Expression of mouse telomerase catalytic subunit in embryos and adult tissues

Telomerase is a ribonucleoprotein complex that elongates telomeres, allowing the stable maintenance of chromosomes during multiple cell divisions. Here, we describe the isolation and characterization of the catalytic subunit of mouse telomerase, mTERT (mouse telomerase reverse transcriptase), an essential protein component of the telomerase complex. During embryonic development, mTERT mRNA is abundantly expressed in the whole embryo, especially in regions of intense proliferation. We found that the mTERT mRNA expression in both embryonic and adult tissues is independent of the essential RNA component of telomerase, mTR, and therefore, of the formation of active telomerase complexes. mTERT protein is present exclusively in tissues with telomerase activity, such as testis, spleen, and thymus. mTERT protein is barely detectable in the thymus of mTR−/− mice, suggesting that mTERT protein stability in this tissue may depend on the actual assembly of active telomerase complexes. Finally, we found that mouse and human telomerase catalytic subunit is located in the cell nucleus, and its localization is not regulated during cell cycle progression.

Prolonged activation of the N-methyl-d-aspartate receptor–Ca2+ transduction pathway causes spontaneous recurrent epileptiform discharges in hippocampal neurons in culture

The molecular basis for developing symptomatic epilepsy (epileptogenesis) remains ill defined. We show here in a well characterized hippocampal culture model of epilepsy that the induction of epileptogenesis is Ca2+-dependent. The concentration of intracellular free Ca2+ ([Ca2+]i) was monitored during the induction of epileptogenesis by prolonged electrographic seizure activity induced through low-Mg2+ treatment by confocal laser-scanning fluorescent microscopy to directly correlate changes in [Ca2+]i with alterations in membrane excitability measured by intracellular recording using whole-cell current–clamp techniques. The induction of long-lasting spontaneous recurrent epileptiform discharges, but not the Mg2+-induced spike discharges, was prevented in low-Ca2+ solutions and was dependent on activation of the N-methyl-d-aspartate (NMDA) receptor. The results provide direct evidence that prolonged activation of the NMDA–Ca2+ transduction pathway causes a long-lasting plasticity change in hippocampal neurons causing increased excitability leading to the occurrence of spontaneous, recurrent epileptiform discharges.

Telomerase expression in chickens: Constitutive activity in somatic tissues and down-regulation in culture

Although human and rodent telomeres have been studied extensively, very little is known about telomere dynamics in other vertebrates. Moreover, our current dependence on mice as a model for human tumorigenesis and aging poses a problem because human and mouse telomere biology is very different. To explore whether chickens might provide a more useful model, we have examined telomerase activity and telomere length in chicken tissues as well as in primary cell cultures. Although chicken telomeres resemble human telomeres in that they are 8–20 kb in length, the distribution of telomerase activity in chickens resembles what is found in mice. Active enzyme is present in germline tissue as well as in a wide range of somatic tissues. Because chicken cells exhibit extremely low rates of spontaneous immortalization, this finding indicates that constitutive telomerase expression does not necessarily lead to an increased immortalization frequency. Finally, we found that telomerase activity is greatly down-regulated when primary cultures are established from chicken embryos. Although this down-regulation explains the telomere loss and replicative senescence that we observed in fibroblast cultures, it raises questions concerning how relevant studies of senescence in primary cell cultures are to aging in whole animals.

From cell death to embryo arrest: Mathematical models of human preimplantation embryo development

Human preimplantation embryos exhibit high levels of apoptotic cells and high rates of developmental arrest during the first week in vitro. The relation between the two is unclear and difficult to determine by conventional experimental approaches, partly because of limited numbers of embryos. We apply a mixture of experiment and mathematical modeling to show that observed levels of cell death can be reconciled with the high levels of embryo arrest seen in the human only if the developmental competence of embryos is already established at the zygote stage, and environmental factors merely modulate this. This suggests that research on improving in vitro fertilization success rates should move from its current concentration on optimizing culture media to focus more on the generation of a healthy zygote and on understanding the mechanisms that cause chromosomal and other abnormalities during early cleavage stages.

Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F1 genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F1 ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F1 ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F1 ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F1 ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F1 ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.

The hybrid PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma transforms fibroblasts in culture.

Pediatric alveolar rhabdomyosarcoma is characterized by a chromosomal translocation that fuses parts of the PAX3 and FKHR genes. PAX3 codes for a transcriptional regulator that controls developmental programs, and FKHR codes for a forkhead-winged helix protein, also a likely transcription factor. The PAX3-FKHR fusion product retains the DNA binding domains of the PAX3 protein and the putative activator domain of the FKHR protein. The PAX3-FKHR protein has been shown to function as a transcriptional activator. Using the RCAS retroviral vector, we have introduced the PAX3-FKHR gene into chicken embryo fibroblasts. Expression of the PAX3-FKHR protein in these cells leads to transformation: the cells become enlarged, grow tightly packed and in multiple layers, and acquire the ability for anchorage-independent growth. This cellular transformation in vitro will facilitate studies on the mechanism of PAX3-FKHR-induced oncogenesis.

Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72. Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins.

The preimplantation mouse embryo is a target for cannabinoid ligand-receptor signaling.

Using a reverse transcription-coupled PCR, we demonstrated that both brain and spleen type cannabinoid receptor (CB1-R and CB2-R, respectively) mRNAs are expressed in the preimplantation mouse embryo. The CB1-R mRNA expression was coincident with the activation of the embryonic genome late in the two-cell stage, whereas the CB2-R mRNA was present from the one-cell through the blastocyst stages. The major psychoactive component of marijuana (-)-delta-9-tetrahydrocannabinol [(-)-THC] inhibited forskolin-stimulated cAMP generation in the blastocyst, and this inhibition was prevented by pertussis toxin. However, the inactive cannabinoid cannabidiol (CBD) failed to influence this response. These results suggest that cannabinoid receptors in the embryo are coupled to inhibitory guanine nucleotide binding proteins. Further, the oviduct and uterus exhibited the enzymatic capacity to synthesize the putative endogenous cannabinoid ligand arachidonylethanolamide (anandamide). Synthetic and natural cannabinoid agonists [WIN 55,212-2, CP 55,940, (-)-THC, and anandamide], but not CBD or arachidonic acid, arrested the development of two-cell embryos primarily between the four-cell and eight-cell stages in vitro in a dose-dependent manner. Anandamide also interfered with the development of eight-cell embryos to blastocysts in culture. The autoradiographic studies readily detected binding of [3H]anandamide in embryos at all stages of development. Positive signals were present in one-cell embryos and all blastomeres of two-cell through four-cell embryos. However, most of the binding sites in eight-cell embryos and morulae were present in the outer cells. In the blastocyst, these signals were primarily localized in the mural trophectoderm with low levels of signals in the polar trophectoderm, while little or no signals were noted in inner cell mass cells.These results establish that the preimplantation mouse embryo is a target for cannabinoid ligands. Consequently, many of the adverse effects of cannabinoids observed during pregnancy could be mediated via these cannabinoid receptors. Although the physiological significance of the cannabinoid ligand-receptor signaling in normal preimplantation embryo development is not yet clear, the regulation of embryonic cAMP and/or Ca2+ levels via this signaling pathway may be important for normal embryonic development and/or implantation.

Homeobox-containing gene transiently expressed in a spatially restricted pattern in the early sea urchin embryo.

In the sea urchin embryo, the lineage founder cells whose polyclonal progenies will give rise to five different territories are segregated at the sixth division. To investigate the mechanisms by which the fates of embryonic cells are first established, we looked for temporal and spatial expression of homeobox genes in the very early cleavage embryos. We report evidence that PlHbox12, a paired homeobox-containing gene, is expressed in the embryo from the 4-cell stage. The abundance of the transcripts reaches its maximum when the embryo has been divided into the five polyclonal territories--namely at the 64-cell stage--and it abruptly declines at later stages of development. Blastomere dissociation experiments indicate that maximal expression of PlHbox12 is dependent on intercellular interactions, thus suggesting that signal transduction mechanisms are responsible for its transcriptional activation in the early cleavage embryo. Spatial expression of PlHbox12 was determined by whole-mount in situ hybridization. PlHbox12 transcripts in embryos at the fourth, fifth, and sixth divisions seem to be restricted to the conditionally specified ectodermal lineages. These results suggest a possible role of the PlHbox12 gene in the early events of cell specification of the presumptive ectodermal territories.

Reproduction and sera embryotoxicity after immunization of monkeys with the laminin peptides YIGSR, RGD, and IKVAV.

Monkeys with excellent reproductive histories were immunized with the laminin peptides YIGSR, RGD, IKVAV, and YD, a control sequence with no known biological function. Sera from the YIGSR-immunized monkey became toxic, causing neural tube defects in whole rat embryo cultures, and this monkey experienced fetal loss after immunization. Sera from the RGD-immunized monkey also became embryotoxic in culture after immunization, but this monkey appeared to become infertile as she failed to initiate a pregnancy for at least 2 years after immunization. In contrast, embryos cultured on sera from the IKVAV- or YD-immunized monkeys were predominantly normal and both monkeys completed successful pregnancies. Antibody levels to the respective peptides or to laminin were not predictive of embryotoxicity, but antibody binding to homogenized yolk sacs as well as to yolk sacs of cultured embryos was associated with sera embryotoxicity and reproductive outcomes in vivo. These observations suggested that the laminin sequences YIGSR and RGD may play a role in immune-mediated reproductive failure by reacting directly with embryonic tissue and could provide a basis for identifying individuals at risk for both spontaneous abortion and infertility.