[Comparative estimation of the EGFP effects on the development of embryos obtained by reciprocal crossing of C57BL/6-Tgn(ACTbEGFP01Osb/J and C57BL/6 mice].

The effect of enhanced green fluorescent protein (EGFP) on the in vitro viability of early embryos of C57BL/6--Tgn(ACTbEGFP010sb/J mice has been studied. The number of viable ova in hemizygous females (-/egfp) has been shown to decrease. Irrespective of the EGFP level, it has no deleterious effect on the early development of embryos obtained by reciprocal crossing of hemizygous (-/egfp) and wild-type (-/-) mice.

[Comparative embryology and mammalian cloning].

A hypothesis has been advanced that logically combines "contradictory" facts concerning the early mammalian development and shows a natural relationship between the embryos developing from a fertilized ovum and from cells of the inner cell mass of blastocyst. When studying the theoretical questions of cloning, it is necessary to take into consideration the peculiarities of prenatal mammalian ontogenesis, which make themselves evident upon comparison with other animals. The absence of yolk in the mammalian ovum defines sharp differences in the early development between mammals and other Amniota. The whole asynchronic cleavage results in the formation of the morula followed by the blastocyst, which hatches from zona pellucida and is implanted into the uterus tissue. This fact allows us to consider the blastocyst as a mammalian larva, which is fed thanks to maternal organism. It is known that, in the body of a larva (blastocyst), a new embryo develops from some somatic cells. This process is known as a polyembryony, which is typical for the development of some parasitic insects. The polyembryony in turn is a variant of somatic embryogenesis, which is a form of asexual reproduction. Thus, two different embryos, "conceptus" and "embryo proper", have different origin: the first forms by the sexual way and the second, by the asexual. The investigation of the mechanisms of somatic embryogenesis in mammals will help us to find conditions necessary for the full reprograming of donor somatic nuclei and provide the successful development of reconstructed embryos.

[Green fluorescent protein has no effect on blastocyst development in C57BL/6-Tg(ACTB-EGFP)1Osb/J mice].

The changes in the state of long-term culture of blastocysts derived from female C57BL/6 mice after crossing with C57BL/6-Tg(ACTB-EGFP)1Osb/J males with a green fluorescent protein transgene EGFP on chromosome 15 were studied. Possible causes of different culture results were analyzed: the preservation of undifferentiated cells as dense clusters in the inner cell mass or their differentiation into extraembryonic endoderm. Comparison of the events going in blastocysts with the -/- or -/EGFP genotypes demonstrated that the GFP presence has no effect on cell processes. This allows us to use embryonic material from this mouse line in experiments that require long-term vital observation of embryonic cells.

[The effect of mutation dominant spotting-Yurlovo (KitW-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-Y/+ 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-Y/+ 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 wildtype 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.

[3-D reconstruction of early preimplantation mouse embryo].

Method of 3-D reconstruction approaches for early mouse embryo in preimplantation stages was modified. The developed technique is based on application of light microscopy of serial thin sections and well known soft operating. The designed method enabled us 1) to get serial sections of a single mouse embryo; 2) to create an orthogonal system independent on the sample for orientation of virtual sections. The adequacy of 3-DR protocol was checked on reconstruction of air bubbles embedded in epoxy resin as a model of sphere.

[Regulation of asexual reproduction in the planarian Dugesia tigrina]. / Reguliatsiia besplologo razmnozheniia planarii 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.

[Effect of homologous peptides of differentiation factors HLDF and PEDF on preimplantation development of mice in vitro]. / Vliianie gomologichnykh peptidov factorov differentsirovki HLDF i PEDF na doimplantatsionnoe razvitie myshei 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.

[The action of catecholamine-synthesis inhibitors and of spiperone on sea urchin and mouse embryos]. / Deistvie ingibitorov sinteza katekholaminov i spiperona na zarodyshi morskikh ezhei i myshei.

We studied the effects of three inhibitors of catecholamine synthesis on the development of sea urchins Sphaerechinus granularis and Paracentrotus lividus. These drugs affected the early embryogenesis, which was expressed in inhibition of the cleavage divisions, appearance of abnormal embryos, and developmental arrest. The addition of arachidonic acid amide and dopamine to the incubation medium weakened the effects of the inhibitors. Spiperone induced developmental defects in preimplantation mouse embryos and sea urchin embryos. Arachidonic acid amide with dopamine exerted a protective effect against spiperone when introduced to sea urchin embryos at the blastula or late gastrula stages, rather than after fertilization. In murine embryos, this amide induced developmental defects and arrest itself and its effect was reversible. Possible mechanisms underlying the effects of these drugs are discussed.

[Non-Mendelian inheritance of the Un(s) mutant gene in mice]. / Nemendelevskoe nasledovanie mutantnogo gena Un(s) u myshei.

The phenotypic expression of the mutation Uns (gene Pax-1) were studied in the progeny of heterozygous mice. In all crosses, the sex ratio close to 1:1 was observed in the progeny. However, among sons and daughters the segregation for the mutant character depended on the variant of the cross variant. When the Uns/+ mice were intercrossed, the ratio of mutant to normal females was 3:1, while the corresponding ratio among males was 0.9:1. Studying preimplantation development in vitro and postimplantation development in vivo revealed no significant mortality in embryos obtained by crossing heterozygous mice. Ultrastructural analysis revealed no distortions in synapsis and desynapsis of axial elements of the autosomal chromosomes in Uns/+ mice. The segregation found seems to result either from different fertilizing ability of normal sperm and sperm carrying the Uns mutation or, which is more probable, from the genetic imprinting of the gene Uns.

[The positive effect of pretreatment with serotonin and gangliosides on the development of early mouse embryos after cryopreservation]. / Polozhitel'noe vliianie predobrabotki serotoninom i gangliozidami na razvitie rannikh zarodyshei myshei posle kriokonservatsii.

We have demonstrated that viability of preimplantation mouse embryos F1 (C57B1/6 x CBA) after cryoconservation at the stage of four blastomeres improves after pretreatment with serotonin (5 HT, 5 microM) or total gangliosides of bovine brain gangliosides (TG, 3 microM) added to the cultivation medium. After thawing, 64% of embryos preincubated with total brain gangliosides and 49% of embryos preincubated with serotonin developed to the stage of blastocyst during the cultivation in vitro; in the control, no more than 25% of embryos reaches this stage, and all these embryos were abnormal. Possible mechanisms of protective action of these compounds is discussed. We conclude that mouse embryos subjected to freezing-thawing procedure can be used to examine the role of serotonin and gangliosides in the regulatory processes of mammalian preimplantation development.

[The effect of the mutant genes Ta, Ra and Sd on early embryogenesis in mice]. / Vliianie mutantnykh genov Ta, Ra, Sd na rannii émbriogenez myshei.

We studied early embryonic mortality of mice from mutant stocks Tabby (Ta, X-chromosome) and RaSd (RaSd/++, chromosome 2) maintained in the heterozygous state in F1 CBA x C57B1/6 hybrid. Tabby and RaSd mice were reciprocally crossed with F1 mice and examined for the morphological status of embryos washed from the oviduct on the third day of pregnancy, when the stage of eight blastomeres is normally attained. Mortality was evaluated from the number of embryos which did not reach the expected stage by this time. The results have shown that 2-4 cell embryos, which have received gene Ta with the X-chromosome of the female parent, differed from embryos with F1 genotype at the same stage of development by their increased mortality rate, whereas among embryos obtained from RaSd, the mortality was mainly observed before cleavage. Death of embryos receiving the mutant gene from hemizygous Ta males or heterozygous RaSd/++ males was not significantly different from the mortality of embryos without these mutations.

[The organ culture of rabbit bladder epithelium using a perfluorocarbon emulsion]. / Organnoe kul'tivirovanie épiteliia mochevogo puzyria krolika s ispol'zovaniem perftoruglerodnoi émul'sii.

The influence of perfluorocarbon emulsion on the development of the growth zone in the organ culture of rabbit bladder epithelium was studied. The results of histological investigations suggest that the preliminary treatment of epithelium explants with perfluorocarbon emulsion for 4 h not only preserves the explants' viability, but enhances the growth zone development. The treatment of explants with perfluorocarbon emulsion before deep freezing increases the cryoresistance of tissue explants and enhances development of the growth zone. The data obtained indicate that perfluorocarbon emulsion can be used for both the storage of tissue explants at +4 degrees C and their cryoconservation.

[The action of acetylcholine antagonists on preimplantation mouse embryos cultured in vitro]. / Deistvie antagonistov atsetilkholina na doimplantatsionnykh zarodyshei myshei, kul'tiviruemykh in vitro.

The effects of acetylcholine antagonists on development of the two-cell mouse embryos was studied. Etpenal-3 and etpenal-14 were shown to delay cleavage, affect cytokinesis, induce fusion of the blastomeres and arrest development at the stage of two-eight blastomeres with respect to the concentration used. Atropine, an M-cholinolytic, affects the early embryogenesis at a higher concentration than the cholinolytics with mixed effect. Acetylcholine does not prevent cleavage and compactization of the embryos and exerts a protective effect against the cholinolytics with mixed effect at the early developmental stages. The effect of atropine is weaker. The data obtained suggest the presence of structures sensitive to cholinolytics in the early mouse embryos and a possible involvement of acetylcholine in the early development of mammals.

[The effect of cyclic nucleotides on the sensitivity of early mouse embryos to biogenic monoamine antagonists]. / Vliianie tsiklicheskikh nukleotidov na chuvstvitel'nost' rannikh zarodyshei myshei k antagonistam biogennykh monoaminov.

The effect of lipophilic cAMP analogs on the sensitivity of preimplantation mouse embryos of two strains to cytotoxic serotonin and adrenalin antagonists was studied. Dioctanoyl-cAMP significantly decreased the sensitivity of embryos to inmecarb and cyproheptadine: experimental embryos developed to the stage of morula or blastocyst, in contrast to control embryos incubated without this protector. A somewhat weaker effect was observed in experiments with propranolol: embryos incubated in the propranolol-containing medium after the addition of dioctanoyl-cAMP were capable of one to two cleavage divisions. 8-bromomonobutyryl-cAMP partially suppressed the inhibitory effect of cyproheptadine and did not affect the sensitivity of embryos to propranolol. These data suggest cAMP involvement in the regulatory activity of neurotransmitters in the early mouse embryos.

[The effect of biogenic monoamine antagonists on the development of preimplantation mouse embryos cultured in vitro]. / Vliianie antagonistov biogennykh monoaminov na razvitie doimplantatsionnykh zarodyshei myshei, kul'tiviruemykh in vitro.

The effect of serotonin and adrenaline antagonists was tested on the early embryos of mice of three lines. All the substances tested produced an arrest or inhibition of cleavage division and the appearance of anomalies. Serotonin introduced in the incubation medium was effective against some serotoninolytics. We were unable to test the protective effect of adrenaline, as in the concentrations used it has its own effect on the development. From the data obtained, a conclusion is made of the existence in early mouse embryos of the structures sensitive to serotonin and adrenaline antagonists. The assumptions is made from the previously obtained data on the presence of biogenic monoamines in early mouse embryos, of functional activity of prospective mediators of the nervous system at the earliest stages of embryonic development of mammals.

[Some regulatory mechanisms of asexual reproduction in planaria]. / Nekotorye reguliatornye mekhanizmy bespologo razmnozheniia planarii

The conditions were studied which influenced the reproduction of an asexual race Dugesia tigrina. The decapitation was shown to arrest divisions until the restroration of a new ganglion. The division of intact planarians does not depend on the presence of additional cut head regions and is inhibited in the presence of other intact or decapitated specimens. These data suggest that two antagonistic factors influence the division of planarians: a stimulating factor is related to the activity of the ganglion and is propagated endogenously; an inhibiting factor is localized in the trunk or tail regions. The latter may be secreted outside and influence other specimens.