Stem cells giving rise to extraembryonic tissues.

The review is devoted to characterization of stem cells involved in the formation of extraembryonic tissues during the early development of mammalian embryos. Here we present our results of characterization of stem cells from the trophoblast and extraembryonic endoderm of voles and comparative analysis of these cells and the corresponding mouse cells and discuss possible signal pathways maintaining these cells in undifferentiated state.

Immunocytological analysis of meiotic recombination in the American mink (Mustela vison).

Using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites along synaptonemal complexes, we estimated the total length of the genetic map, the recombination rate and crossover distribution in the American mink (Mustela vison). We prepared spreads from 130 spermatocytes of five male minks and mapped 3320 MLH1 foci along 1820 bivalents. The total recombination length of the male mink genome, based on the mean number of MLH1 foci for all chromosomes, was 1327 cM. The overall recombination rate was estimated to be 0.48 cM/Mb. In all bivalents, we observed prominent peaks of MLH1 foci near the distal ends and a paucity of them near the centromeres. This indicates that genes located at proximal regions of the chromosomes should display much tighter genetic linkage than physically equidistant markers located near the telomeres.

Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras.

Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76-80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11-13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells.

[Extraembryonic endoderm stem cell lines from common voles of the genus Microtus].

Twenty-eight independent extraembryonic endoderm (XEN) stem cell lines have been obtained from morula and blastocyst cells of common voles. Most cell lines form very few cell-cell contacts when growing and morphologically correspond to the XEN that were earlier described in mice. In addition, XEN cell lines with atypical morphology forming colonies have been obtained for the first time. Both types of XEN lines rapidly proliferate, retain their morphology and karyotype during more than 25 passages in cell culture, and express genes characteristic of XEN. One of two X chromosomes in XEN lines with karyotype XX has been shown to be inactive and associated with the Xist gene transcript. It has been demonstrated that the paternal X chromosome is inactive.

[Genetic control of chromosome synapsis in mice heterozygous for a paracentric inversion].

Frequencies of formation of inversion loops and their relative sizes were studied in laboratory mice heterozygous at paracentric inversion In1(1)Rk in chromosome 1, depending on the genetic background. Homozygotes In1/In1 were crossed with mice from five inbred strains (A/HeJ, BALB/cJ, C3H/HeJ, C57BL/6J, DBA2/J). The frequency of formation of inversion loops, their relative sizes, and the dependence of these parameters on the stage of pachitene were analyzed on electron-microscopic slides of spread spermatocytes in first-generation hybrids. It was shown that the genetic background and cross direction statistically significantly influenced the duration of individual pachitene stages and the frequency of inversion loops, but not relative loop size. Using a database on SNP distribution in the inbred strains examined, we carried out in silico mapping of genes affecting the genotype-dependent characters. We have found that the efficiency of synapsis in the inversion does not depend on interstrain differences in homology of the chromosome 1 region involved in the inversion. Genes controlling the inversion loop frequency in the inversion heterozygotes were mapped to chromosome 7, and genes controlling the duration of individual pachitene stages, to chromosomes 2 and 5.

Induced Pluripotent Stem Cells of Microtus levis x Microtus arvalis Vole Hybrids: Conditions Necessary for Their Generation and Self-Renewal.

Every year, the list of mammalian species for which cultures of pluripotent stem cells (PSCs) are generated increases. PSCs are a unique tool for extending the limits of experimental studies and modeling different biological processes. In this work, induced pluripotent stem cells (iPSCs) from the hybrids of common voles Microtus levis and Microtus arvalis, which are used as model objects to study genome organization on the molecular-genetic level and the mechanisms of X-chromosome inactivation, have been generated. Vole iPSCs were isolated and cultured in a medium containing cytokine LIF, basic fibroblast growth factor (bFGF), ascorbic acid, and fetal bovine serum. Undifferentiated state of vole iPSCs is maintained by activation of their endogenous pluripotency genes - Nanog, Oct4, Sox2, Sall4, and Esrrb. The cells were able to maintain undifferentiated state for at least 28 passages without change in their morphology and give rise to three germ layers (ectoderm, mesoderm and endoderm) upon differentiation.

Polyploidization in the trophoblast and uterine glandular epithelium of the endotheliochorial placenta of silver fox (Vulpes fulvus Desm.), as revealed by the DNA content.

Dynamics of genome multiplication during establishment of interrelations between trophoblast and glandular epithelium of the endometrium has been studied in the course of formation of placenta in the silver fox. During formation of the placenta, penetration of the trophoblast into the zone of the endometrial glandular epithelium and of endometrial blood vessels into the zone of expanding trophoblast occurs. The trophoblast, which gradually replaces epithelium and a part of the stroma of the endometrium, closely adjoins endometrial vessels but does not disrupt them, thereby the endotheliochorial placenta is formed. Cytophotometric measurements of the DNA content in trophoblast nuclei have shown that most of them are polyploid: predominantly 4-64c, occasionally 128c and 256c. Polyploidy of the trophoblast may be a consequence of various types of polyploidizing mitoses. Cytophotometric measurements of the DNA content in mitotic figures have revealed the presence of mitoses of diploid cells, i.e. with the DNA amount of 4c (2n), and polyploid cells, i.e. 8c (4n), and 16c (8n), therefore trophoblast cells in the silver fox placenta are able to enter mitosis up to the octaploid level. Higher degrees of polyploidy in the trophoblast cells seem to be achieved by endoreduplication. Polyploidization of the uterine glandular epithelial cells during placentation in the silver fox occurs until the level of 8c. Thus, the tissue-specific response of the uterus to the implanting embryo consists of active proliferation and polyploidization of the glandular epithelium, which may compensate formation of prominent population of decidual cells (i.e., connective tissue cells). In the endotheliochorial placenta of the silver fox the regularity is confirmed that cells of both maternal and fetal origin are, as a rule, polyploid in sites of their contact in placenta, which may be of protective significance in the contact of allogenic organisms.

[Characteristics of nuclear structure and karyosphere formation in mink oogenesis]. / Osobennosti struktury iadra i formirovaniia kariosfery v oogeneze norki.

Changes in nuclear structures have been followed in oocytes of adult minks, from the primordium follicle stage to the Graaf vesicle stage. The followed dynamics well compared with what has been known for other mammals studied. With the mink oogenesis, an intensive production of nucleolus-like bodies occurs: these may be as many as 100 per nucleus. On later stages of occyte growth, ring-like extranuclear bodies were found which have not been observed for other mammals. With the mink, karyosphere formation is generally similar to that in other mammalian species examined, being, however, observed a bit earlier than usually, i.e. on the stage of multilayered follicle with the antrum. Due to this fact, the karyosphere, with the mink, persists longer. With the mink, contrary to other species examined, the formation of the karyosphere as a dense Feulgen-positive body is not accompanied with a total disappearance of the nuclear envelope and nucleolus-like bodies.

[Genome multiplication in the trophoblasts and glandular epithelium of endometrium during embryo implantation and placentation of silver fox]. / Umnozhenie genoma v kletkakh trofoblasta i zhelezistogo épiteliia éndometriia pri implantatsii zarodysha i platsentarii u serebristo- cherno'i lisitsy.

Dynamics of genome multiplication during establishment of interrelations between the trophoblast and the glandular epithelium of endometrium was studied in the course of placenta formation in the silver fox. Endometrium response on the embryo implantation exhibits some features of inflammation. In the course of placenta formation the trophoblast gains access to the endometrial glandular epithelium zone, while the endometrial blood vessels grow the other way into the expanding trophoblast zone. The trophoblast gradually replaces the whole epithelium and part of the stroma of the endometrium, closely adjoining the endometrial vessels but not disrupting them. Cytophometric DNA measurements in the trophoblast nuclei have shown that most of the nuclei are polyploid: predominantly 4c-64c, occasionally 128c and 256c. Polyploidy of the trophoblast may result from various types of polyploidizing mitoses. Cytophotometric DNA measurements in mitotic figures have revealed mitoses with DNA amounts equal to 4c (2n), 8c (4n), and 16c (8n), which indicates that trophoblast cells in the silver fox placenta are able to enter mitosis prior to the octaploid level. Higher degrees of polyploidy in the trophoblast cells may be achieved presumably by endoreduplication. In the silver fox polyploidization of uterine grandular epithelial cells during placentation occurs until the level of 8c. Thus, the tissue-specific response of the uterus to the implanting embryo is an active proliferation and polyploidization of the glandular epithelium, rather than formation of a population of polyploid decidual cells (i.e. connective tissue cells). Using the silver fox endotheliochorial placenta as an example, a regularity has been confirmed that cells of both maternal and fetal origin are polyploid in sites of their contact in placenta, which might be of protective significance in the contact of allogenic organisms.

[Analysis of chimeric mice bearing the fused mutation]. / Analiz khimernykh myshei, nesushchikh mutatsiiu fused.

Interaction of cells with different genotypes and expression of Fused (Fu) gene in chimaeric mice were studied. Genetic analysis of 20 chimaeras showed that gonads of chimaeras in our experiments consisted of white component cells. The analysis of Fu gene expression in chimaeric mice supported the conclusions of the previous papers that mutations of Fu gene appeared not in the result of the hypofunction of normal allele. Two hypotheses were proposed: 1) the product of Fu gene migrates out of the mutant cells and changes the phenotype of the cells with normal allele; 2) suppressors from C57BL/6 mice influence on the penetrance of Fu gene only on the intracellular level.

[The visualization of the pronuclei in zygotes of the American mink]. / Vizualizatsiia pronukleusov v zigotakh amerikanskoi korki.

Visualization of the pronuclei in optically nontransparent mink zygotes was achieved after centrifugation at 15,000 g. Under these conditions, the lipid fraction was concentrated in the light hemisphere of the zygote, while the pronuclei were localized in the equatorial area. Centrifugation did not affect the viability of zygotes, and no reliable differences in the rate of birth were found after transplantation of the native and centrifuge zygotes to the recipient females. Treatment of the zygotes with cytochalasin B before centrifugation leads to the condensation of lipids in the light area and allows distinct visualization of the pronuclei in the free zone. However, when these zygotes are cultivated, cleavage is irregular. Organization of the cytoskeleton in the mink zygotes is discussed.

[Embryonic development of 2 strains of mice in the early cleavage period]. / Issledovanie razvitiia émbrionov myshei dvukh linii v rannii period drobleniia.

RNA metabolism at 1-, 2- and 8-celled stages was studied in C3H and C57Bl mice by means of detection of RNA content in individual embryos and microcolumnal chromatography of lysate of the embryos labelled with 3H-uridine. The increase of RNA content in the 8-celled embryos of the both strains is due to active synthesis of high and low molecular weight RNAs during this period. A comparison of 3H-uridine incorporation in RNA, and nucleotide fractions of 2-celled embryos has shown that the embryonic genome per se is activated earlier in C3H mice. The embryonic development and RNA changes in them are similar in the pure bred and hybrid embryos with common mothers. This serves as an additional evidence of the leading role of maternal factors in embryonic development during the first cleavage divisions.

[The interaction of homotypic and heterotypic embryonic stem cells with developing embryos]. / Vzaimodeistvie gomo- i geterotipcheskikh émbrional'nykh stvolovykh kletok s razvivaiushchimisia zarodyshami.

It has been demonstrated that embryonic stem cells form adhesive contacts with external blastomeres of mouse morula, while there is no such contact with blastocysts. The development of morula in the blastocysts is delayed inside a dense layer of such cells; however, in some cases, external blastomeres of the morula begin to differentiate into trophoblastic cells. The introduction of an excessive number of embryonic stem cells (15-20) into a 4-8-cell embryo results in abnormal development. When heterotypic embryonic mink stem cells are co-cultivated, they show only very weak adhesion with mouse blastomeres and are displaced as a result of compactization. When blastocysts are formed after the injection of heterotypic embryonic stem cells, such cells remain in the perivitelline space. In some cases, heterotypic embryonic stem cells continue to be determined in the trophoblastic direction and produce trophoblastic vesicles autonomously. The role of cell interaction in the determination of cells during early mammalian development is discussed.

[Isolation of ES-like lines of common voles of the genus Microtus from blastocysts and germ cells and as a result of the fusion of somatic cells with mouse embryonic stem cells]. / Poluchenie ES-podobnykh linii obyknovennykh polevok roda Microtus iz blastotsist, germinal'nykh kletok i ot sliianiia somaticheskikh kletok s émbryonal'nymi stvolovymi kletkami myshi.

Three and four independent cell lines with limited pluripotency were obtained from the inner cell mass cells of blastocysts and primordial germ cells of common voles, respectively. The results of cytogenetic analysis suggest that all these lines originated from the embryos of F1 Microtus rossiaemeridionalis x M. arvalis males and had a great number of near-triploid cells already during the early passages. The cells of these lines, like those of the inner cell mass, were characterized by the alkaline phosphatase activity. Nine independent cell lines were obtained as a result of hybridization of the mouse embryonic stem cells and vole splenocytes: eight lines and one line from hybridization with the M. kirgisorum and M. rossiaemeridionalis splenocytes, respectively. The cells of these lines expressed some properties of embryonic stem lines had a chromosome complement similar to the sum of two initial diploid sets of the mouse and vole.

["Chromosome memory" of parental genomes in embryonic hybrid cells]. / "Khromosomnaia pamiat'" roditel'skikh genomov v émbrional'nykh gibridnykh kletkakh.

In the hybrid cells obtained by fusion of embryonic stem cells with adult differentiated cells, homologous chromosomes are in two ontogenetic configurations: pluripotent and differentiated. In order to assess the role of cis- and trans-regulation in the maintenance of these states, we studied a set of clones of hybrid cells of the type embryonic stem cells-splenocytes and used two approaches: segregation of parental chromosomes and comparison of pluripotency of the past hybrid cells and embryonic stem cells. The segregation test showed that the hybrid cells lost only the homologs of the somatic partner and this process was sharply accelerated when the cells were cultivated in nonselective conditions, thus suggesting the full or partial preservation of the initial differences in the organization of parental homologs. The descendants of the former hybrid cells, which had the karyotype similar to that of embryonic stem cells, demonstrated the level of pluripotency, comparable with that of embryonic stem cells despite the long-term effect of trans-acting factors from the somatic partner in the genome of hybrid cells. The data obtained are interpreted in the framework of the concept of "chromosome memory", in the maintenance of which the key role is played by cis-regulatory factors.

[The preimplantation embryonic development of 2 species of mammals from the family Mustelidae (Mustela erminea and Mustela vison)]. / Doimplantatsionnoe razvitie zarodyshei dvukh vidov mlekopitaiushchikh iz semeistva kunitseobraznykh (Mustela erminea i Mustela vison).

The preimplantation development of common weasel and American mink embryos was studied using light microscopy. Oocytes and blastomeres of these embryos are rich with lipids synthesized during oogenesis. Apparently, most lipids are utilized during the trophoblast formation. Large spherical blastomeres protecting zona pellucida are formed by the stage of implantation. The shape of the blastocyst, as well as central superficial implantation, are typical for carnivores and distinguish the studied order from other ones (e.g., from rodents or artiodactyls). Several aspects of evolution of mammals are discussed. A suggestion is made that differences between orders in the shape of the blastocyst and ways of their implantation reflect poly-phyletic origin of mammals.

[Age-related aspects of male reproductive function in rats with normal and accelerated senescence].

The study was performed on male rats with normal rate of aging (Wistar) and senescence-accelerated OXYS rats. Sexual motivation behaviour, activation of endocrine testicle function under condition of sexual activation, gravimetric rates were studied in male rats of different age. Potential ability to spermatogenesis in testis was studied in 14-month male rats. Males underwent the partition test: a receptive female was introduced into the male's cage, but the male and female were separated by a transparent partition. The number of approaches to the partition and total time spent near partition during the test served as an index of sexual motivation. Activation of hypothalamic-pituitary-testicular complex was estimated by plasma testosterone level. A decrease of sexual motivation in the 14-month OXYS males was observed in comparison both with 3- and 6-month OXYS rats and with 14-month Wistar rats. However, no decrease in hormonal component of sexual arousal in aged OXYS males rats was detected. No interstrain differences in potential ability to spermatogenesis in 14-month rats were observed. However, no interstrain differences in the weights of androgen-dependent reproductive organs (testes and preputial glands) were observed under 18-month age. However, the weight of epididymisis in 24-month OXYS rats was significantly smaller than in Wistar rats.

Reproductive isolation due to the genetic incompatibilities between Thrichomys pachyurus and two subspecies of Thrichomys apereoides (Rodentia, Echimyidae).

We tested intrinsic reproductive isolation between 3 taxa of the South American caviomorph rodent Thrichomys (Rodentia, Echimyidae): T. pachyurus, T. apereoides subsp. apereoides and T. apereoides subsp. laurentius. They were mated in captivity and produced viable progeny. Some F1 hybrid females were fertile, whereas all F1 males were sterile. Histological examination revealed meiotic arrest at the primary spermatocyte stage. No sperm was detected in testes or epididymes. Electron microscopic analysis of surface spread synaptonemal complexes revealed a complete failure of chromosome pairing in F1 hybrids of T. pachyurus with T. apereoides subsp. laurentius and T. apereoides subsp. apereoides. In the male hybrids between T. apereoides subsp. apereoides and T. apereoides subsp. laurentius, meiosis did not proceed beyond diplotene, although all of the chromosomes, including heteromorphic ones, paired in an orderly fashion. Backcross males with homomorphic karyotypes showed segregation in meiosis progression. This indicates that male hybrid sterility is due to genetic, but not chromosomal, incompatibility of the parental taxa.

[Effect of genetic factors on the rate of embryonic growth in mink]. / Vliianie geneticheskikh faktorov na skorost' émbrional'nogo rosta norok.

A comparative analysis of the growth rate of embryos of two groups of minks differing in the coat colour was carried out. For comparison, 10 development stages with progressively more complicated morphology were taken. It has been demonstrated that the difference in the emmbryo growth rate between the standard and "sapphire" colour forms appear at the "pre-fetal" and, especially, "fetal" period of development.