Reference genes selection for real-time quantitative PCR analysis in mouse germinal vesicle oocytes.

Reference gene selection in mouse oocytes is an important task required to perform further adequate analysis of target gene expression levels. In the current work we have analyzed expression stability of the seven most commonly used reference genes (Actb, Eef1e1, Gapdh, H2afz, Ppia, Rpl4 and Ubc) in mouse oocytes at the germinal vesicle (GV) stage. We have performed analysis of expression stability of the above-mentioned reference genes with the three most commonly used software tools: geNorm, BestKeeper and NormFinder. Taking into account the results obtained from all of these programmes Gapdh, Rpl4 and H2afz seem to be suitable candidate reference genes in GV oocytes of mouse.

Selection of stable expressed reference genes in native and vitrified/thawed human ovarian tissue for analysis by qRT-PCR and Western blot.

PURPOSE: To select reference genes with stable messenger RNA (mRNA) expression for quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis of vitrified/thawed human ovarian tissue and to evaluate in human ovarian tissue the levels of key proteins which are commonly used as reference proteins. METHODS: Pieces of ovarian tissue were obtained during laparoscopy from patients (n = 10, 24-36 years old) who suffered from types of cancer that does not affect reproductive system. Tissue strips from the intact group were immediately placed into liquid nitrogen. Tissue strips from the second group were successively placed into solutions with cryoprotective agents. Then, these strips were rapidly placed into liquid nitrogen. After thawing, ovarian tissue strips were cultured during 2 h in complete growth medium. Gene expression levels were measured using quantitative RT-PCR. Also, protein levels of three key reference genes were measured using Western blot. Statistical analysis of obtained data was performed by BestKeeper, NormFinder, and geNorm software utilities; correlation coefficients were also calculated. RESULTS: The most suitable reference genes for qRT-PCR analysis of human cortical ovarian tissue after cryopreservation by vitrification are genes of ribosomal proteins RPL4, RPLP0, RPS18, and heat shock protein HSP90AB1. The protein levels of three commonly used reference genes (ACTB, GAPDH, and HSP90) were measured in two groups of samples of human ovarian tissue: intact and vitrified/thawed. The levels of ACTB, GAPDH, and HSP90 proteins were similar in native and vitrified/thawed samples. CONCLUSION: Selection of suitable reference genes is the first aim of any research dedicated to the investigation of gene expression, because the interpretation of obtained results largely depends on selection of appropriate reference genes. Nowadays, there are many mathematical approaches allowing to select not only single reference gene but also a group of the most stably expressed reference genes. The use of mathematical models which take into account multiple reference genes will allow to obtain more accurate data on the expression of target genes.

Angiogenic potential of spheroids from umbilical cord and adipose-derived multipotent mesenchymal stromal cells within fibrin gel.

One of the essential goals in regenerative medicine is microvascularization which enables an effective blood supply within de novo constructed tissues and organs. In our study, we used two common multipotent mesenchymal stromal cell (MMSC) sources (subcutaneous adipose tissue and Wharton's jelly of the umbilical cord) where is a subpopulation of endothelial precursors. In the medium supplemented with VEGF, the 3D cultures of UC MMSCs and ADSCs promoted the endothelial cell differentiation. To evaluate their ability to form a capillary-like network, we encapsulated spheroids within non-modified and PEGylated fibrin hydrogels. The PEGylated hydrogel supported better the formation of multibranched cords than the pure fibrin gel. Analysis of tubule growth rate, length, and branching showed that the differentiated ADSCs had higher angiogenic potential than the differentiated hUC MMSCs. Our study can be a basis for the development of new strategies in tissue engineering and treatment of vascular diseases.

Synthesis and Membrane Transport of Serotonin in the Developing Ovarian Follicle of Mouse.

Using RT-PCR, we showed the presence of Tph1 mRNA in follicular cells and Tph2 mRNA in oocytes isolated from primary multilayer ovarian follicles of mouse and the absence of Ddc expression, which indicates that serotonin cannot be synthesized in a developing ovarian follicle. The membrane serotonin transporter Sert is expressed in follicular cells and oocytes. Experiments on the cultivation of follicles in vitro confirmed the absence of serotonin synthesis from 5-hydroxytryptophan and the presence of membrane transport activity in the oocyte.

[Morphomechanical Factors in Gastrulation Process and Differentiation of Embryonic Tissue of Xenopus laevis].

Effect of mechanical stretch on the differentiation of axial anlages and Chordin gene expression was studied in sandwich explants prepared from embryonic tissues of Xenopus laevis at the early gastrula stage in two variants: with dissected or intact dorso-medial region. In the first case, convergent cell movements were suppressed and properly organized axial organs (notochord and somites) were almost completely absent. However, they developed if the explants of such type were artificially stretched in the ventro-dorsal direction. In this case, axial organs elongated in the line of stretching, that is in the direction vertical to their normal orientation. Segmented mesoderm was always in contact with the chord anlage. In situ hybridization revealed that the area of Chordin gene expression was also extended in the direction of stretching. PCR showed that Chordin gene expression in stretched explants with disrupted dorso-medial region was statistically at the same level as in the explants with intact dorso-medial region. At the same time, the corresponding gene expression in unstretched explants with disrupted dorso-medial region was statistically higher. The obtained data indicate that mechanical stretch and associated cell movements are a necessary and sufficient condition for the formation of proper histological structure of axial organs and regulation of Chordin gene expression.

Effect of Prolyl-Glycyl-Proline (PGP) and Its Acetylated Form (N-AcPGP) on Calcium Level in the Cytoplasm of Rat Peritoneal Mast Cells.

Tripeptide glycyl-prolyl-proline (PGP), a regulatory peptide of the glyproline family, possesses a pronounced anti-inflammatory effect primarily due to its ability to prevent secretion of the proinflammatory mediator histamine by rat peritoneal mast cells. Activation of mast cell with synacthen (ACTH1-24) and substance 48/80 leads to an increase in intracellular calcium concentration. Pretreatment of mast cells with PGP prevented calcium entry into the cytoplasm from both intercellular space and intracellular stores. Acetylated peptide (N-AcPGP) produced a similar effect on histamine release and intracellular calcium content in mast cells activated with synacthen. These findings indicate that both forms of the peptide can stabilize mast cells and prevent intracellular calcium increase.

The technology of obtaining multipotent spheroids from limbal mesenchymal stromal cells for reparation of injured eye tissues.

It is known that stem and progenitor cells open new possibilities for restoring injured eye tissues. Limbal eye zone, formed mainly by derivatives of neural crest, is the main source of stem cells for regeneration. The current study considers development of innovative technology for obtaining 3D spheroids from L-MMSC. It was shown that under 3D conditions L-MMSC due to compactization and mesenchymal-epithelial transition self-organize into cellular reparative modules. Formed L-MMSC spheroids retain and promote undifferentiated population of stem and progenitor limbal cells, as supported by expression of pluripotency markers - Oct4, Sox2, Nanog. Extracellular matrix synthetized by cells in spheroids allows retaining the functional potential of L-MMSC that are involved in regeneration of both anterior and, probably, posterior eye segment.

[Expression of transmitter receptor genes in early development of sea urchin Paracentrotus lividus].

Neurotransmitters (including serotonin and acetylcholine) perform a number of prenervous functions in early sea urchin development. To detect the particular receptor components involved in these processes, we carried out a database search and nucleotide sequences homologous to serotonin receptor type 4, and the alpha6- and alpha10-subunits of nicotinic acetylcholine receptor were found among EST-clones from early Paracentrotus lividus embryos. Expression of these transcripts during early development was demonstrated using RT-PCR. These results are the first molecular biology evidence ofserotonin and acetylcholine receptor expression in sea urchin early embryogenesis.