Stable bull fertility protein markers in seminal plasma.

Bull fertility is an important trait in breeding as the semen of one bull can, potentially, be used to perform thousands of inseminations. The high number of inseminations needed to obtain reliable measures from Non-Return Rates to oestrus creates difficulties in assessing fertility accurately. Improving molecular knowledge of seminal properties may provide ways to facilitate selection of bulls with good semen quality. In this study, liquid chromatography mass spectrometry (LC-MS/MS) was used to analyze the protein content from the seminal plasma of 20 bulls with Non-Return Rates between 35 and 60%, sampled across three seasons. Overall, 1343 proteins were identified and proteins with consistent correlation to fertility across multiple seasons found. From these, nine protein groups had a significant Pearson correlation (p < 0.1) with fertility in all three seasons and 34 protein groups had a similar correlation in at least two seasons. Among notable proteins showing a high and consistent correlation across seasons were Osteopontin, a lipase (LIPA) and N-acetylglucosamine-1phosphotransferase subunit gamma. Three proteins were combined in a multiple linear regression to predict fertility (r = 0.81). These sets of proteins represent potential markers, which could be used by the breeding industry to phenotype bull fertility. SIGNIFICANCE: The ability of bull spermatozoa to fertilize oocytes is crucial for breeding efficiency. However, the reliability of this trait from field measures is relatively low and the prediction of fertility given by conventional methods to evaluate sperm quality is currently not very accurate. In this work, we identify sets of proteins in bull seminal plasma from repeated samples collected at different times of the year that correlate to fertility in a consistent way. We combined these individual proteins to build a molecular signature predictive of fertility. This study provides an overview of proteins linked to fertility in seminal plasma, thereby increasing knowledge of the bull seminal plasma proteome. Protein signatures from the latter, potentially related to fertility, may be of use to predict fertility for individual bulls.

Comparative proteomic analysis of hyphae and germinating cysts of Phytophthora pisi and Phytophthora sojae.

The recently described oomycete pathogen Phytophthora pisi causes root rot on pea and faba bean, while the closely related Phytophthora sojae is the causal agent of soybean root and stem rot. Differences in the pathogenicity factor repertoires that enable the two species to have distinct host specificity towards pea and soybean, were studied using tandem mass spectrometry in a global proteome study of hyphae and germinating cysts in P. pisi and P. sojae. In total 2775 proteins from P. pisi and 2891 proteins from P. sojae were identified. Fifty-eight orthologous proteins were more abundant in germinated cysts of both pathogens and thus identified as candidate proteins for the infective stage. Several of these proteins were associated with lipid transport and metabolism, and energy production. Twenty-three orthologous proteins were more abundant in hyphae of both pathogens and thus identified as candidate proteins for vegetative growth. Proteins uniquely present in germinating cysts of either P. pisi or P. sojae were considered as candidates for species-specific pathogenicity factors that may be involved in host specificity. Among these proteins were serine proteases, membrane transporters and a berberine-like protein. These results significantly expand the knowledge of the expressed proteome in P. pisi and P. sojae. BIOLOGICAL SIGNIFICANCE: P. sojae and P. pisi are closely related species that specifically cause root rot on soybean and pea, respectively. The pathogenicity factors contributing to their host specificity remained unknown. We carried out a comparative large-scale proteome analysis of vegetative (hyphae) and infective (germinating cysts) life stages in P. pisi and P. sojae. This study provides knowledge of the common factors and mechanism involved in initiation of infection and species-specific proteins that may contribute to the host specificity of these pathogens. This knowledge will lead to a better understanding of the infection biology of these pathogens, allowing new possibilities towards developing alternative and effective plant protection measures.

Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A.

Phosphodiesterase type 3B (PDE3B) has been shown to be activated and phosphorylated in response to insulin and hormones that increase cAMP. In order to study serine/threonine protein phosphatases involved in the regulation of rat adipocyte PDE3B, we investigated the phosphorylation and activation of PDE3B in vivo in response to phosphatase inhibitors and the dephosphorylation and deactivation of PDE3B in vitro by phosphatases purified from rat adipocyte homogenates. Okadaic acid and calyculin A induced dose- and time-dependent activation of PDE3B. Maximal effects were obtained after 30 min using 1 microM okadaic acid (1.8-fold activation) and 300 nM calyculin A (4-fold activation), respectively. Tautomycin and cyclosporin A did not induce activation of PDE3B. Incubation of adipocytes with 300 nM calyculin A inhibited protein phosphatase (PP) 1 and PP2A completely. Okadaic acid (1 microM) reduced PP2A activity by approx. 50% but did not affect PP1 activity, and 1 microM tautomycin reduced PP1 activity by approx. 60% but PP2A activity by only 11%. This indicates an important role for PP2A in the regulation of PDE3B. Furthermore, rat adipocyte PDE3B phosphatase activity co-purified with PP2A but not with PP1 during MonoQ chromatography. As compared with insulin, okadaic acid and calyculin A induced phosphorylation of PDE3B by 2.8- and 14-fold respectively, whereas tautomycin and cyclosporin A had no effect. Both calyculin A and okadaic acid induced phosphorylation on serine 302, the site known to be phosphorylated on PDE3B in response to insulin and isoproterenol (isoprenaline), as well as on sites not identified previously. In summary, PP2A seems to be involved in the regulation of PDE3B in vivo and can act as a PDE3B phosphatase in vitro. In comparison with insulin, calyculin A induced a dramatic activation of PDE3B and both calyculin A and okadaic acid induced phosphorylation on additional sites, which could have a role in signalling pathways not yet identified.

Regulation of protein kinase B in rat adipocytes by insulin, vanadate, and peroxovanadate. Membrane translocation in response to peroxovanadate.

Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB.