Calpain 2 expression pattern and sub-cellular localization during mouse embryogenesis

Regulation of migration and proliferation by calpain has been shown in various cell types; however, no data are available concerning calpain 2 (capn2) localization in embryonic tissues. Here, we report the expression pattern of capn2 during mouse embryonic development. Expression of the capn2 gene is observed throughout embryonic development. From ES cells and the 8-cell stage to late neurulation stages, CAPN2 is expressed in the cytoplasm and nuclear compartments, with a clear co-localisation with chromatin. Whole-mount in situ hybridization analysis from E8.5 to 14.5 stages indicates high levels of capn2 expression in the nervous system, heart and mesodermal tissues. Up-regulation is maintained during later developmental stages in proliferating cells and in precursor cells involved in muscle (myoblasts) or bone formation (chondrocytes). At later developmental stages, elevated mRNA levels coincided with CAPN2 nuclear localization in these cell types, while differentiated cells maintained cytoplasmic expression. This detailed analysis reveals dynamic expression: nuclear localization was associated either with active cell mitosis in embryonic stem cells and early developmental stages or with precursor cells later during organogenesis. Thus, these data indicate that CAPN2 may represent a key factor in development from the first cell division.     

BEN, a novel surface molecule of the immunoglobulin superfamily on avian hemopoietic progenitor cells shared with neural cells.

BEN is a novel molecule of the immunoglobulin superfamily that we previously identified by means of a monoclonal antibody on neural cell populations during avian development and epithelial cells of the bursa of Fabricius. In this paper, we describe the expression of BEN by hemopoietic cells during ontogeny. In the thymus, BEN is expressed as early as E9, and from E12 until just after hatching 30-60% of thymocytes are BEN positive. Thus the cells expressing BEN are immature thymocytes and not yet differentiated T cells. In the spleen, BEN expression parallels the myelopoietic activity. It is present on 75% of splenocytes during embryonic development and falls rapidly to 20% of cells during the first week after hatching when the spleen is becoming a secondary lymphoid organ. BEN is also found on a large proportion (about 80% positive cells) of bone marrow cells during ontogeny. Post hatching, BEN is present on 40-50% of bone marrow cells. The population of BEN-positive cells in the bone marrow includes myeloid and erythroid progenitor cells, identified by their ability to form colonies in vitro. BEN expression is lost as progenitor cells proliferate and differentiate to develop mature colonies in the clonal assay. Mature myeloid cells, such as macrophages, granulocytes, thrombocytes, and erythrocytes do not express the BEN antigen. Taken together, these data demonstrated that BEN is a stage-specific rather than a lineage-specific differentiation antigen expressed by immature hemopoietic cells.     

Nuclear localization of beta-catenin in vegetal pole cells during early embryogenesis of the starfish Asterina pectinifera

Recently, beta-catenin has been reported to control the expression of morphogenetic genes through the Wnt signaling pathway in invertebrate embryogenesis. In this study, the distribution pattern of beta-catenin during starfish embryogenesis was investigated using immunohistochemistry. In 16-cell stage embryos, beta-catenin began to accumulate in some nuclei at the vegetal pole. During the early cleavage stage, the cells expressing nuclear beta-catenin increased in number in the vegetal pole region of the embryos, and the beta-catenin signal increased in intensity in each nucleus. At the blastula stage, signal for beta-catenin was also found in the cytoplasm of the cells with nuclear beta-catenin. At the vegetal plate stage, almost all vegetal plate cells expressed beta-catenin in both the nucleus and cytoplasm. When the embryos developed to early gastrulae, cells with nuclear beta-catenin were restricted to the archenteron tip, and the signal gradually faded in later stages. The localization and temporal change of beta-catenin expression suggests that beta-catenin has a pivotal role in archenteron formation in starfish embryos.     

Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos.

The present study characterized the profile of nuclear remodeling in nuclear transplant rabbit embryos and investigated the relationship between chromatin behavior after transfer and embryo development. The developmental potential and pattern of remodeling of donor nuclei from cleavage-, morula-, and blastocyst- (inner cell mass ICM, and trophectoderm, TE) stage donors were evaluated. In addition, we determined whether a modification in the synchrony between blastomere fusion and oocyte activation altered the profile of nuclear remodeling and affected development of reconstituted embryos. Development to blastocysts was similar with 8- and 32-cell-stage donor nuclei (42% and 33%, respectively, p greater than 0.1). However, it was reduced with ICM transplants (17%, p less than 0.05), and development of TE transplants did not progress beyond the 8-cell stage. Upon blastomere fusion into nonactivated oocyte cytoplasm, nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by pronuclear (PN) formation and swelling. PCC occurred synchronously within 1.2-1.5 h post-fusion with all stages of donor nuclei (p greater than 0.1). PN formation in 8- and 32-cell transplants occurred approximately 4 h after fusion, and was synchronous to that of female pronuclei in activated oocytes; however, it was delayed in ICM and TE transplants (p less than 0.01). With all stages of donor nuclei, final nuclear diameter was similar to, or larger than, that of female pronuclei. Fusion to activated oocyte cytoplasm, as opposed to nonactivated cytoplasm, prevented PCC and extensive nuclear swelling (16.0 +/- 0.7 vs. 30 +/- 0.7 microns, respectively, p less than 0.01). Nuclear diameter in early embryos was smaller (p less than 0.01), and development to blastocysts was reduced (p less than 0.05). The results indicate that remodeling of the donor nucleus is not essential for development to blastocysts; however, it is beneficial. Furthermore, complete reprogramming seems possible only after remodeling of the donor nucleus, i.e., PCC in nonactivated cytoplasm, followed by nuclear swelling upon activation of the oocyte.     

Temporal and spatial expression of MMP-2,-9,-14 and their inhibitors TIMP-1,-2,-3 in the corpus luteum of the cycling rhesus monkey

The corpus luteum (CL) is a transient endocrine organ that secretes progesterone to support early pregnancy. If implantation is unsuccessful, luteolysis is initiated. Extensive tissue remodeling occurs during CL formation and luteolysis. In this study, we have studied the possible involvement of MMP-2,-9,-14, and their inhibitors, TIMP-1,-2,-3 in the CL of cycling rhesus monkey at various stages by in situ hybridization, immunohistochemistry and microscopic assessment. The results showed that the MMP-2 mRNA and protein were mainly expressed in the endothelial cells at the early and middle stages of the CL development, while their expressions were observed in the luteal cells at the late stage during luteal regression. MMP-9 protein was detected in the CL at the early and middle stages, and obviously increased at the late stage. The expressions of MMP-14 and TIMP-1 mRNA were high at the early and late stages, and low at the middle stage. TIMP-2 mRNA was high throughout all the stages, the highest level could be observed at the late stage. The TIMP-3 production was detected throughout all the stages, but obviously declined during CL regression. MMP-9,-14 and TIMP-1,-2,-3 were mainly localized in the cytoplasm of the steroidogenic cells. The results suggest that the MMP/TIMP system is involved in regulation of CL development in the primate, and the coordinated expression of MMP-2,-14 and TIMP-1,-3 may have a potential role in the CL formation and the functional maintaining, while the interaction of MMP-2,-9,-14 and TIMP-1,-2,-3 might also play a role in CL regression at the late stage of CL development in the primate.     

Temporal and spatial expression of MMP-2,-9,-14 and their inhibitors TIMP-1,-2,-3 in the corpus luteum of the cycling rhesus monkey

A CL develops by extensive cellular reorganization and neovascularization of the remnants of the evacu-ated follicle following ovulation. In both rodent and primate, the development of CL is a rapid process with very high cellular turnover[1,2]. A CL is u…     

An ultracytochemical study of the respiratory potency, integrity, and fate of the sea urchin sperm mitochondria during early embryogenesis

Cytochrome oxidase activity via cytochrome c, as demonstrated by the diaminobenzidine procedure, has been employed in this electron microscope cytochemical study to determine the respiratory potency, integrity and fate of the Arbacia sperm mitochondrion at fertilization and during early embryogenesis. The sperm mitochondrion remained intact and was intensely positive for cytochrome oxidase activity both during and after penetration into the egg. The mitochondrion remained highly reactive throughout zygote formation, up to the eight-cell stage. The sperm mitochondrion formed many projections and buds in the cytoplasm of immature oocytes, monospermic and polyspermic eggs, and in blastomeres. At all stages of early embryogenesis, close juxtaposition and structural contact were observed between the highly reactive sperm mitochondrion and the less reactive egg mitochondria. The results suggest that following fertilization the mitochondrion of the sea urchin spermatozoon retains some degree of metabolic autonomy within the ooplasm. The structural integrity of the paternal mitochondrion is maintained along with a functional respiratory enzyme system (cytochrome c-a3). The hypothesis that the fertilizing sperm mitochondrion may have some relevance to sea urchin development is discussed.     

Reconstitution of endoplasmic reticulum in rapidly dividing cells of early Xenopus embryos

The cytology of early blastomeres of Xenopus laevis embryos was examined. Particular attention was given to the organization of the nuclear envelope of karyomeres (chromosome vesicles) and the endoplasmic reticulum (ER) at different stages in early cleavage cycles of frog development. Nuclear envelope formation was observed to occur rapidly around individual chromosomes during early anaphase, and karyomeres fused subsequently to yield the final nucleus during telophase. Endoplasmic reticulum in the perinuclear cytoplasm was observed to be vesicular during metaphase and cisternal in form during telophase. Following microinjection of rat liver rough microsomes into early blastomeres, heterologous ER components were identified by electron microscope immunocytochemistry. The foreign ER was observed as large, reconstituted cisternae at stages in the cell cycle when the nuclear envelope was intact. Therefore, transplanted ER maintained the capacity to reconstitute in the cytoplasm of a rapidly dividing cell. In an attempt to better assess ER structure at the metaphase stage of the cell cycle, we next slowed down the division process by treating Xenopus embryos with anti-microtubule agents. Treatment with critical concentrations of colchicine, nocodazole, or vinblastine led to cleavage arrest but not to inhibition of the nuclear cycle. Following such treatment, homologous ER was observed in a vesicular form at all stages of the nuclear cycle. Heterologous ER, however, identified by immunocytochemistry in microinjected cells treated with nocodazole, displayed both vesicular and cisternal forms. We conclude that microinjected ER membranes exhibit cell-cycle-specific behavior, which is different from that of the host cell ER.     

Inhibition of endoplasmic reticulum stress improves mouse embryo development

X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development.     

Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer

Active demethylation of cytosine residues in the sperm genome before forming a functional zygotic nucleus is thought to be an important function of the oocyte cytoplasm for subsequent embryonic development in the mouse. Conversely, this event does not occur in the sheep or rabbit zygote and occurs only partially in the cow. The aim of this study was to investigate the effect of limited methylation reprogramming in the normal sheep embryo on reprogramming somatic nuclei. Sheep fibroblast somatic nuclei were partially demethylated after electrofusion with recipient sheep oocytes and undergo a stepwise passive loss of DNA methylation during early development, as determined by 5-methylcytosine immunostaining on interphase embryonic nuclei. A similar decrease takes place with in vivo-derived sheep embryos up to the eight-cell stage, although nuclear transfer embryos exhibit a consistently higher level of methylation at each stage. Between the eight-cell and blastocyst stages, DNA methylation levels in nuclear transfer embryos are comparable with those derived in vivo, but the distribution of methylated DNA is abnormal in a high proportion. By correlating DNA methylation with developmental potential at individual stages, our results suggest that somatic nuclei that do not undergo rapid reorganization of their DNA before the first mitosis fail to develop within two to three cell cycles and that the observed methylation defects in early cleavage stages more likely occur as a direct consequence of failed nuclear reorganization than in failed demethylation capacity. However, because only embryos with reorganized chromatin appear to survive the 16-cell and morula stages, failure to demethylate the trophectoderm cells of the blastocyst is likely to directly impact on developmental potential by altering programmed patterns of gene expression in extra-embryonic tissues. Thus, both remodeling of DNA and epigenetic reprogramming appear critical for development of both fertilized and nuclear transfer embryos.     

Morphogenetic function of the nuclei in the early development of the common freshwater snail, Limnaea stagnalis]

The early embryos L. stagnalis were placed in the actinomycin solution at the successive developmental stages. The permeability to actinomycin was previously increased by the pricking through egg capsules. The inactivation of nuclei by actinomycin up to the stage of 12 blastomeres resulted in the arrest of development at the 22 cell stage. The inactivation of nuclei at the subsequent development stages resulted in the developmental arrest at later stages. These data suggest that the embryonic development up to the 22 cell stage is provided by the nuclear function during oogenesis. The morphogenetic nuclear function of the embryo begins at the stage of 12 blastomeres and provides the embryonic development beyond the 22 cell stage.