N. Yu. Sakharova

Interaction of living cells with fluorescent derivatives of biogenic amines.

In nonsynaptic systems, biogenic amines (catecholamines and serotonin) may regulate the development of animal and plant cells [1, 2]. The mechanisms of action of these compounds are poorly studied, which is partly due to insufficient development of adequate experimental methods. One of these methods is fluorescent analysis, which requires the use of corresponding fluorescent probes. The purpose of this study was to test the possibility of using synthetic fluorescent lipophilic analogues of dopamine and serotonin for the analysis of the location of binding of neurotransmitters by living animal and plant cells. The study was performed with early mouse embryo cells and field horsetail ( Equisetum arvense ) vegetative microspores, collected in the floodplain of the Oka River. In the modeling of the fluorescent compounds binding by certain cell components, bovine serum albumin (BSA), DNA and RNA from Escherichia coli (Serva, Germany), and β -carotene (Medprom, Russia) were used. The fluorescence intensity was measured using two types of microspectrofluorometers. One of them served for recording the fluorescence spectra, as described in [3]; the other one (Radikal DMF-2), connected with a personal computer, was used for measuring fluorescence in the green part of spectrum (510–530 nm) [4]. The resolving capacity of this technique allowed recording the fluorescence of individual parts of the cell.

Stimulation of the Viability of Early Mouse Embryos Cultured in Vitro by β-Endorphin-Like Peptides

The growth of a mammalian embryo is accompanied by complex interactions between the embryo and the mother, as well as between the embryo cells themselves. Peptide growth factors play an important role in cell division, differentiation, and morphogenesis during mammalian embryogenesis. Several families of growth factors of various origins influence preimplantation of mammalian embryos. These are insulin-like (IGF), epidermal (EGF), and platelet-derived (PDGF) growth factors, as well as transforming growth factor b (TGF-b), fibroblast growth factor (FGF), and leukemia-inhibiting factor (LIF) [1]. The growth of the blastocyst, which is the final stage of preimplantation, proved to depend on EGF and LIF [2]. These two factors promote the formation of a favorable conditions for blastocyst implantation and the subsequent growth of the embryo. β -Endorphin can be assigned to the peptide growth factors that regulate embryo growth [3]. Follicular cells around the oocyte are known to secrete β -endorphin. This seems to influence oocyte maturation and ovulation [4]; the fact that the endometrium cells secrete β -endorphin [5] suggests that this substance has an effect on the interaction between the embryo and the mother. The in vitro sensitivity of growing preimplantation mouse embryos to the agonists and antagonists of opioid receptors suggests that the latter are present on the embryos and bind β -endorphin [6]. In the 1980s, the fragment 364–377 of the heavy chain of immunoglobulin G (IgG) similar to the central part of the β -endorphin molecule has been identified in extracts of the human placenta [7]. In the Pushchino Branch of the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Institute of Biochemistry of the Russian Academy of Sciences, β -endorphin-like peptides were synthesized. These are immunorphin (SLTCLVKGFY), which is the fragment 364–373 of the heavy chain of human IgG, and its fragments: pentarphin (VKGFY), the immunorphin 6–10 fragment; cyclopentarphin, cyclo(VKGFY); and cyclodipentarphin, cyclo(VKGFYVKGFY). These substances were shown to be selective antagonists of non-opioid (naloxon-insensitive) β -endorphin receptors that were detected on cells of the rat immune system and on membranes from the rat myocardium, adrenal glands, spleen, and brain [8, 9]. We have showed previously that immunorphin at a concentration of 10 –7 I stimulated early growth of mouse embryos in vitro [10]. Therefore, immunorphin may be considered a β -endorphin-like nonspecific growth factor. In this study, the effects of the β -endorphin-like peptides pentarphin ( 10 –7 I), cyclopentarphin ( 10 –7 I), and cyclodipentarphin 10 –6 I) on the growth of two-, four-, and eight-cell mouse embryos were studied in vitro. In the control group, none of the above peptides were added to the growing embryos. The specificity of the peptide effect was confirmed in experiments on the effect of a “cocktail” of amino acids constituting these peptides (Val, Lys, Gly, Phe, and Tyr; 10 –7 M) on mouse embryo preimplantation. In total, 344 and 215 embryos were used in the experiment and control, respectively.

Regulation of Asexual Reproduction of the Planarians Dugesia tigrina

We studied asexual reproduction of planarians under the natural and artificial photoperiodic conditions. It was shown that light inhibits the fission of planarians, while darkness stimulates it. The diurnal dynamics of the fission of planarians demonstrated a circadian rhythm. This rhythm is stable, which is expressed when the conditions are experimentally changed: constant darkness, unnatural rhythm of light-darkness succession). However, this stability is affected at the time zone change. The planarians are adapted to new conditions and begin to fission at once in correspondence with the new diurnal regime.

Succinate-Based Preparation Alleviates Manifestations of the Climacteric Syndrome in Women

Clinical placebo-controlled study of Enerlit-Clima (bioactive succinate-based food additive) a showed positive effect of the preparation on general clinical and psychoemotional manifestations of the climacteric syndrome. A trend to an increase in estradiol level in early pathological climacteric and normalization of the endometrial status were observed.

Analysis of the Effects of Blue Light on Morphofunctional Status of In Vitro Cultured Blastocysts from Mice Carrying Gene of Enhanced Green Fluorescent Protein (EGFP)

We studied the effect of blue light (440-490 nm) on the development of late blastocysts of mice carrying the gene of enhanced green fluorescent protein (EGFP). Exposure to blue light for 20 min reduced adhesive properties of blastocysts and their capacity to form primary colonies consisting of the cells of inner cell mass, trophoblast, and extraembryonic endoderm. The negative effects of blue light manifested in morphological changes in the primary colonies and impairment of differentiation and migration of cells of the trophoblast and extraembryonic endoderm. The problems of cell–cell interaction and inductive influences of the inner cell mass on other cell subpopulations are discussed. EGFP blastocysts were proposed as the model for evaluation of the mechanisms underlying the effects of blue light as the major negative factor of visible light used in in vitro experiments on mammalian embryos.

Effect of docosahexaenoyl dopamine on the in vitro development of early mouse embryos

38 Involvement of neurotransmitters in early (preneu ral) embryogenesis was mainly studied on embryos of marine invertebrates and low vertebrates [1, 2]. As for mammals, it was demonstrated that there are struc tures in preimplantation mouse embryos that are sen sible to antagonists of serotonin, adrenalin, and ace tylcholine [3–6], and cAMP is involved in the func tional activity of these substances [7]. Not only their receptors, but also other components of transmitter systems (specific transporter proteins, metabolic enzymes) were detected in oocytes and embryos of mammals [8, 9]. The affect of dopamine is the least studied, since it is extremely unstable in the incuba tion medium. Due to the creation of artificially func tionalized amides of polyene fatty acids with biogenic monoamides, it became possible to use amides of dopamine with fatty acids instead of dopamine itself. These substances were the most suitable for studying the role of classical neurotransmitters in biological systems, since they easily penetrate through the cell membrane and are stable in the incubation medium. Their action is also of independent interest, since they have their own biological activity not limited to the activity of their biogenic amines and fatty acids [10, 11].

Localization of green fluorescent protein in mouse preimplantation embryos

The localization of enhanced green fluorescent protein (EGFP) was studied in preimplantation embryos obtained from reciprocal mating of hemizygous C57Bl/6-Tgn (ACTbEGFP)1Osb/J mice with C57Bl/6 mice. Specific fluorescence of EGFP was observed in all oocytes and embryos obtained from transgenic females during all preimplantation stages and in embryos inheriting the EGFP gene from transgenic males starting from the 8 blastomere stage during the compactization period. EGFP mRNA or EGFP synthesized during oogenesis can be retained in embryos during the entire preimplantation period, while expression of EGFP gene transferred from the father coincides with the onset of compactization. The possibility of using these embryos in experimental mammalian embryology is discussed.

The Effect of Mutation dominant spotting-Yurlovo (Kit W-Y ) on Spermatogenesis, Early Embryogenesis, and Fertility of C57BL/6JY Mice

The effect of mutation KitW-Y found in C57BL/6 mice on fertility, spermatogenesis, and early embryogenesis of mice have been studied. If heterozygotes KitW/+ are crossed with wild-type mice, fertility decreases by 20%. Homozygotes KitW-Y/KitW-Y and compounds KitW-Y/KitSsm are nonviable. The study of spermatogenesis in KitW/+ mice has demonstrated a negative effect of this mutation on spermatocytes. Histological examination of the testes of mutant males has shown local empty spaces in seminal ducts. Electron microscopic examination of synaptonemal complexes have demonstrated desynapsis disturbance in some nuclei at the diplotene stage of meiotic prophase I. However, these disturbances do not cause a decrease in the number of fertilized oocytes/ova. The decrease in fertility is accounted for disturbances of early embryogenesis. In vivo and in vitro analyses of early embryogenesis have demonstrated that cleavage divisions are asynchronous in KitW-Y/+ heterozygous embryos. Some of these embryos die before implantation, and others cleave more rapidly than wild-type embryos, which give them selective advantage during the postimplantation period of embryogenesis. The pattern of KitW-Y expression during spermatogenesis and embryogenesis mimics potential human pathology, which makes these mutants an interesting and valuable object for genetics and developmental biology.

The use of light microscopy for three-dimensional reconstruction of early embryos of mammals.

Studying the mechanisms of interaction between embryonic cells at the earliest stages of embryo development is necessary for successful progress in cloning technology [1]. The problem is to determine the number of cells involved in the formation of the embryo proper in mammals [2]. It was shown in experiments with chimera embryos at the stage of four blastomeres that only two blastomeres provided the pool of cells of the embryo proper [2]. However, it is difficult to identify the cells involved in the formation of the embryo proper and to follow the processes of transformation of each individual blastomere.

Effect of Homologous Peptides of Differentiation Factors HLDF and PEDF on Preimplantation Development of Mice in vitro

We studied the effect of synthetic peptides PEDF-6 and HLDF-6 on preimplantation development of mouse embryos in vitro. PEDF-6 peptide corresponds to fragment 351–356 and of pigment epithelium-derived differentiation factor (PEDF), while HLDF-6 peptide corresponds to fragment 84–89 of differentiation factor HLDF isolated from HL-60 cell line. Despite high homology, these peptides had different effects on the early development. PEDF-6 had no effect on the cleavage of 2–4-cell embryos but decelerated blastocyst formation from such embryos and disturbed their structure. In the presence of HLDF-6 the blastomeres divided more actively as compared to the control and a higher number of embryos developed to the blastocyst stage. The effects of both peptides were stage-specific: the affect the embryos at early cleavage stages and, apparently, determine their further development at that moment although do not directly affect formation of the blastocysts.