Haploidy in preattachment bovine embryos

Four of twenty-four bovine embryos (day 5-7) subjected to chromosome analysis had haploid cells in metaphase. Two were clearly n/2n. The origin of the haploid cells was determined to be male in two embryos, owing to the presence of a Y chromosome or a male marker chromosome suggesting spermatozoa in division.     

Porcine conceptuses secrete an interferon during the preattachment period of early pregnancy

Maternal recognition of pregnancy in sheep and cattle is thought to be initiated by the conceptus secretory proteins ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1), respectively. Recently, these proteins have been shown to be members of the interferon-alpha (IFN-alpha) family. In this study, we have examined whether pig conceptuses also produce IFN during early pregnancy. conceptuses were collected at Days 8, 10, 11, 12, 13, 15, and 17 of pregnancy and cultured in serum-free medium for 24 h. Day 11 conceptuses secreted a dominant 22,000-24,000 Mr cluster of acidic proteins (pI 5.2-5.4), that appeared to cross-react on immunoblots with antiserum against human IFN-alpha but not against oTP-1. Antiviral activity characteristic of an IFN was present in conceptus culture medium and uterine flushings from Day 11 through Day 17 of pregnancy, but was absent in flushings prior to Day 11 of pregnancy and in flushings from Day 12 nonpregnant gilts. The antiviral activity coeluted with a 22,000-24,000 Mr protein during partial purification through a gel filtration column. The activity was extremely labile, but could be restored by sequential protein denaturation, reduction, and renaturation. We conclude that production of IFN by early conceptuses is not restricted to ruminant species, and may therefore represent a more general phenomenon.     

Proteomic analysis of the porcine platelet proteome and alterations induced by thrombin activation

Platelets are enucleated cells derived from bone marrow megakaryocytes and defects in platelet functions could be involved in many cardiovascular diseases. Proteomics can be used to provide a new insight in the study of these platelet functions and can help to identify the biochemical events underlying platelet activation. In this study, we have obtained a reference 2-DE map of porcine platelet proteins. A large number of cytoskeletal and metabolic proteins were found as well as some proteins related to cell mobility and immunological functions. Other proteins implicated in the cell signalling process, transport or apoptosis were also identified. Moreover, we have analysed, by 2D-DIGE methodology, quantitative modifications of platelet proteins following their activation by thrombin. 26 spots exhibited statistically significant differences, and a total of 16 spots corresponding to 13 different proteins were successfully identified. Using Ingenuity Pathway Analysis, the association of the deregulated proteins with canonical pathways highlighted two major pathways; coagulation system and integrin signalling. These results confirm that this proteomic approach (based on 2D-DIGE, mass spectrometry and bioinformatic and pathway databases) has proved to be a powerful tool when applied to studying signalling pathways that could play a relevant role in the activation of platelets.     

Comparative analysis of proteome differential regulation during cell dedifferentiation in Arabidopsis

Cell dedifferentiation is a cell fate switching process in which differentiated cells undergo genome reprogramming to regain the competency of cell division and organ regeneration. The molecular mechanism underlying the cell dedifferentiation process remains obscure. In this report, we investigate the cell dedifferentiation process in Arabidopsis using a shotgun proteomics approach. A total of 758 proteins are identified by two or more matched peptides. Comparative analyses at four time points using two label-free methods reveal that 193 proteins display up-regulation and 183 proteins display down-regulation within 48 h. While the results of the two label-free quantification methods match well with each other, comparison with previously published 2-DE gel results reveal that label-free quantification results differ substantially from those of the 2-DE method for proteins with peptides common to multiple proteins, suggesting a limitation of the label-free methods in quantifying proteins with closely related family members in complex samples. Our results show that the shotgun approach and the traditional 2-DE gel approach complement each other in both protein identification and quantification. An interesting observation is that core histones and histone variants are subjected to extensive down-regulation, indicating that there is a dramatic change in the chromatin during cell differentiation.     

Inhibitor profiles of alkaline phosphatases in bovine preattachment embryos and adult tissues.

The alkaline phosphatases are a small family of isozymes. Bovine preattachment embryos transcribe mRNA for two tissue-specific alkaline phosphatases (TSAP2 and TSAP3) beginning at the 4- and 8-cell stages. Whereas no mRNA has been detected in oocytes, there is maternally inherited alkaline phosphatase activity. It is not known which isozyme(s) is responsible for the maternal activity or when TSAP2 and TSAP3 form functional protein. No antibodies are available that recognize the relevant bovine alkaline phosphatases. Therefore, sensitivity to heat and chemical inhibition was used to separate the different isozymes. By screening tissues, it was determined that the bovine tissue-nonspecific alkaline phosphatase (TNAP) is inactivated by low temperatures (65C) and low concentrations of levamisole (<1 mM), whereas bovine tissue-specific isozymes require higher temperatures (90C) and levamisole concentrations (>5 mM). Inhibition by L-homoarginine and L-phenylalanine was less informative. Cumulus cells transcribe two isozymes and the pattern of inhibition suggested heterodimer formation. Inhibition of alkaline phosphatase in bovine embryos before the 8-cell stage indicated the presence of only TNAP. At the 16-cell stage the pattern was consistent with TNAP plus TSAP2 or -3 activity, and in morulae and blastocysts the pattern indicated that the maternal TNAP is fully supplanted by TSAP2 or TSAP3.     

Changes in EGF concentrations during estrous cycle in bovine endometrium and their alterations in repeat breeder cows

The objective of the present study was to determine if abnormalities in the cyclic changes of endometrial EGF concentrations can be a diagnostic tool for repeat breeder cows. First, the profile of EGF concentrations during the estrous cycle was determined using endometrial tissues obtained from 31 Holstein cows after slaughter. Cyclic cows had two peaks of EGF concentrations. Then, endometrial tissues were obtained from 12 control and 20 repeat breeder cows by biopsy on Days 3, 7, and 14 of the same estrous cycle. Endometrial EGF concentrations in biopsied samples of the controls were similar to those found in slaughterhouse materials; they were high on Days 3 and 14 (9.2 and 10.4 ng/g tissue, respectively) and low on Day 7 (3.8 ng/g tissue). Concentrations of EGF in repeat breeder cows had a different profile; they were similar on Days 3, 7, and 14 (4.4, 3.4, and 4.0 ng/g tissue, respectively). In conclusion, changes in endometrial EGF concentrations were altered in repeat breeders; these alterations may be a potential diagnostic marker for repeat breeder cows.     

2-D differential membrane proteome analysis of scarce protein samples

Proteome studies with small sample amounts are difficult to perform, especially when membrane proteins are the focus of interest. In our study a new method for the analysis of scarce membrane protein samples combining large gel 2-D-CTAB/SDS-PAGE with fluorescence dye saturation labelling (satDIGE) was developed, allowing a highly sensitive differential analysis of different cell states. After Triton X-114 phase partitioning, enriched membrane protein samples of T cells were labelled at cysteine residues using fluorescence dyes and separated by large gel 2D-CTAB/SDS-PAGE. For a differential analysis 3 mug protein was found to be sufficient to detect proteins in a widespread well-separated diagonal spot pattern.     

Heat stress-induced alterations in the synthesis and secretion of proteins and prostaglandins by cultured bovine conceptuses and uterine endometrium

Effect of in vitro heat stress on protein and prostaglandin synthesis and secretion by bovine conceptuses and endometrium was examined. Conceptuses (n = 11) and endometrium (n = 10) obtained on Day 17 of pregnancy were cultured at thermoneutral (39 degrees C, 24 h) or heat stress (39 degrees C, 6 h; 43 degrees C, 18 h) temperatures in medium supplemented with L-[4,5-3H]leucine (100 microCi) and arachidonic acid (10 micrograms/ml). Radiolabeled protein secreted into culture medium increased with time in both groups. Heat stress reduced (p less than 0.001) incorporation of [3H]leucine into intracellular and secreted proteins by conceptuses but did not alter incorporation of [3H]leucine by endometrium. In particular, heat stress reduced by 72% the secretion of bovine trophoblast protein-1, the conceptus polypeptide believed to cause extension of luteal lifespan. Two-dimensional, sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that heat stress altered the array of proteins in endometrial and conceptus tissues, as evidenced by the induction of "heat-shock proteins." Endometrial secretion of prostaglandin F (p less than 0.001) and conceptus secretion of prostaglandin E2 (p less than 0.05) increased in response to heat stress. Sensitivity of bovine conceptuses and endometrium to heat stress in vitro suggests that infertility associated with maternal heat stress may be caused, partially by alterations in signals required for maintenance of the corpus luteum during early pregnancy.     

Oviductal cell proteome alterations during the reproductive cycle in pigs

The mammalian oviduct plays a crucial role in events leading to the establishment of pregnancy. During the reproductive cycle, the reproductive system undergoes various changes, including alterations in the number of different cell types in the oviductal epithelium and changes in the height of oviductal cells. Maintaining the unique oviductal environment required for the fertilization and early embryonic development comes with an energy cost to the organism. Therefore, it is hypothesized that structural and functional changes to the oviduct during the reproductive cycle represent vital preparations for the development of suitable environments for conception and embryo support. Here, we aimed to identify the changes in protein expression profile that occur during the follicular and luteal stages of the reproductive cycle in oviductal epithelial cells. The porcine oviductal epithelial cell proteomes from the follicular and luteal stages of the reproductive cycle were contrasted after separation by 2-D gel electrophoresis. Several oviductal epithelial cell proteins were up- or down-regulated during the reproductive cycle. We checked the quantitative changes of two of these molecules during different stages of the reproductive cycle using Western blot analysis. Finally, a number of these proteins were identified using tandem mass spectrometry. The results demonstrated distinctive differences in the proteomic profiles of the oviduct between follicular and luteal phases of the reproductive cycle.     

Profiling the metabolic proteome of bovine mammary tissue

2-DE and MALDI mass fingerprinting were used to analyse mammary tissue from lactating Friesian cows. The goal was detection of enzymes in metabolic pathways for synthesis of milk molecules including fatty acids and lactose. Of 418 protein spots analysed by PMF, 328 were matched to database sequences, resulting in 215 unique proteins. We detected 11 out of the 15 enzymes in the direct pathways for conversion of glucose to fatty acids, two of the pentose phosphate pathway enzymes and two of the enzymes for lactose synthesis from glucose. We did not detect enzymes that catalyse the first three reactions of glycolysis. Our results are typical of enzyme detection using 2-DE of mammalian tissues. We therefore advocate caution when relating enzyme abundances measured by 2-DE to metabolic output as not all relevant proteins are detected. 2-D DIGE was used to measure interindividual variation in enzyme abundance from eight animals. We extracted relative protein abundances from 2-D DIGE data and used a logratio transformation that is appropriate for compositional data of the kind represented in many proteomics experiments. Coefficients of variation for abundances of detected enzymes were 3-8%. We recommend use of this transformation for DIGE and other compositional data.     

Investigation of serum proteome alterations in human glioblastoma multiforme

Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most common and lethal adult malignant brain tumor. The present study was conducted to investigate the alterations in the serum proteome in GBM patients compared to healthy controls. Comparative proteomic analysis was performed employing classical 2DE and 2D‐DIGE combined with MALDI TOF/TOF MS and results were further validated through Western blotting and immunoturbidimetric assay. Comparison of the serum proteome of GBM and healthy subjects revealed 55 differentially expressed and statistically significant (p <0.05) protein spots. Among the identified proteins, haptoglobin, plasminogen precursor, apolipoprotein A‐1 and M, and transthyretin are very significant due to their functional consequences in glioma tumor growth and migration, and could further be studied as glioma biomarkers and grade‐specific protein signatures. Analysis of the lipoprotein pattern indicated elevated serum levels of cholesterol, triacylglycerol, and low‐density lipoproteins in GBM patients. Functional pathway analysis was performed using multiple software including ingenuity pathway analysis (IPA), protein analysis through evolutionary relationships (PANTHER), database for annotation, visualization and integrated discovery (DAVID), and GeneSpring to investigate the biological context of the identified proteins, which revealed the association of candidate proteins in a few essential physiological pathways such as intrinsic prothrombin activation pathway, plasminogen activating cascade, coagulation system, glioma invasiveness signaling, and PI3K signaling in B lymphocytes. A subset of the differentially expressed proteins was applied to build statistical sample class prediction models for discrimination of GBM patients and healthy controls employing partial least squares discriminant analysis (PLS‐DA) and other machine learning methods such as support vector machine (SVM), Decision Tree and Naïve Bayes, and excellent discrimination between GBM and control groups was accomplished.     

Genistein blocks homocysteine-induced alterations in the proteome of human endothelial cells

Dietary isoflavones from soy are suggested to protect endothelial cells from damaging effects of endothelial stressors and thereby to prevent atherosclerosis. In search of the molecular targets of isoflavone action, we analyzed the effects of the major soy isoflavone, genistein, on changes in protein expression levels induced by the endothelial stressor homocysteine (Hcy) in EA.hy 926 endothelial cells. Proteins from cells exposed for 24 h to 25 microM Hcy alone or in combination with 2.5 microM genistein were separated by two-dimensional gel electrophoresis and those with altered spot intensities were identified by peptide mass fingerprinting. Genistein reversed Hcy-induced changes of proteins involved in metabolism, detoxification, and gene regulation; and some of those effects can be linked functionally to the antiatherosclerotic properties of the soy isoflavone. Alterations of steady-state levels of cytoskeletal proteins by genistein suggested an effect on apoptosis. As a matter of fact genistein caused inhibition of Hcy-mediated apoptotic cell death as indicated by inhibition of DNA fragmentation and chromatin condensation. In conclusion, proteome analysis allows the rapid identification of cellular target proteins of genistein action in endothelial cells exposed to the endothelial stressor Hcy and therefore enables the identification of molecular pathways of its antiatherosclerotic action.