumulus cells. Before ICSI, straws of frozen sperm were cut under liquid nitrogen, with a small section thawed directly in medium. Oocytes were selected based on normal morphology and an extruded polar body. Before injection, individual sperm were selected at 200× based on normal morphology and progressive motility. Oocytes were injected with sperm from High (n=62 oocytes) and Low (n=56). A third group of oocytes (n=43) were sham injected (no sperm) to determine the rate of parthenogenetic cleavage. Cleavage rates were compared using chi-squared test. Cleavage rates differed (P&lt;0.0001) among groups, with 53% (33/62) for High, 34% (19/56) for Low, and 0% (0/43) for sham injections. Sperm populations from the High group had higher (P&lt;0.04) cleavage rates than those from the Low group. We concluded that PLCz in stallion sperm populations is a valuable indicator of ICSI success, and this protein is important factor for oocyte activation and initiation of embryo development after assisted fertilization.

scholarly journals Quantitative Flow Cytometric Evaluation of MaximalCryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model

2000 ◽  
Vol 66 (10) ◽  
pp. 4315-4317 ◽  
Author(s):  
Agnès Delaunay ◽  
Gilles Gargala ◽  
Xunde Li ◽  
Loic Favennec ◽  
Jean Jacques Ballet
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Risk Evaluation ◽  
Detection Sensitivity ◽  
Contaminated Water ◽  
Suckling Mouse ◽  
Flow Cytometric ◽  
Waterborne Transmission ◽  
Flow Cytometric Evaluation

ABSTRACT The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.

scholarly journals Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes

2012 ◽  
Vol 27 (6) ◽  
pp. 1768-1780 ◽  
Author(s):  
S.-Y. Yoon ◽  
J. H. Eum ◽  
J. E. Lee ◽  
H. C. Lee ◽  
Y. S. Kim ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Phospholipase C ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Human Oocytes

Localisation of phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (WBP2 N-terminal like) on equine spermatozoa and flow cytometry quantification of PLCZ1 and association with cleavage in vitro

2019 ◽  
Vol 31 (12) ◽  
pp. 1778
Author(s):  
Raul A. Gonzalez-Castro ◽  
Fabio Amoroso-Sanches ◽  
JoAnne E. Stokes ◽  
James K. Graham ◽  
Elaine M. Carnevale
Keyword(s):  
Flow Cytometry ◽  
Dna Fragmentation ◽  
Fluorescence Intensity ◽  
Intracytoplasmic Sperm Injection ◽  
Binding Protein ◽  
Sperm Parameters ◽  
Oocyte Activation ◽  
Oocyte Cytoplasm ◽  
Principal Piece

Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal piece of the tail. Anti-WBP2NL antibody identified two WBP2NL bands (~28 and ~32kDa) in the postacrosomal region, midpiece and principal piece of the tail. PLCZ1 and WBP2NL expression was positively correlated (P=0.04) in sperm heads. Flow cytometry evaluation of PLCZ1 revealed large variations in fluorescence intensity and the percentage of positively labelled spermatozoa among stallions. PLCZ1 expression was significantly higher in viable than non-viable spermatozoa, and DNA fragmentation was negatively correlated with PLCZ1 expression and the percentage of positively labelled spermatozoa (P&lt;0.05). The use of equine sperm populations considered to have high versus low PLCZ1 content resulted in significantly higher cleavage rates after ICSI of bovine and equine oocytes, supporting the importance of PLCZ1 for oocyte activation.

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