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.

[Blastocyst Hatching in Humans].

The human oocyte is surrounded by the zona pellucida­an elastic, transparent extracellular matrix consisting of specific glycoproteins. The zona pellucida is preserved after fertilization and surrounds the developing human embryo for a few days. The embryo needs to get out of the zona pellucida before implantation to establish cell contacts between the trophectoderm and endometrial epithelium. The release of the embryo from the zona pellucida is carried out at the stage of the blastocyst and called zona hatching. During zona hatching the blastocyst breaks the zona pellucida and performs active movements to escape through a gap formed in the zona. While microscopic description of zone hatching is well known, biochemical and cytological basis of zone hatching remains poorly understood. The break of the zona pellucida occurs under the influence of two forces: mechanical pressure of the growing blastocyst on the zone and chemical dissolution of the zone material with secreted lytic enzymes. There is only one paper (Sathananthan et al., 2003), which describes the specialized cells in the trophectoderm that locally dissolve the zona pellucida, promoting the emergence of the hole for blastocyst release. Taking into account the singleness of the paper and the absence of further development of this subject by the authors in the following decade, the existence of specialized cells for zone hatching should be assumed with great care. Lytic enzymes, secreted by cells of the trophectoderm for dissolving the zona pellucida, are different. Depending on the species of the mammal, different classes of proteases participate in the zone hatching process: serine proteases, cysteine proteases, metalloproteinases. Proteases, secreted by human trophectoderm, are not described. The mechanisms of the active movement during blastocyst hatching are investigated to a lesser degree. Only the involvement of the cytoskeleton of trophectoderm cells in the mechanism of blastocyst compression was shown, and the participation of desmosomes in the coordinated change in the form of trophectoderm cells during compression is suggested. This review summarizes literature data on the possible mechanisms of zone hatching in the development of human embryos, obtained in experiments in vitro, as well as in animal models.

Female fertility preservation strategies: cryopreservation and ovarian tissue in vitro culture, current state of the art and future perspectives.

In the present review, the main strategies of female fertility preservation are covered. Procedures of fertility preservation are necessary for women who suffer from diseases whose treatment requires the use of aggressive therapies, such as chemotherapy and radiotherapy. These kinds of therapy negatively influence the health of gametes and their progenitors. The most commonly used method of female fertility preservation is ovarian tissue cryopreservation, followed by the retransplantation of thawed tissue. Another approach to female fertility preservation that has been actively developed lately is the ovarian tissue in vitro culture. The principal methods, advantages and drawbacks of these two strategies are discussed in this article.

Behavior of Transplanted Multipotent Cells after in Vitro Transplantation into the Damaged Retina.

The use of stem cell technologies in retinal defect reparation therapy has produced beneficial results. Nowadays, numerous protocols exist which provide a neural differentiation of the stem cells transplanted into the retina. However, questions concerning the functional replacement of the missing retinal neurons by transplanted cells thus far remain unanswered. The organotypic culture protocol was used in this study in order to prove the possibility of transdifferentiation of bone marrow stromal cells (MMSCs) and neural stem/progenitor cells (NSPCs) from EGFP-positive mice and the functional integration of these cells. This technique enables a detailed characterization of cell behavior post-transplantation. Using atomic force microscopy, we reliably demonstrated the difference (p < 0.01) between the thickness of the outgrowths formed by glial and endothelial retina cells and the thickness of neurites and neuro-like transplanted MMSC outgrowths. MMSCs are also shown to form synapses up to 2.5 ± 0.06 µm in diameter on day 4 after the transplantation. Following electrical stimulation (20V, 0.5Hz, 200ms), clear depolarization of retinal neurons and their outgrowths is detected. It is shown that some of these GFP+ MMSCs, which changed their morphology after the transplantation in retinal explants to neuro-like MMSCs, are capable of depolarizing after exogenous stimulation.

Cell technology employing femtosecond laser pulses.

Practical advantages of using femtosecond laser pulses for manipulations in cell surgery were demonstrated. The use of femtosecond laser pulses enables precision punching of the zona pellucida of the embryo without damaging its cells. With the help of femtosecond laser tweezers/scalpel, auxillary laser hatching was performed and a technique of optical biopsy of mammalian embryo was developed, which enabled non-contact sampling of embryonic material for preimplantation diagnostics. Our findings suggest that about 90% embryos retained the ability to develop at least to the blastula stage after this manipulation.

Carnosine-induced changes in the development of spermatogenic epithelial cells in senescence accelerated SAM mice.

Carnosine significantly increased the number of spermatogonia and Sertoli cells in mice prone (SAMP1) and resistant (SAMR1) to accelerated aging and appreciably reduced cell yield in meiosis and spermiogenesis in SAMP1 mice. In experimental SAMP1 mice catastrophic changes in the number of gametes were paralleled by intensive degradation of the spermatogenic epithelium. In SAMR1 mice treated with carnosine highly ordered spermatogenic structure was preserved.

Transplantation of human embryonic myoblasts and bone marrow stromal cells into skeletal muscle of C57BL/10J-mdx mice.

We studied expression of dystrophin in skeletal muscles of C57BL/10J-mdx mice after transplantation of human embryonic and fetal myoblasts and bone marrow stromal cells. Dystrophin-positive areas corresponding to the location of transplanted cell were detected in muscles of all recipient mice after transplantation of different cell cultures, but the distribution of dystrophin characteristic of normal muscle fibers was detected only after transplantation of embryonic myoblasts. Dystrophin distribution in muscle fibers after transplantation of fetal myoblasts and bone marrow stromal cells was atypical.

[Cytogenetic aberrations in the cells of liver and spermatogenetic epithelium in senescence accelerated SAMP1 and SAMR1 mice]. / Tsitogeneticheskie aberratsii v kletkakh pecheni i spermatogennogo épiteliia u uskorenno stareiushchikh myshei linii SAMP1 i SAMR1.

It was shown that during ontogenesis, the mice prone to (SAMP1) and resistant against accelerates senescence did not differ substantially in the frequency of cytogenetic aberrations in the hepatocytes and spermatogenic cells (spermatozoa and circular spermatids). These data suggest that in the mice of both lines, the processes of appearance, development, and functioning of complex biological systems, such as liver and testis, take place against the background of high genetic instability. The role of genetic instability in senescence is discussed.

Bacterial beta-galactosidase and human dystrophin genes are expressed in mouse skeletal muscle fibers after ballistic transfection.

Ballistic transfection, based on cell and tissue bombardment by the tungsten and gold microparticles covered with the gene DNA, was used for the delivery of a bacterial beta-galactosidase and a full-length cDNA copy of the human dystrophin genes into mouse skeletal muscles. CMV-lacZ, SV40-lacZ, LTR-lacZneo and full-length cDNA dystrophin (pDMD-1, approximately 16 kb) in eukaryotic expression vector pJ OMEGA driven by mouse leukaemia virus promotor (pMLVDy) were used throughout the studies. Musculus glutaeus superficialis of C57BL/6J and quadriceps femoris of mdx male mice were opened surgically under anesthesia and bombarded by means of the gene-gun technique originally developed by us. Different mixtures of gold and tungsten particles at ratios of 4:1, 1:1, 1:4 were applied. X-gal assay revealed marked beta-gal activity, both in total muscles and whole muscle fibers on histological sections, up to three months after transfection. The most intensive staining was observed after SV40-lacZ delivery. No staining was detected with LTR-lacZneo DNA as well as in untreated muscles. The higher tungsten particle concentration in the bombardment mixture correlated with more intense X-gal staining. At the gold/tungsten ratio of 1:4 the microparticles penetrated the musculus glutaeus superficialis and transfected the underlying musculus glutaeus medius as well. Immuno-cytochemical assay for human dystrophin revealed dystrophin positive myofibers (DPM) in the bombarded area up to two months after transfection. The proportion of DMP varied from 2.5% on day 17 up two 5% on day 60 after bombardment compared to only 0.5% in the control mdx mice. These results suggest the applicability of particle bombardment for gene delivery into muscle fibers.

Development of day-8 colony-forming unit-spleen hematopoietic progenitors during early murine embryogenesis: spatial and temporal mapping.

The ontogeny of the hematopoietic system in mammalian embryos occurs during the yolk sac (YS) and the fetal liver (FL) stages. Events leading to the establishment of hematopoiesis in the FL remain obscure. The appearance of colony-forming units-spleen (CFU-S) in the FL is preceded by a gradual increase of CFU-S in the YS and a more rapid increase in the AGM region (area comprising dorsal aorta, gonads, and mesonephros) during day 10 of development (Medvinsky et al, Nature 364:64, 1993). By this time, the AGM CFU-S attain a high frequency equivalent to that found in the adult bone marrow. The analogous area gives rise to adult hematopoiesis in amphibians and probably in birds. We present here a more complete picture of CFU-S development during transition from the pre-liver to liver stage of hematopoiesis. (1) Dissectional analysis of the mouse AGM region shows the presence of CFU-S both around the dorsal aorta and in the uro-genital ridges. (2) The embryonic gut also shows low but distinctive CFU-S activity. This initial intrabody pattern of CFU-S distribution in murine embryogenesis parallels that found for primordial germ cells. (3) The beginning of definitive liver hematopoiesis is accompanied by wide dissemination of CFU-S in the embryonic tissues. (4) Comparison of spleen colonies arising from the AGM and YS has shown morphologic differences. In contrast to simple erythroid constitution of the YS colonies, a broader variety of cells are found within the AGM-derived colonies that are similar to those derived from 11-day FL. These data suggest a lineage relationship for hematopoietic progenitors between the AGM region and the FL.