Role of membrane phosphotyrosine proteins in human spermatozoal function

1991 ◽  
Vol 99 (1) ◽  
pp. 157-165
Author(s):  
R.K. Naz ◽  
K. Ahmad ◽  
R. Kumar
Keyword(s):  
Physiological State ◽  
Human Sperm ◽  
Kinase Assay ◽  
Relative Molecular Mass ◽  
Sperm Cells ◽  
Western Blots ◽  
Tyrosine Residues ◽  
Sperm Extract ◽  
32P Incorporation

The monoclonal anti-phosphotyrosine antibody (PTA) recognized proteins related to relative molecular mass regions of 94,000 +/− 3000 and 46,000 +/− 3000 Mr on Western blots of detergent-solubilized non-capacitated human sperm extract (HSE). The pattern of phosphorylation at tyrosine residues depended upon the physiological state of the sperm cells. At least six protein bands corresponding to four molecular regions of 94,000 +/− 3000, 46,000 +/− 3000, 25,000 +/− 7000 and 12,000 +/− 2000 Mr, respectively, were labeled with 32P when human sperm were capacitated in vitro; the proteins belonging to the former three regions were phosphotyrosine proteins as they were precipitable by PTA. In vitro kinase assay performed directly on HSE indicated autophosphorylation of proteins of the same four molecular regions, with the capacitated sperm preparations having 30% higher 32P incorporation into 94,000 +/− 3000 Mr proteins and 17% less incorporation into 12,000 +/− 2000 Mr proteins as compared to the non-capacitated sperm preparations. Both of these protein regions were also autophosphorylated at tyrosine residues when immunoprecipitated phosphotyrosine proteins were used for the kinase assay. Phosphorylation of tyrosine residues of 94,000 +/− 3000 Mr proteins was further stimulated by 1.38- to 1.46-fold in response to exposure to zona pellucida proteins, namely the porcine ZP3 and human zona proteins (HZP); the HZP induced the highest response. Immunofluorescence observations on fixed human sperm demonstrated that capacitation as well as exposure to zona proteins increased the degree of tyrosine-specific fluorescence per sperm cell as well as the number of sperm cells that showed fluorescence at the acrosomal region of the spermhead.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src

1994 ◽  
Vol 299 (3) ◽  
pp. 613-621 ◽  
Author(s):  
P W Modderman ◽  
A E G K von dem Borne ◽  
A Sonnenberg
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cell Activation ◽  
Secretory Granules ◽  
Protein S ◽  
Kinase Assay ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Reducing Conditions ◽  
Sds Page

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src.

scholarly journals Holographic virtual staining of individual biological cells

2020 ◽  
Vol 117 (17) ◽  
pp. 9223-9231 ◽  
Author(s):  
Yoav N. Nygate ◽  
Mattan Levi ◽  
Simcha K. Mirsky ◽  
Nir A. Turko ◽  
Moran Rubin ◽  
...  
Keyword(s):  
Human Sperm ◽  
Sperm Cells ◽  
Clinical Settings ◽  
Biological Cells ◽  
Cell Organelles ◽  
Vitro Fertilization ◽  
Cell Staining ◽  
Human In Vitro Fertilization ◽  
Global Standardization

Many medical and biological protocols for analyzing individual biological cells involve morphological evaluation based on cell staining, designed to enhance imaging contrast and enable clinicians and biologists to differentiate between various cell organelles. However, cell staining is not always allowed in certain medical procedures. In other cases, staining may be time-consuming or expensive to implement. Staining protocols may be operator-sensitive, and hence may lead to varying analytical results, as well as cause artificial imaging artifacts or false heterogeneity. We present a deep-learning approach, called HoloStain, which converts images of isolated biological cells acquired without staining by holographic microscopy to their virtually stained images. We demonstrate this approach for human sperm cells, as there is a well-established protocol and global standardization for characterizing the morphology of stained human sperm cells for fertility evaluation, but, on the other hand, staining might be cytotoxic and thus is not allowed during human in vitro fertilization (IVF). After a training process, the deep neural network can take images of unseen sperm cells retrieved from holograms acquired without staining and convert them to their stainlike images. We obtained a fivefold recall improvement in the analysis results, demonstrating the advantage of using virtual staining for sperm cell analysis. With the introduction of simple holographic imaging methods in clinical settings, the proposed method has a great potential to become a common practice in human IVF procedures, as well as to significantly simplify and radically change other cell analyses and techniques such as imaging flow cytometry.

P–033 In vitro protective effect of α -tocopherol and anthocyanin against TiO2-NPs induced genotoxicity on human spermatozoa

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Santonastaso ◽  
F Mottola ◽  
C Iovine ◽  
N Colacurci ◽  
L Rocco
Keyword(s):  
Genomic Stability ◽  
Human Sperm ◽  
Negative Control ◽  
Sperm Cells ◽  
Tio2 Nps ◽  
Sperm Dna Damage ◽  
Sperm Dna ◽  
Rapd Pcr ◽  
Intracellular Ros

Abstract Study question Do α -tocopherol and anthocyanin counteract human sperm DNA damage provoked by titanium dioxide nanoparticles (TiO2-NPs)? Summary answer: ↑-tocopherol and anthocyanin are able to counteract TiO2-NPs genotoxicity on human sperm cells reducing oxidative stress. What is known already The environmental release and the extensive use of TiO2-NPs have been implicated in poor human sperm functionality.TiO2-NPs is genotoxic on human sperm cells causing a loss of sperm DNA integrity, an increase of apoptotic process and a reduction of genomic stability related to an over production of intracellular ROS.Antioxidants are the substances that can scavenge free radicals. α -tocopherol, present in vegetables, is the most important lipophilic antioxidant involved in restore sperm parameters in several experimental models. Anthocyanin, present in Aronia melanocarpaand belonging to the flavonoid family, is able to prevent damage caused by varicocele-induced ROS in rats. Study design, size, duration Semen samples from 132 men were obtained by masturbation following 3–5 days sexual abstinence and were examined for sperm concentration, viability, motility and morphology according to WHO 2010. The sperm cells, after purification with 45–90% double density gradient, were exposed in vitro to 1 µg/L of TiO2-NPs, 1 µg/L of TiO2-NPs whit 1 mg/L of anthocyanin and 1 µg/L of TiO2-NPs plus 1 mg/L of α -tocopherolfor 15,30,45 and 90 minutes at 37 °C. Participants/materials, setting, methods Sperm motility and concentration were analyzed with Makler camber while sperm viability and morphology were evaluated by Eosin-Nigrosin Test and by Testsimplets® prestained slides respectively. Antigenotoxicity was evaluated by Comet assay, TUNEL test and RAPD-PCR technique and Genomic Template Stability (GTS,%) calculation. The intracellular ROS level was assessed by DFC Assay. The data were analyzed using ANOVA test by GraphPad Prism 6 and considered significant if p-value ≤ 0.05. Main results and the role of chance Sperm analyses showed none statistically significant changes in sperm viability and motility (progressive and non-progressive) for each treatment. Anthocyanin and α -tocopherol counteracted sperm DNA damage induced in vitro by TiO2-NPs neutralizing ROS in a time-dependent way. Comet assay displayed that both antioxidants reduced sperm DNA strand breaks produced by TiO2-NPs, in particular the damage was no longer statistically significant starting from 30 and 90 minutes of anthocyanin-TiO2-NPs and α-tocopherol-TiO2-NPs co-exposure respectively. The antioxidant supplementation induced a statistically decrease of sperm DNA fragmentation provoked by TiO2-NPs after 45 co-treatment minutes.The RAPD-PCR technique evidenced variations of bands number in the TiO2-NPs treated sperm compared to the negative control and anthocyanin and α -tocopherol-TiO2-NPs co-treated samples. Human sperm genomic stability increased after anthocyanin and α -tocopherol TiO2-NPs co-exposure respect to the TiO2-NPs single treatment, until it almost reaches the negative control at 90 minutes. Intracellular ROS percentage was significantly lower both in anthocyanin and α -tocopherol TiO2-NPs co-treated compared to TiO2-NPs alone starting from 45 minutes. Limitations, reasons for caution In vitro study. Wider implications of the findings: Our results showed a protective effect of anthocyanin and α -tocopherol on human DNA by neutralizing intracellular ROS induced by TiO2NPs. We suggest anthocyanin and α -tocopherol as suitable molecules to defend human sperm DNA from oxidative stress, with a potentially role in treatmentof male infertility due to environmental factors. Trial registration number None

scholarly journals In Vitro Effects of Titanium Dioxide Nanoparticles (TiO2NPs) on Cadmium Chloride (CdCl2) Genotoxicity in Human Sperm Cells

Nanomaterials ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 1118 ◽  
Author(s):  
Marianna Santonastaso ◽  
Filomena Mottola ◽  
Concetta Iovine ◽  
Fulvio Cesaroni ◽  
Nicola Colacurci ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Titanium Dioxide ◽  
Dna Fragmentation ◽  
Titanium Dioxide Nanoparticles ◽  
Human Sperm ◽  
Sperm Cells ◽  
Sperm Dna Fragmentation ◽  
Sperm Dna ◽  
And Oxidative Stress

The environmental release of titanium dioxide nanoparticles (TiO2NPs) associated with their intensive use has been reported to have a genotoxic effect on male fertility. TiO2NP is able to bind and transport environmental pollutants, such as cadmium (Cd), modifying their availability and/or toxicity. The aim of this work is to assess the in vitro effect of TiO2NPs and cadmium interaction in human sperm cells. Semen parameters, apoptotic cells, sperm DNA fragmentation, genomic stability and oxidative stress were investigated after sperm incubation in cadmium alone and in combination with TiO2NPs at different times (15, 30, 45 and 90 min). Our results showed that cadmium reduced sperm DNA integrity, and increased sperm DNA fragmentation and oxidative stress. The genotoxicity induced by TiO2NPs-cadmium co-exposure was lower compared to single cadmium exposure, suggesting an interaction of the substances to modulate their reactivity. The Quantitative Structure-Activity Relationship (QSAR) computational method showed that the interaction between TiO2NPs and cadmium leads to the formation of a sandwich-like structure, with cadmium in the middle, which results in the inhibition of its genotoxicity by TiO2NPs in human sperm cells.

scholarly journals Biparental Inheritance of γ-Tubulin during Human Fertilization: Molecular Reconstitution of Functional Zygotic Centrosomes in Inseminated Human Oocytes and in Cell-free Extracts Nucleated by Human Sperm

1999 ◽  
Vol 10 (9) ◽  
pp. 2955-2969 ◽  
Author(s):  
Calvin Simerly ◽  
Sara S. Zoran ◽  
Chris Payne ◽  
Tanja Dominko ◽  
Peter Sutovsky ◽  
...  
Keyword(s):  
Human Sperm ◽  
Calcium Sensitivity ◽  
Microtubule Assembly ◽  
Biparental Inheritance ◽  
Western Blots ◽  
Human Fertilization ◽  
Egg Extracts ◽  
Sperm Aster

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents γ-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. γ-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal γ-tubulin. Recruitment of maternal γ-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm “priming” induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.

In vitro genotoxic effects of titanium dioxide nanoparticles (n‐TiO 2 ) in human sperm cells

2019 ◽  
Vol 86 (10) ◽  
pp. 1369-1377 ◽  
Author(s):  
Marianna Santonastaso ◽  
Filomena Mottola ◽  
Nicola Colacurci ◽  
Concetta Iovine ◽  
Severina Pacifico ◽  
...  
Keyword(s):  
Titanium Dioxide ◽  
Titanium Dioxide Nanoparticles ◽  
Human Sperm ◽  
Sperm Cells ◽  
Genotoxic Effects

scholarly journals Simultaneous Morphology, Motility and Fragmentation Analysis of Live Individual Sperm Cells for Male Fertility Evaluation

2021 ◽  
Author(s):  
Keren Ben-Yehuda ◽  
Simcha K Mirsky ◽  
Mattan Levi ◽  
Itay Barnea ◽  
Inbal Meshulach ◽  
...  
Keyword(s):  
Deep Learning ◽  
Dna Fragmentation ◽  
Male Fertility ◽  
Human Sperm ◽  
Sperm Cells ◽  
Interferometric Imaging ◽  
Vitro Fertilization ◽  
Lack Of Information ◽  
Common Clinical Practice

We present a new technique for simultaneously analyzing morphology, motility and DNA fragmentation of live human sperm cells at the single-cell level for male fertility evaluation. It relies on quantitative stain-free interferometric imaging and multiple deep-learning frameworks. In the common clinical practice, only motility evaluation is carried out on live human cells, while full morphological evaluation and DNA fragmentation assays require different staining protocols, and therefore cannot be performed simultaneously on the same cell. This results in a lack of information regarding the intersection of these scores. We use a clinic-ready interferometric module and deep learning to acquire dynamic sperm cells without chemical staining, and evaluate all three scores per each cell together with virtual staining. We show that the number of cells that pass each criterion separately does not accurately predict how many would pass all criteria, thus the triple evaluation per cell is necessary for accurate fertility grading. This stain-free evaluation is expected to decrease the uncertainty in male fertility evaluation, as well as be applied for sperm selection during in vitro fertilization.

scholarly journals Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability

2003 ◽  
Vol 95 (5) ◽  
pp. 1889-1895 ◽  
Author(s):  
Fiemu E. Nwariaku ◽  
Patricia Rothenbach ◽  
Zijuan Liu ◽  
Xudong Zhu ◽  
Richard H. Turnage ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Endothelial Permeability ◽  
Kinase Assay ◽  
Western Blots ◽  
Mapk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Mapk Activity ◽  
Gtp Binding ◽  
Extracellular Signal

Our laboratory previously demonstrated that MAPK activation is an important signal during cytokine-induced endothelial permeability (Nwariaku FE, Liu Z, Terada L, Duffy S, Sarosi G, and Turnage R. Shock 18: 82-85, 2002). Because GTP-binding proteins have been implicated in MAPK activation, we now hypothesize that the GTP-binding protein Rho is a mediator of TNF-induced MAPK activation and increased endothelial permeability. Transmonolayer permeability was assessed in human lung microvascular cells by measuring transmonolayer electrical resistance. MAPK activity was assessed by using a phospho-specific immunoprecipitation kinase assay and by comparing Western blots for phospho-MAPK with total MAPK. MAPK inhibitors used were SB-202190 and PD-098059, whereas Clostridium botulinum C3 transferase was used as a Rho inactivator. Rho-associated coiled-coil kinase was inhibited with Y-27632. TNF increased pulmonary endothelial permeability in vitro and caused a rapid, sustained increase in endothelial p38 and extracellular signal-regulated kinase MAPK activity. Inhibition of p38 and extracellular signal-regulated kinase MAPK with SB-202190 and PD-098059, respectively, decreased TNF-induced endothelial permeability. C3 transferase attenuated TNF-induced MAPK activation and blocked TNF-induced endothelial permeability. Finally, inhibition of Rho-associated coiled-coil kinase with Y-27632 prevented both MAPK activation and TNF-induced decreases in transmonolayer resistance. Rho acts upstream of mitogen-activated protein kinases in mediating TNF-induced pulmonary endothelial leak.

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