Proteomic profile alterations in porcine conceptuses during early stages of development.

The dynamic embryo development during the early stages of gestation requires precise molecular changes, including proteomic ones. We aimed to find unique proteins for porcine conceptuses specifically during the peri-implantation period, i.e. on days 15-16 of pregnancy. The proteomic profile of these conceptuses was compared with conceptuses at an earlier stage of the development, i.e. collected during maternal recognition of pregnancy on days 12-13 of pregnancy. The 2DE, gel image analysis, and MALDI TOF mass spectrometry were used 500 protein spots were annotated as common to conceptuses harvested during both studied periods. Proteomic profile of the conceptuses collected during the peri-implantation period contains 24 unique proteins. Identified unique for the peri-implantation period proteins are involved in adhesion processes, cadherin, and actin-binding, and actin filament organization, extracellular matrix organization, and cytoskeleton organization. Systemic analysis of identified proteins confirmed their involvement in cell adhesion and cytoskeletal organization as being two major affected functions. The unique proteins might be recognized as factors conditioning the proper peri-implantation embryo development and gaining competences for implantation. In further studies, BRCA1 might be considered as a candidate for a potential marker of embryonic competences for implantation in pigs.

Proteomic analysis of the endometrium during early pregnancy in the domestic pig.

Reproductive processes in domestic pigs have been studied extensively. Pigs are one of the main sources of meat for human consumption and are an established model for investigations into mammalian, including human, reproductive physiology. Studies of the uterus during early pregnancy will lead to a better understanding of mechanisms governing pregnancy. Proteomics provides the possibility to explore endometrial functions in an unbiased way. The aim of the study was to compare endometrium harvested from Days 12-13 and 15-16 of pregnancy with the corresponding days of the oestrous cycle. We identified endometrial proteins that are unique to the early stages of pregnancy (Days 12-13 and 15-16). Twenty-one proteins were identified that were uniquely expressed on the selected days of pregnancy or the oestrous cycle. Out of 21 identified proteins, 14 referred to the pregnancy periods. Systemic analysis of the identified proteins revealed cell adhesion and cytoskeletal organisation as two of the major functions, both of which are important for the establishment and maintenance of pregnancy. Thrombospondin 1 expression was validated using western blotting analysis and the results suggest its involvement in the adhesiveness of the embryo during the peri-implantation period in pigs.

Proteomics of dedifferentiation of SK-N-BE2 neuroblastoma cells.

Neuroblastoma develops through processes which include cellular dedifferentiation. Ability of tumors to form spheroids is one of the manifestations of dedifferentiation and carcinogenic transformation. To study mechanisms of dedifferentiation of neuroblastoma cells, we generated spheroids and performed a proteomics study to compare the spheroids with parental SK-N-BE2 cells. We observed that dedifferentiation induced extensive changes in the proteome profiles of the cells, which affected more than 30% of detected cellular proteins. Using mass spectrometry, we identified 239 proteins affected by dedifferentiation into spheroids as compared to the parental cells. These proteins represented such regulatory processes as transcription, cell cycle regulation, apoptosis, cell adhesion, metabolism, intracellular transport, stress response, and angiogenesis. A number of potent regulators of stemness, differentiation and cancer were detected as subnetworks formed by the identified proteins. Our validation tissue microarray study of 30 neuroblastoma cases confirmed that two of the identified proteins, DISC1 and DNA-PKcs, had their expression increased in advanced malignancies. Thus, our report unveiled extensive changes of the cellular proteome upon dedifferentiation of neuroblastoma cells, indicated top subnetworks and clusters of molecular mechanisms involved in dedifferentiation, and provided candidate biomarkers for clinical studies.

Transcriptomic analysis of the myometrium during peri-implantation period and luteolysis--the study on the pig model.

In pigs, implantation begins with the attachment of embryos to the endometrium. As the process is regulated by the expression of numerous genes, endometrial transcriptomic profiles have been extensively studied in early gravid pigs. However, the myometrium, a secretory tissue, should not be neglected, as it can also participate in the regulation of implantation in early pregnant pigs. To clarify this issue, the transcriptomic profile of the porcine myometrium during the peri-implantation period (i.e. on days 15 to 16 of pregnancy) was compared with the profile observed during luteolysis (i.e. on days 15 to 16 of the oestrous cycle) with an Agilent's Porcine (V2) Two-Colour Gene Expression Microarray 4 × 44 (Agilent, USA). Analysis of the microarray data revealed that of 526 unique, accurately annotated genes, the expression of 271 unique genes was upregulated, while the expression of 255 genes was downregulated in pregnant versus cyclic myometrium. The in-depth data analysis revealed differential expression of genes encoding for factors involved in immunomodulation, tissue growth and differentiation, and prostaglandin and steroid biosynthesis and action. Moreover, the comparison of the obtained data on the myometrial transcriptome with our previously published results on the endometrial transcriptome allowed us to determine substantial differences in the regulatory function of both tissues. The new insights into the function of the myometrium of early pregnant pigs obtained here are in agreement with our previous results that suggest that this tissue plays an important role in providing optimal conditions for developing embryos. Therefore, the importance of the myometrium as an active embryo signal-responsive tissue during early pregnancy cannot be underestimated.

The activity and localization of 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase and release of androstenedione and progesterone by uterine tissues during early pregnancy and the estrous cycle in pigs.

Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3ß-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3ß-HSD mRNA, 2) uterine 3ß-HSD protein activity and 3) in vitro production of A(4) and P(4) by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3ß-HSD and the presence and activity of 3ß-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A(4) by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P(4) did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P(4) in pregnant pigs on days 12 to 13. The release of P(4) by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs.