scholarly journals A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula)

Reproduction ◽  
2004 ◽  
Vol 127 (1) ◽  
pp. 95-103 ◽  
Author(s):  
K S Sidhu ◽  
K E Mate ◽  
T Gunasekera ◽  
D Veal ◽  
L Hetherington ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Fluorescein Isothiocyanate ◽  
Tammar Wallaby ◽  
Progressive Increase ◽  
Brushtail Possum ◽  
Sperm Capacitation ◽  
Flow Cytometric ◽  
Signal Transduction Event ◽  
Marsupial Species

The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a ‘T’-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the ‘T’-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of samples and in other species.

scholarly journals Ancient Viral Integrations in Marsupials: A Potential Antiviral Defence

2021 ◽  
Author(s):  
Emma F Harding ◽  
Alice G Russo ◽  
Grace J H Yan ◽  
Paul D Waters ◽  
Peter A White
Keyword(s):  
Viral Infections ◽  
Active Role ◽  
Tammar Wallaby ◽  
Eutherian Mammal ◽  
Brushtail Possum ◽  
Replicase Gene ◽  
Tasmanian Devil ◽  
Available N ◽  
Marsupial Species ◽  
Australian Marsupial

Abstract Marsupial viruses are understudied compared to their eutherian mammal counterparts, although they may pose severe threats to vulnerable marsupial populations. Genomic viral integrations, termed endogenous viral elements (EVEs) could protect the host from infection. It is widely known past viral infections and EVEs play an active role in antiviral defence in invertebrates and plants. This study aimed to characterise actively transcribed EVEs in Australian marsupial species, because they may play an integral role in cellular defence against viruses. This study screened publicly available RNA sequencing datasets (n=35) and characterised 200 viral transcripts from thirteen Australian marsupial species. Of the 200 transcripts, 188 originated from either Bornaviridae, Filoviridae or Parvoviridae EVEs. The other 12 transcripts were from putative active infections from members of the Herpesviridae and Anelloviridae, and Hepadnaviridae. EVE transcripts (n=188) were mapped to marsupial genomes (where available, n=5/13) to identify the genomic insertion sites. Of the 188 transcripts, 117 mapped to 39 EVEs within the koala, bare-nosed wombat, tammar wallaby, brushtail possum and Tasmanian devil genomes. The remaining eight animals had no available genome (transcripts n=71). Every marsupial have Bornaviridae, Filoviridae and Parvoviridae EVEs, a trend widely observed in eutherian mammals. Whilst eutherian bornavirus EVEs are predominantly nucleoprotein-derived, marsupial bornavirus EVEs demonstrate a surprising replicase gene bias. We predicted these widely distributed EVEs were conserved within marsupials from ancient germline integrations, as many were over 65 million years old. One bornavirus replicase EVE, present in six marsupial genomes, was estimated to be 160 million years old, predating the American-Australian marsupial split. We considered transcription of these EVEs through small non-coding RNA as an ancient viral defence. Consistent with this, in koala small RNA sequence datasets we detected Bornaviridae replicase and Filoviridae nucleoprotein produced piRNA. These were enriched in testis tissue, suggesting they could protect marsupials from vertically transmitted viral integrations.

Ontogeny and pathway of formation of 5α-androstane-3α,17β-diol in the testes of the immature brushtail possum Trichosurus vulpecula

2005 ◽  
Vol 17 (6) ◽  
pp. 603 ◽  
Author(s):  
Jean D. Wilson ◽  
Geoffrey Shaw ◽  
Marilyn B. Renfree ◽  
Richard J. Auchus ◽  
Michael W. Leihy ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Tammar Wallaby ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Mature Adults ◽  
Immature Male ◽  
Marsupial Species ◽  
Age Range

The testicular androgen 5α-androstane-3α,17β-diol (androstanediol) mediates virilisation in pouch young of a marsupial, the tammar wallaby, and is the principal androgen formed in immature rodent testes. To chart the pattern of androstanediol formation in another marsupial species, the testes or fragments of testes from brushtail possums (Trichosurus vulpecula) that spanned the age range from early pouch young to mature adults were incubated with 3H-progesterone and the products were identified by high-performance liquid chromatography. The only 19-carbon steroids identified in pouch young and adult testes were the Δ4-3-keto-steroids testosterone and androstenedione. However, androstanediol and another 5α-reduced androgen (androsterone) were synthesised by testes from Day 87–200 males and these appeared to be formed from the 5α-reduction and 3-keto reduction of testosterone and androstenedione. In the prostate and glans penis of the immature male, 3H-androstanediol was converted to dihydrotestosterone. We conclude that the timing of androstanediol formation in the possum testis resembles the process in rodents rather than in the tammar wallaby and that any androstanediol in the circulation probably acts in target tissues via conversion to dihydrotestosterone.

Calcium entry activated by store depletion in coronary endothelium is promoted by tyrosine phosphorylation

1996 ◽  
Vol 270 (1) ◽  
pp. H267-H274 ◽  
Author(s):  
N. R. Sharma ◽  
M. J. Davis
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Fluorescein Isothiocyanate ◽  
Fold Increase ◽  
Porcine Coronary Artery ◽  
Store Depletion ◽  
Patch Technique

Application of substance P (SP), a potent endothelium-dependent vasodilator, to porcine coronary artery endothelial cells (PCAECs) results in release of Ca2+ from intracellular stores followed by extracellular Ca2+ influx. We tested the hypothesis that intracellular store depletion results in tyrosine phosphorylation, which promotes Ca2+ influx. PCAECs labeled with antiphosphotyrosine antibody conjugated to fluorescein isothiocyanate showed a 3.3- to 3.4-fold increase in fluorescence in response to SP or 2,5-di-tert-butylhydroquinone (BHQ), an agent that depletes intracellular stores by inhibiting the endoplasmic reticulum Ca(2+)-adenosinetriphosphatase. In both cases, the tyrosine kinase inhibitor, genistein, reduced the fluorescence intensity to near-basal levels. Pretreatment of PCAECs with the tyrosine kinase inhibitors, genistein or tyrphostin, induced a significant reduction in the plateau phase of SP-induced Ca2+ elevation with no effect on the release of Ca2+ from stores. Neither daidzein, a structurally similar but inactive analogue of genistein, nor H-7, a serine-threonine kinase inhibitor, affected SP-induced Ca2+ influx. Voltage-clamp recordings using the perforated patch technique with simultaneous Ca2+ measurements showed that intracellular Ca2+ elevation and inward current activated by SP and BHQ were reduced by 60-70% in response to genistein. These data indicate that the link between store depletion and Ca2+ influx in endothelial cells requires tyrosine phosphorylation.

Protein-tyrosine Kinase p72 syk Is Activated by Platelet Activating Factor in Platelets

1994 ◽  
Vol 72 (06) ◽  
pp. 937-941 ◽  
Author(s):  
Karim Rezaul ◽  
Shigeru Yanagi ◽  
Kiyonao Sada ◽  
Takanobu Taniguchi ◽  
Hirohei Yamamura
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Platelet Activating Factor ◽  
Kinase Assay ◽  
Potential Candidate ◽  
Protein Tyrosine ◽  
Induced Aggregation

SummaryIt has been demonstrated that activation of platelets by platelet-activating factor (PAF) results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. We report here that p72 syk is a potential candidate for the protein-tyrosine phosphorylation following PAF stimulation in porcine platelets. Immunoprecipitation kinase assay revealed that PAF stimulation resulted in a rapid activation of p72 syk which peaked at 10 s. The level of activation was found to be dose dependent and could be completely inhibited by the PAF receptor antagonist, CV3988. Phosphorylation at the tyrosine residues of p72 syk coincided with activation of yllsyk. Pretreatment of platelets with aspirin and apyrase did not affect PAF induced activation of p72 syk .Furthermore, genistein, a potent protein-tyrosine-kinase inhibitor, diminished PAF-induced p72 syk activation and Ca2+ mobilization as well as platelet aggregation. These results suggest that p72 syk may play a critical role in PAF-induced aggregation, possibly through regulation of Ca2+ mobilization.

Interspecific variation of zona pellucida glycoconjugates in several species of marsupial

Reproduction ◽  
2000 ◽  
pp. 111-120 ◽  
Author(s):  
JA Chapman ◽  
OW Wiebkin ◽  
WG Breed
Keyword(s):  
Glycine Max ◽  
Sialic Acid ◽  
Zona Pellucida ◽  
Fluorescein Isothiocyanate ◽  
Interspecific Variation ◽  
Lectin Histochemistry ◽  
Sialic Acids ◽  
Arachis Hypogea ◽  
Marsupial Species ◽  
Similar Intensity

The zona pellucida glycoconjugate content of several marsupial species was investigated using differential lectin histochemistry. Ovaries from fat-tailed dunnarts, a southern brown bandicoot, grey short-tailed opossums, brushtail possums, ringtail possums, koalas and eastern grey kangaroos were fixed, embedded in paraffin wax, sectioned and stained with ten fluorescein isothiocyanate-conjugated lectins. Sections were also incubated with either neuraminidase or saponified, respectively, before incubation with the lectins to identify saccharide residues masked by sialic acids or O-acetyl groups on sialic acids. The zonae pellucidae surrounding the oocytes of the marsupials demonstrated interspecific variation in glycoconjugate content, with mannose-containing glycoconjugates exhibiting the greatest variation. Some of the zona pellucida glycoconjugates of all species, except those of the opossums, were masked by sialic acid with an increase in fluorescence with lectins from Arachis hypogea (PNA), and Glycine max (SBA), after desialylation. The disaccharide beta-galactose(1-4)N-acetyl-D-glucosamine appeared to be conformationally masked by O-acetyl groups of sialic acids in the zonae pellucidae of all species, with an increase in fluorescence with the lectin from Erythrina cristagalli (ECA), after saponification. Similar intensity and localization of beta-(1-4)-N-acetyl-D-glucosamine, as shown by staining of the lectin from Triticum vulgaris (WGA), to the inner and outer regions of the zona pellucida, were found to those reported in eutherian species. WGA fluorescence became uniform throughout the zonae pellucidae after saponification, indicating differential O-acetylation of sialic acids on the internal compartment of the zonae pellucidae.

Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells

2006 ◽  
Vol 290 (2) ◽  
pp. C463-C471 ◽  
Author(s):  
Shutang Zhou ◽  
Bradley A. Webb ◽  
Robert Eves ◽  
Alan S. Mak
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Tyrosine Phosphorylation ◽  
Phorbol Ester ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Muscle Cells ◽  
Phosphorylation Sites ◽  
A7r5 Cells ◽  
Sirna Knockdown

Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKCα inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKCα acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation.

Secretin regulates paracellular permeability in canine gastric monolayers by a Src kinase-dependent pathway

2002 ◽  
Vol 283 (4) ◽  
pp. G893-G899 ◽  
Author(s):  
Monica C. Chen ◽  
Travis E. Solomon ◽  
Eduardo Perez Salazar ◽  
Robert Kui ◽  
Enrique Rozengurt ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Paracellular Permeability ◽  
Maximal Response ◽  
Src Tyrosine Kinase

Previous studies found that epidermal growth factor (EGF) decreased paracellular permeability in gastric mucosa, but the other physiological regulators and the molecular mechanisms mediating these responses remain undefined. We investigated the role of secretin and Src in regulating paracellular permeability because secretin regulates gastric chief cell function and Src mediates events involving the cytoskeletal-membrane interface, respectively. Confluent monolayers were formed from canine gastric epithelial cells in short-term culture on Transwell filter inserts. Resistance was monitored in the presence of secretin with or without specific kinase inhibitors. Tyrosine phosphorylation of Src at Tyr416 was measured with a site-specific phosphotyrosine antibody. Basolateral, but not apical, secretin at concentrations from 1 to 100 nM dose dependently increased resistance; this response was rapid and sustained over hours. PP2 (10 μM), a selective Src tyrosine kinase inhibitor, but not the inactive isomer PP3, abolished the increase in resistance by secretin but only modestly attenuated apical EGF effects. AG-1478 (100 nM), a specific EGF receptor tyrosine kinase inhibitor, attenuated the resistance increase to EGF but not secretin. Secretin, but not EGF, induced tyrosine phosphorylation of Src at Tyr416 in a dose-dependent fashion, with the maximal response observed at 1 min. PP2, but not PP3, dramatically inhibited this tyrosine phosphorylation. Secretin increases paracellular resistance in gastric mucosa through a Src-mediated pathway, while the effect of EGF is Src independent. Src appears to mediate the physiological effects of this Gs-coupled receptor in primary epithelial cells.

scholarly journals Signal transduction mechanisms involved in in vitro ram sperm capacitation

Reproduction ◽  
2006 ◽  
Vol 132 (5) ◽  
pp. 721-732 ◽  
Author(s):  
Patricia Grasa ◽  
José Álvaro Cebrián-Pérez ◽  
Teresa Muiño-Blanco
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Influx ◽  
Calcium Entry ◽  
Calcium Entry Blocker ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine ◽  
Entry Blocker ◽  
Ram Sperm

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca2+, NaHCO3and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca2+. cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.

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