Chromosomal Aberrations As a Biological Phenomenon in Human Embryonic Development.

Frequent chromosomal abnormalities are a distinctive feature of early embryonic development in mammals, especially humans. Aneuploidy is considered as a contributing factor to failed embryo implantation and spontaneous abortions. In the case of chromosomal mosaicism, its effect on the potency of embryos to normally develop has not been sufficiently studied. Although, a significant percentage of chromosomal defects in early human embryos are currently believed to be associated with the features of clinical and laboratory protocols, in this review, we focus on the biological mechanisms associated with chromosomal abnormalities. In particular, we address the main events in oocyte meiosis that affects not only the genetic status of an unfertilized oocyte, but also further embryo viability, and analyze the features of first cleavage divisions and the causes of frequent chromosomal errors in early embryonic development. In addition, we discuss current data on self-correction of the chromosomal status in early embryos.

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.

In Vitro Mouse Ovarian Follicle Growth and Maturation in Alginate Hydrogel: Current State of the Art.

This review describes the main factors affecting the in vitro development of mouse ovarian follicles under conditions of three-dimensional alginate hydrogel system. The factors discussed include concentration of alginate hydrogel, presence of additives (collagen, fibrin) influencing substrate rigidity; culture conditions; composition of culture media; substances that act like antioxidants (salts of ascorbic acid, glutathione) and contribute to the improvement of lipid metabolism (L-carnitine), hormones and growth factors. The methods for follicle group cultivation in alginate hydrogel and cocultivation of different cell populations with follicles encapsulated in alginate hydrogel are covered in the present article.

Detection of T-Cadherin Expression in Mouse Embryos.

The aim of the present study was to evaluate T-cadherin expression at the early developmental stages of the mouse embryo. Using in situ hybridization and immunofluorescent staining of whole embryos in combination with confocal microscopy, we found that T-cadherin expression is detected in the developing brain, starting with the E8.75 stage, and in the heart, starting with the E11.5 stage. These data suggest a possible involvement of T-cadherin in the formation of blood vessels during embryogenesis.

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.

[The effect of antisense oligonucleoties specific to the harakiri mRNA on spontaneous and induced defects of mouse preimplantation embryo development].

The effect of antisense oligonucleotides specific to mRNA of the proapoptotic gene harakiri (Hrk) on the development of mouse SAMP1 (senescence-accelerated mouse prone) and (C57BL/6J x DBA/2J)F1 preimplantation embryos cultured in vitro was investigated. The SAMP1 mice are characterized by genetically determined decrease of fertility along with the highly frequent perturbations of embryonic development. Reproduction indices of the (C57BL/6J x DBA/2J) hybrids lie within the normal range. Because of this, preimplantation abnormalities in this line were induced by the action of proapoptotic agent bleomycine. It was demonstrated that antisense inhibition of the Hrk expression had no effect on the frequency of genetically determined abnormalities of early embryonic development in SAMP1 mice. In case of induced abnormalities, addition of oligonucleotides specific to mRNA of proapoptotic Hrk gene influenced the number of abnormalities, and at the same time, improved the quality of survived embryos via increasing the blastocyst hatching.

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.

[Hereditary muscular dystrophy: bioengineering approaches to muscle fiber repair].

Restoration of disturbed functions of the organs and tissues is the main task of contemporary genetic and cellular biotechnology, including genetic and cellular therapy. Duchenne dystrophy, one of the most widespread human genetic diseases, is at the same time the most extensively studied from the viewpoint of both genetic and histological changes leading to muscle fiber degeneration. Although many studies carried out on models, recognized analogous to Duchenne dystrophy, gave hopeful results, clinical tests with the use of developed methods gave no expected success and the rate of mortality from this disease amounts to 100%. Based on the world experience and analysis of the authors' data, possible influence of the intensity of regeneration on success of genetic and cellular therapy has been considered.

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.

Carnosine modifies the incidence of genetically abnormal sex cells in the testes of senescence-accelerated mice.

The quantitative micronucleus test showed that the natural dipeptide carnosine increases the count of aberrant spermatogonia and round spermatids in the testes in SAMR1 mice resistant to accelerated aging by 64 and 85%, respectively, compared to the control. However, this agent did not modify the incidence of chromosome mutations in spermatogenic cells in SAMP1 mice predisposed to accelerated aging.

[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.

[Activity of exogenous genetic designs, introduced into cells by a ballistic transfection method, in murine ontogenesis]. / Aktivnost' ékzogennykh geneticheskikh konstruktsii, vvedennykh v kletki metodom ballisticheskoi transfektsii, v ontogeneze myshi.

We used the method of particle bombardment (ballistic transfection) to introduce beta-galactosidase and human dystrophin genes into mouse embryos and skeletal muscles of adult mice. We examined the mechanisms of DNA transfer into skeletal muscle cells, the biological processes accompanying and following this transfer, the susceptibility of various types of muscle cells to transfection, and the duration of expression of and conditions affecting the introduced genes. We have also developed an effective, convenient, and practical methods of skeletal muscles transfection.