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PubMed2006

Infertility in murine acute Trypanosoma cruzi infection is associated with inhibition of pre-implantation embryo development.

We previously showed that Trypanosoma cruzi acute infection induced infertility in a great proportion of female mice, which resulted from a defect taking place before implantation. In this study, we have analyzed every step of reproduction from mating to implantation to identify the most sensitive event. Our results show that mating, ovulation, fertilization, and first division of the zygote of infected mice take place normally compared with uninfected mice, indicating that the defect occurred after the two-cell stage. In vivo development of two-cell embryos to the blastocyst stage was indeed dramatically delayed; some embryos even arrested their development before having reached the eight-cell stage while others degenerated. The effect was less pronounced when embryos were allowed to develop in vitro, indicating that the infectious context of the mother plays a role in maintaining growth retardation. The delay of embryonic development was associated with insufficient divisions of the blastomeres and led to abnormal blastocyst outgrowth that may explain implantation failure. Inhibition of cell division was correlated with the maternal parasitemia. This work clearly shows that T. cruzi infection dramatically impedes embryonic development, offering a model for further in vivo studies of embryotrophic factors produced by the oviduct of infected females.

Acute Disease↗
PubMed1994

Telomere shortening in renal cell carcinoma.

The ends of human chromosomes consist of a specialized structure, the telomere, composed of repeats of TTAGGG making up a total of 5-15 kilobase pairs, depending on age and proliferative activity of the tissue. The major function of telomeres is to provide stability to chromosomes and protect underlying unique coding sequences from degradation. There is a loss of telomeric sequences following every cell division estimated to be between 50 and 65 basepairs/cell division in human fibroblasts and embryonic kidney cells in vitro. This loss is due to the fact that DNA replication is incomplete for one strand at each telomere end. In lower eukaryotes there is a compensation mechanism provided by the enzyme telomerase, which is inactive in human somatic cells. Telomerase activation has also been detected in vitro immortalized human cells. In this study we analyzed renal cell carcinoma for the occurrence of telomere shortening using the probe (TTAGGG)4. Southern blots of HinfI-digested DNA revealed a shortening of mean telomere restriction fragment (TRF) length of 0.4 to 2.5 kilobase pairs in 2 or 3 intratumoral samples in all 10 tumors analyzed. No obvious intratumoral heterogeneity was found in mean TRF length values. However, heterogeneity was shown by the occurrence of at least two separate peak TRF values in 7 of 10 tumors, indicating the presence of different tumor cell clones. A conflicting observation was made when we evaluated the intensity of the hybridization signals, where three of the tumors showed an increase in hybridization signals despite concomitant TRF reduction. We found no correlation between tumor size and calculated tumor cell divisions undergone. In two tumors, the calculated cell division cycles were unrealistically low compared to the tumor size. These data suggest that telomerase activation might occur in human renal cell carcinoma.

Base Sequence↗
PubMed2000

[Features of the development of homo- and heterotopic allotransplants of rat embryonal neocortex].

Mechanisms of regulation of cell division in the developing neocortex are largely unknown. The aim of the present study was to investigate the influence of a microenvironment on the fetal neocortex histogenesis. The fetal neocortex from 15-day old Wistar rat embryo was grafted into the neocortex, crushed sciatic nerve and anterior chamber of eye of adult rats. A comparative study of graft development was carried out on 1, 3, 7, 10, 30 days using histological (Nissl stain, hematoxylin-eosin) and immunohistochemical (monoclonal antibody to proliferating cell nuclear antigen, and to glial fibrillary acidic protein) methods. Grafted neuroepithelial cells proliferated in grafts that developed in the neocortex and the anterior chamber of eye for 7 days, and in the sciatic nerve for 10 days. In all grafts differentiating neuroblasts, young neurons and mature neurons were observed 7, 10 and 30 days later, respectively. In 10 days, transplants in the nerve have a glial capsule, in contrast to other sites of grafting. The capsule consists of ependymocytes with microvilli and cilia 30 days later. These cells are GFAP-positive. Our results indicate epigenetic influence on the development of neuroepithelial precursors. The microenvironment of the peripheral nerve is suggested to promote glyogenesis in developing grafts. Afferent inputs do not influence the proliferative potency of brain cell precursors.

Animals↗
PubMed1993

Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, NCCIT.

BACKGROUND: Germ cell tumors are empirically divided into seminomas and nonseminomatous germ cell tumors (NSGCT). Some authorities consider seminomas to be the precursors of NSGCT, whereas others consider them as distinct and unrelated neoplasms. Here, we report that the human NSGCT-derived stem cell line, NCCIT has hybrid features of seminoma and embryonal carcinoma, and suggest that this cell line could be useful for studying the relationship of seminoma to NSGCT. EXPERIMENTAL DESIGN: NCCIT, a developmentally pluripotent permanent cell line derived from a mediastinal NSGCT was karyotyped and characterized morphologically, immunochemically, and biochemically. The cells were grown under standard tissue culture conditions and were also exposed to retinoic acid to induce differentiation. RESULTS: The dividing NCCIT stem cell populations consist of vimentin-positive, keratin-negative cells that do not express desmoplakin or cadherin E (uvomorulin) and are not interconnected with one another. These cells have a high nucleocytoplasmic ratio and contain few cytoplasmic organelles, except for free ribosomes and a small number of mitochondria. Lacto- and globoseries oligosaccharide antigens recognized with antibodies to murine stage specific antigens 1, 3 and 4 (SSEA-1, SSEA-3 and SSEA-4), and human teratocarcinoma mucin-like antigen TRA-1-60 and TRA-1-81 are coexpressed on the cell membranes of a considerable number of stem cells. On most cells alkaline phosphatase can be detected by enzyme histochemistry. The placental isoenzyme of alkaline phosphatase was demonstrated by Western blotting in cell extracts. The liver/bone/kidney isoenzyme of alkaline phosphatase is immunochemically detected on 40% of cells. The culture supernatants also contain chorionic gonadotropin and alpha-fetoprotein, presumably derived from trophoblastic and yolk sac-like cells. The cells are hyperdiploid (chromosome range from 54 to 64) and show prominent structural chromosomal aberrations, mostly deletions and isochromosomes. Retinoic acid treatment inhibited the growth of NCCIT cells and induced stem cell differentiation into keratin, glial fibrillary acid protein, and neurofilament-positive somatic cells. The differentiation was associated with the disappearance of oligosaccharide surface antigens typical of the undifferentiated stem cells; a loss of proteins typical of undifferentiated cells and the appearance of new proteins; and the deposition of extracellular matrix. CONCLUSIONS: NCCIT is a developmentally pluripotent cell line that can differentiate into derivatives of all three embryonic germ layers (i.e., ectoderm, mesoderm, and endoderm) and extraembryonic cell lineages. We suggest that this cell line could be a malignant replica of human cleavage stage embryonic cells with features intermediate between seminoma and embryonal carcinoma.

Adult↗
PubMed1995

Transitional differentiation patterns of principal and intercalated cells during renal collecting duct development.

The developing renal collecting duct epithelium of neonatal rabbits exhibits 3 different zones. The ampullary tip epithelium acts as an embryonic inducer and is responsible for the generation of all of the nephron anlagen. It pilots the whole microarchitecture of the kidney. In the ampullary neck epithelium multiple cell divisions cause the elongation of the embryonic collecting duct so that the organ can grow. Finally, the cells in the ampullar shaft transdifferentiate into the functional collecting duct epithelium (CD) consisting of Principal (P) and various kinds of Intercalated (IC) cells. It is unknown by which morphogenic mechanisms the ampullar cells develop into the heterogeneously composed collecting duct epithelium. Using both morphological and immunohistochemical methods, we investigated the transdifferentiation patterns leading from the ampullar epithelium to the P and IC cells in the neonatal kidney. An electron microscope analysis of the cortico-medullary course of the developing collecting duct revealed that conspicuous morphological alterations start in the neck of the ampulla. The lumen of the neck region is narrowed to a slit. While most of the cells in the ampullar tip exhibit few, short microvilli, the neck cells bear numerous, extremely long microvilli at their apical cell poles. All of the neck cells exhibit the same cytoplasmic staining pattern and the same number of mitochondria. Farther down in the shaft, clearly recognizable P and IC cells are found. Thus, differentiation into P and IC cells starts with a transitional precursor cell type in the ampullar neck. Perfusion culture experiments with the embryonic collecting duct epithelium made it possible to generate transitional and differentiated cell types for the first time under in vitro conditions. The cultured epithelial cells showed characteristics common to both P and IC cells. Immunohistochemical findings revealed that morphological differentiation starts before the functional properties of P and IC cells can be detected.

Animals↗
PubMed1978

Embryonic mouse thymus development: stem cell entry and differentiation.

The entry and subsequent division and differentiation of blood-borne stem cells within the embryonic thymus has been studied in a system where the inflowing cells were either chromosomally or antigenically marked. H-2 positive stem cells enter the thymus within 5 days of grafting but may wait for up to 14 days before proliferating to give rise to Thy-1 positive thymocytes.

Animals↗
PubMed1989

An increase in the intracellular pH of fertilized eggs of Xenopus laevis is associated with inhibition of protein and DNA syntheses and followed by an arrest of embryonic development.

In many systems, events participating in cell division are controlled by intracellular pH (pHi). In Xenopus eggs, fertilization is accompanied by an increase in pHi which occurs concomitantly with an increase in protein synthesis and a reinitiation of DNA synthesis, leading the embryo to cell division. In this paper, we have shown that increasing pHi of fertilized eggs from 7.8 to 8.2 by using weak bases produced an arrest in embryonic development. Such a change in pHi was accompanied by a severe inhibition of both protein and DNA syntheses. In order to discriminate between a direct effect of pHi and a pH-independent effect of weak bases on these biosyntheses, the situation was studied in vitro. For this purpose, cytoplasmic extracts were used in which weak base addition did not produce any change in pH. Under these conditions, protein synthesis was not inhibited, suggesting that pH is probably one of the events implicated in the regulation of protein synthesis. On the other hand, DNA synthesis was inhibited by weak bases in vitro, without any change in pH intervening.

Animals↗
PubMed2003

Trisomy 8 mosaicism: selective growth advantage of normal cells vs. growth disadvantage of trisomy 8 cells.

A fetus with trisomy 8 mosaicism was identified prenatally due to an abnormal maternal serum triple screen. Tissue samples were taken at birth to determine the level of trisomy 8 mosaicism found within embryonic and extra-embryonic tissues, rates of cell division for the two cell lines, and the effect of mosaicism on the phenotype. The level of trisomy 8 cells in blood and fibroblasts was higher than in placental tissue. Cell cycle kinetics, by incorporation of bromodeoxyuridine for 48 hr, was not significantly different between the trisomy 8 and normal cells for blood or amnion. Fluorescent in situ hybridization (FISH) using centromeric probe for chromosome 8 showed significantly more trisomy 8 in interphase vs. metaphase in lymphoblasts, umbilical cord fibroblasts, and chorion. The loss of trisomy 8 cells is not due to anaphase lag, as determined by micronuclei analysis. The similarity of cell cycle kinetics between trisomy 8 cells and normal diploid cells suggests some trisomy 8 cells are exiting the cell cycle prematurely. This growth disadvantage of trisomy 8 cells results in the appearance of growth advantage for diploid cells.

Amnion↗
PubMed1998

Cytostatic activity develops during meiosis I in oocytes of LT/Sv mice.

Oocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that during in vitro maturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H x C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase II oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity.

Anaphase↗
PubMed1986

Growth of mouse embryos in bicarbonate media buffered by carbon dioxide, hepes, or phosphate.

The purpose of this study was to determine if mouse embryos could be grown successfully in a culture medium devoid of the carbon dioxide phase (CO2). Mouse embryos fertilized in vivo were collected and cultured in Hepes medium with and without bicarbonate (HCO3-) and a phosphate medium with and without HCO3-. In these experiments no CO2 gas phase was used. Further embryos were cultured in Whittingham's modified Tyrode's (T6) medium with a CO2 gas phase and served as controls. The degree of embryonic development was noted. Surviving blastocysts were transferred to the uteri of pseudopregnant mice and delivery at term was allowed to occur. There was no significant difference in the degree of embryonic development in those embryos cultured in T6 or Hepes medium (+ HCO3-) or in the number of live offspring obtained when these blastocysts were placed within the mouse uterus. Although embryonic development apparently proceeded successfully in the phosphate (+ HCO3-) medium, none of these blastocysts survived when transferred to mouse uteri. No embryonic growth occurred in either the Hepes or phosphate media which were devoid of HCO3-. It appears that a Hepes medium containing HCO3-, which uses no CO2 gas phase, is as effective as T6 medium, which uses a gas phase, in supporting in vitro mouse embryonic growth.

Animals↗
PubMed1979

Aberrations of holokinetic chromosomes and associated lethality after x-irradiation of meiotic stages in Tetranychus urticae Koch (acari, tetranychidae).

Chromosomes of the holokinetic organization type were irradiated with X-rays in various stages of meiosis in unfertilized eggs of Tetranychus urticae Koch. Visible cytological aberrations, lethality and sterility were investigated in subsequent generations. Chromosome fragments are the most frequently occurring light-microscopically visible chromosome aberrations; bridges are not formed. Contrary to expectations, the presence of fragments appears to be positively correlated with the occurrence of lethality; loss of fragments, mis-segregation and the measure of damage to the broken chromosome parts are involved. In contrast with monokinetic chromosomes the earliest lethality occurs only after about 10 divisions. The ratios between different embryonic lethality types (early vs. late) differ depending on the stage irradiated: in more compact chromatin, more serious damage (i.e. more early lethality syndromes) is induced than in less compact chromatin. In the progeny of the surviving males, neither translocations nor independent fragments are found; indirect evidence indicated the occasional presence of inversions. The presumptive inversions are induced more frequently in a chromatin-compact stage (metaphase I) than in a less compact one (telophase I).

Animals↗
PubMed1986

Anomalies in sea-urchin egg development induced by a novel purine isolated from the sea-anemone Bunodosoma caissarum.

Caissarone (mol. wt 229.5; melting point 285-290 degrees C) is a novel purine isolated and purified from the sea-anemone Bunodosoma caissarum. The purine inhibits the detachment of the vitelline layer from the sea-urchin egg plasma membrane after fertilization and this effect leads to polyspermy. Various abnormalities were detected at various embryonic stages, from multipolar egg division through unequal cleavages and exogastrulation up to teratogenic effects on the sea-urchin larvae (echinopluteus).

Animals↗
PubMed2005

Decatenation checkpoint deficiency in stem and progenitor cells.

The decatenation checkpoint normally delays entry into mitosis until chromosomes have been disentangled through the action of topoisomerase II. We have found that the decatenation checkpoint is highly inefficient in mouse embryonic stem cells, mouse neural progenitor cells, and human CD34+ hematopoietic progenitor cells. Checkpoint efficiency increased when embryonic stem cells were induced to differentiate, which suggests that the deficiency is a feature of the undifferentiated state. Embryonic stem cells completed cell division in the presence of entangled chromosomes, which resulted in severe aneuploidy in the daughter cells. The decatenation checkpoint deficiency is likely to increase the rates of chromosome aberrations in progenitor cells, stem cells, and cancer stem cells.

Animals↗
PubMed1999

Cell memory and cancer--the story of the trithorax and Polycomb group genes.

Most of the cells in an adult organism possess the same DNA content and the same complement of genes, yet we can recognise many different cell types in the adult. Development is the process by which the cells of the embryo progressively acquire their adult fate and position. The genetic network which controls these processes is beginning to be unravelled at an increasing pace, yet one key area is still somewhat neglected--namely, developmental cell memory, the process by which cells record their ontogeny. A significant component of the phenotype of cancer cells may be explained by deregulation of genes whose normal role is to control the division, differentiation and migration of embryonic cells during development. It should therefore not be surprising that genes implicated in cell memory processes during development are also implicated in disease. In this review we outline what is known about the Polycomb and trithorax group of proteins as candidate genes for the memory process, both in terms of basic functions and the roles of abnormalities in these genes resulting in cancer.

Animals↗
PubMed1991

Regeneration of the root pole in surgically transected carrot embryos occurs by position-dependent, proximodistal replacement of missing tissues.

Torpedo-stage carrot embryos were surgically transected at various locations along the shoot-root axis and explants of the cotyledon-bearing shoot pole were sectioned and examined in order to provide a more detailed description of root pole regeneration. When excisions occurred at the sites of the future hypocotyl, the future radicle or the future root apical meristem, the regenerating axial tissues exhibited patterns of cellular organization that were nearly identical to those seen in unsevered controls. To accomplish this restoration, new cells, of the type normally found at each cutting site, were produced behind a regeneration dome that formed over the original surgical site. The regeneration dome was displaced by division and expansion-driven extension of the longitudinal axis, and cells in the renewed region quickly acquired individual anatomical traits and collective tissue morphologies that corresponded to those of cells in the analogous locations in intact embryos. Although no clear mechanism is implied, the results of these experiments suggest that interactions between cells near the surgical margin permit them to retain their sense of location within the original structure, and apprise them of the removal of their basipetally positioned neighbors. With varying-length remnants of the shoot serving as the only vestige of the original size and shape of the embryo, cells close to the site of excision were apparently reconfigured to commence ordered divisions that ultimately reconstituted the embryonic axis.

Microscopy, Electron↗
PubMed1995

Isolation and characterization of a chicken homolog of the E2F-1 transcription factor.

In higher eukaryotes, the E2F-1 transcription factor is an essential and limiting component of cell cycle progression in late G1. E2F-1 heterodimerizes with members of the DP gene family and the resulting heterodimer regulates the expression of several proto-oncogenes and the genetic machinery of DNA replication. Cell cycle regulation of E2F activity is mediated through its association with the tumor suppressor Rb gene product. To examine the evolutionary conservation of the E2F-1 protein sequence and its developmental expression pattern we have isolated and sequenced the chick E2F-1 gene (chE2F-1) cDNA. The chicken protein is 34 amino acids (a.a) shorter than its human counterpart (403/437 a.a.) but has extremely well conserved bHLH and pRb binding domains, with respectively 94% and 83% identity. The position of the leucine zipper is also strictly conserved thereby accounting for ability of E2F-1 to form heterodimers with human and chicken DP-1. E2F-1 expression was analysed in synchronized cells as well as in embryonic or newborn chick tissues and appears to be closely correlated to the cell proliferation rate. In situ hybridization studies have shown very high expression levels in the neuroretina during the early stages of embryonic development when active neuroblast division occurs. In contrast, a sharp down-regulation is observed when cells become postmitotic. Overexpression of the chE2F-1 protein leads to oncogenic transformation only when a truncated version of the transgene lacking the pRb binding domain is used; the full length protein either has no effect or may be deleterious for cell survival.

Adenovirus E2 Proteins↗
PubMed2003

Characterization of Polo-like kinase-1 in rat oocytes and early embryos implies its functional roles in the regulation of meiotic maturation, fertilization, and cleavage.

Polo-like kinase 1 (Plk1) is a family of serine/threonine protein kinases that play important regulatory roles during mitotic cell cycle progression. In this study, Plk1 expression, subcellular localization, and possible functions during rat oocyte meiotic maturation, fertilization, and embryonic cleavages were studied by using RT-PCR, Western blot, confocal microscopy, drug-treatments, and antibody microinjection. Both the mRNA and protein of this kinase were detected in rat maturing oocytes and developing embryos. Confocal microscopy revealed that Plk1 distributed abundantly in the nucleus at the germinal vesicle (GV) stage, was associated with spindle poles during the formation of M-phase spindle, and was translocated to the spindle mid-zone at anaphase. In fertilized eggs, Plk1 was strongly stained in the cytoplasm between the apposing male and female pronuclei, from where microtubules radiated. Throughout cytokinesis, Plk1 was localized to the division plane, both during oocyte meiosis and embryonic mitosis. The specific subcellular distribution of Plk1 was distorted after disrupting the M-phase spindle, while additional aggregation dots could be induced in the cytoplasm by taxol, suggesting its intimate association with active microtubule assembly. Plk1 antibody microinjection delayed the meiotic resumption and blocked the emission of polar bodies. In conclusion, Plk1 may be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during rat oocyte meiotic maturation, fertilization, and early embryonic mitosis.

Animals↗