Diploid,but not haploid,human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells.     

Cytogenetics of human oocytes,zygotes, and embryos after in vitro fertilization

Summary Chromosome errors, inherited or arising de novo during gametogenesis and transmitted at fertilization to the conceptus, may be a major cause of embryonic mortality. The in vitro fertilization and embryo transfer (IVF/ET) procedure provides extra material — oo-cytes, zygotes, and embryos — to investigate the contribution of chromosomal abnormality to implantation failure. This paper reviews the results of cytogenetic studies on such material. Estimates from a total of 1120 oocytes from 11 studies give an overall proportion of chromosomal abnormality of 35%. Single and multiple nullisomies and disomies are found, involving nonrandom chromosome gain or loss. Hypohaploid complements are more frequent than hyperhaploid complements. The higher rate of chromosome loss of hypohaploid karyotypes was found to be largely artifactual. The estimated overall frequency of aneuploidy is 13%. In embryos the level of chromosomal abnormality is 23%–40%. Errors of fertilization are responsible for a substantial number of triploid embryos, many of which develop into mosaics. Factors extrinsic to the conceptus, such as infertility, advanced maternal age, and ovarian hyperstimulation, may increase the level of chromosomal abnormality. More refined methods for accurately recognizing and selecting chromosomally normal embryos for transfer are needed to improve the success rate of this reproductive technology.     

In vitro fertilization and expression of transgenes in gametes and zygotes

The in vitro fertilization system of maize is the well characterized model system for the fertilization process and early zygotic embryogenesis of higher plants. Application of molecular methods to the in vitro fertilization system led to the isolation of new genes and uncovered specific expression patterns of cell cycle regulators. Recent studies showed that expression of transgenes is possible in gametes and zygotes, thus transgenic approaches might offer an opportunity to unravel the roles of genes during fertilization and early development. The competence of gametes and zygotes to express transgenes will also enable the expression of GFP based reporter genes for the visualization of subcellular components in these cells in vivo. This review focuses on the data concerning the expression of transgenes in gametes and zygotes and describes some examples of recent developments in transgenic technology illustrating the emerging possibilities in experimental design by combining this technology with in vitro fertilization. Received: 20 December 2001 / Revision accepted: 6 June 2001     

Morphofunctional characteristics of the endosteum

Basing on the literature data analysis, modern notions concerning endosteum structure, its connections with the bone and bone marrow are presented. Participation of the endosteum in processes of osteogenesis and resorption, as well as its changes with ageing are discussed.     

Morphofunctional changes in human flatfoot

The process of the flatfoot formation is accompanied not only by stretching but also by pressing of the plantar ligaments and muscles. In the plantar aponeurosis and ligaments subjected to pressing, adaptive rearrangements are observed; they are accompanied by transformation of the fibrillar connective tissue into cartilaginous one, that rather prevents the ligaments to affect the heigh of the fornix. In the structure of the plantar muscles rather noticeable changes occur, destructive ones including, that is seen in their microscopical picture.     

Morphofunctional characteristics of cadmium-induced arterial hypertension

Chronic (12 weeks) peroral administration of cadmium chloride to albino rats in a dose of 2.5 mg/100 g body weight results in arterial hypertension characterized by the increase in systolic blood pressure up to 148 +/- 1.8 mm Hg (vs. 115.4 +/- 1.5 mm Hg in the control animals); the increase in vascular resistance, left ventricular cardiomyocyte hypertrophy, as well as by hypertrophy of arterial walls, the decrease in the ventricular index, the activation of synthesizing function of atrial endocrine cardiomyocytes; enhanced secretion of ANP; a more than two-fold increase in plasma myoglobin concentration, as well as by the development of cadmium-induced nephropathy. In the rehabilitation period (9 weeks) a relatively quick fall in the blood pressure is observed, as well as morphological features of myocardial and renal function recovery, suggesting the nonpersistent nature of cadmium-induced hypertension.     

Tamoxifen-induced alterations in meiotic maturation and cytogenetic abnormalities in mouse oocytes and 1-cell zygotes

Alterations in the rate of oocyte meiotic maturation (OM) and the timing of the metaphase-anaphase transition may predispose oocytes to premature centromere separation (PCS) and aneuploidy. Tamoxifen has the potential for perturbing the rate of OM since it can function as a calcium antagonist by binding to calmodulin and inhibiting the formation of a calcium-calmodulin complex which is needed for activating calmodulin-dependent cAMP phosphodiesterase and initiating OM. The objective of this study was to test the hypothesis that tamoxifen alters the rate of OM and predisposes oocytes to PCS and aneuploidy. Different does of tamoxifen were administered by oral gavage to female mice preovulation. Metaphase II oocyte and 1-cell zygote chromosomes were C-banded and cytogenetically analysed. Tamoxifen treatment resulted in a modest, but significant (p < 0.05), increase in oocytes with PCS. Similar frequencies of hyperploidy and oocytes with unpaired, single chromatids (SC) were found. Metaphase I, diploid and premature anaphase (PA) oocytes were not detected. Hyperploidy, polyploidy, PCS, PA and SC were not detected in zygotes. These data indicate that the levels of tamoxifen-induced PCS found in mouse oocytes did not predispose zygotes to aneuploidy. Tamoxifen did, however, reduce the proportion of females exhibiting oestrus.     

Kinetochore appearance during meiosis,fertilization and mitosis in mouse oocytes and zygotes

The events of mammalian fertilization overlap with the completion of meiosis and first mitosis; the pronuclei never fuse, instead the parental genomes first intermix at the mitotic spindle equator at metaphase. Since kinetochores are essential for the attachment of chromosomes to spindle microtubules, this study explores their appearance and behavior in mouse oocytes, zygotes and embryos undergoing the completion of meiosis, fertilization and mitoses. Kinetochores are traced with immunofluorescence microscopy using autoimmune sera from patients with CREST (CREST = calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) scleroderma. These sera cross-react with the 17 kDa centromere protein (CENP-A) and the 80 kDa centromere protein (CENP-B) found at the kinetochores in human cell cultures. The unfertilized oocyte is ovulated arrested at second meiotic metaphase and kinetochores are detectable as paired structures aligned at the spindle equator. At meiotic anaphase, the kinetochores separate and remain aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis is lost. The female pronucleus and the second polar body nucleus each receive a detectable complement of kinetochores. Mature sperm have neither detectable centrosomes nor detectable kinetochores, and shortly after sperm incorporation kinetochores become detectable in the decondensing male pronucleus. In pronuclei, the kinetochores are initially distributed randomly and later found in apposition with nucleoli. At mitosis, the kinetochores behave in a pattern similar to that observed at meiosis or mitosis in somatic cells: irregular distribution at prophase, alignment at metaphase, separation at anaphase and redistribution at telophase. They are also detectable in later stage embryos. Colcemid treatment disrupts the meiotic spindle and results in the dispersion of the meiotic chromosomes along the oocyte cortex; the chromosomes remain condensed with detectable kinetochores. Fertilization of Colcemid-treated oocytes results in the incorporation of a sperm which is unable to decondense into a male pronucleus. Remarkably kinetochores become detectable at 5 h post-insemination, suggesting that the emergence of the paternal kinetochores is not strictly dependent on male pronuclear decondensation.     

Morphofunctional characteristics of the microcirculatory bed of the human spinal dura mater]

Some morphofunctional peculiarities in microcirculatory pathways of the dura mater of the human spinal cord are described. They are concerned with the structure of arteriolo-venular anastomoses through which a rather large amount of arterial blood is transported into the venous bed. Around the vessels of arterial type running at an angle to the longitudinal axis of the vessel connective tissue fibres of the dura mater, there is a tissue layer intensively impregnated with silver salts and stained PAS-positively. The venous part of the dura mater microcirculatory pathways has a large number of accessory reservoirs in the form of venous "lakes". Functional importance of the peculiarities mentioned above for the dura mater and the perimedullar apparatus is clarified.     

Isolation and molecular cytogenetic characteristics of the alpha-satellite DNA of human chromosome 6

In this work a molecular-cytogenetic characteristic has been given to the alpha-satellite DNA of chromosome 6. Using different restrictases made it possible to evaluate structural heterogeneity of the obtained subgroup of the alphoid DNA. Polymorphic restriction fragments of DNA have been found which can be used in determining linkage groups.     

Asymmetric cell division of rice zygotes located in embryo sac and produced by in vitro fertilization

In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. This unequal division of the zygote is a putative first step for formation of the apical–basal axis of plants and is a fundamental feature of early embryogenesis and morphogenesis in angiosperms. Because fertilization and subsequent embryogenesis occur in embryo sacs, which are deeply embedded in ovular tissue, in vitro fertilization of isolated gametes is a powerful system to dissect mechanisms of fertilization and post-fertilization events. Rice is an emerging molecular and experimental model plant, however, profile of the first zygotic division within embryo sac and thus origin of apical–basal embryo polarity has not been closely investigated. Therefore, in the present study, the division pattern of rice zygote in planta was first determined accurately by observations employing serial sections of the egg apparatus, zygotes and two-celled embryos in the embryo sac. The rice zygote divides asymmetrically into a two-celled embryo consisting of a statistically significantly smaller apical cell with dense cytoplasm and a larger vacuolated basal cell. Moreover, detailed observations of division profiles of zygotes prepared by in vitro fertilization indicate that the zygote also divides into an asymmetric two-celled embryo as in planta. Such observations suggest that in vitro-produced rice zygotes and two-celled embryos may be useful as experimental models for further investigations into the mechanism and control of asymmetric division of plant zygotes.     

CA++ in fertilization and mitosis: the phosphatidylinositol cycle in sea urchin gametes and zygotes is involved in control of fertilization and mitosis

We determined that the phosphatidylinositol (PI) cycles in both sea urchin sperm and eggs are necessary for normal fertilization, and that the PI cycle in sea urchin zygotes is involved in control of mitosis. The PI cycle is involved in Ca++ homeostasis so our data are direct evidence that Ca++ is involved with control of mitosis and fertilization. We implicated the PI cycle by adding Li+ to sea urchin eggs, sperm, or zygotes: those effects of Li+ due to effects on the PI cycle were overcome by myo-inositol but not by its optical isomer, scyllitol, and not by mannitol.     

Morphofunctional characteristics of adrenal reactivity in rats of different ages

Ultrastructural peculiarities of the cortical and medullary substance of the adrenals have been studied in mature (8-month-old) and old (26-month-old) intact white male rats and in rats of the same age groups in 3 h after electrical stimulation of the hypothalamic ventromedial nuclei. By means of certain biochemical methods contents of hormones (corticosterone, adrenaline and noradrenaline) have been estimated, as well as that of cholesterin and ascorbic acid in blood and adrenal tissues. After the hypothalamus is stimulated in the mature rats, certain peculiarities in the adrenals reactivity are revealed; they demonstrate positive ultrastructural and functional correlation of secretory cells in the cortex and medulla and are characterized with hypertrophy of the nuclei, increased amount of polyribosomes, hypertrophied Golgi complex and canaliculi of the endoplasmic reticulum and increased contents of corticosteroids and catecholamines in blood. In the old rats after stimulation of the hypothalamus, certain unequal ultrastructural and functional peculiarities are revealed. They demonstrate both disturbance of the hypothalamic regulation of the adrenal function at ageing and decreased adaptive possibilities of the adrenal secretory cells at the subcellular level.     

Centrosome inheritance in starfish zygotes: selective loss of the maternal centrosome after fertilization

The mature egg inherits a centrosome from the second meiotic spindle, and the sperm introduces a second centrosome at fertilization. Since only one of these centrosomes survives to be used in development, specific mechanisms must exist to control centrosome inheritance. To investigate how centrosome inheritance is controlled we used starfish eggs as a model system, because they undergo meiosis after fertilization. As a result, the fate of the maternal and paternal centrosomes can be followed by light microscopy and experimentally manipulated in vivo. We show initially that only the paternal centrosome is used in starfish zygote development; the maternal centrosome retained from meiosis II is functionally lost before first mitosis. We then tested a number of possible ways in which the zygote could exert this differential control over the stability of centrosomes initially residing in the same cytoplasm. The results of these experiments can be summarized as follows: (1) Although the microtubule organizing center activity of the maternal centrosome is not degraded after meiosis, the ability of this centrosome to double at successive mitoses is lost. (2) The sperm centrosome is not "masked" from cytoplasmic conditions which could destabilize all centrosomes during or after the meiotic sequence. (3) The functional loss of the maternal centrosome is not due to its cortical location. (4) The loss of this doubling capacity is determined by the egg, not by putative inhibitory factors from the fertilizing sperm. (5) The destabilization of the maternal centrosome is not due to the complete loss of its centrioles. Together, these results demonstrate that all maternal centrosomes are equivalent and that they are intrinsically different from the paternal centrosome. This intrinsic difference, in concert with a change in cytoplasmic conditions after meiosis, determines the selective loss of the maternal centrosome inherited from the meiosis II spindle.     

Morphofunctional characteristics of young rowers with increased blood pressure

The morphofunctional characteristics of 47 young athletes (rowers) were studied. Stage I essential hypertension was found in 25.6% of the young athletes and high normal blood pressure (BP) in 8.5% of the athletes. Detailed clinical and functional observation has shown that, in the group of rowers with an elevated BP, the weight of the left ventricle myocardium was increased and the functional reserve was reduced as compared to the group of athletes with a normal BP level.