Effects of endosulfan on survival and development of Bombina orientalis (Boulenger) embryos.

The developmental toxicity of endosulfan was examined in the anuran Bombina orientalis embryos. Survival rates of embryos following 50 microM endosulfan treatment was significantly lower than vehicle control at 96 h onward. When the embryos develop to the tail fin circulation stage, embryonic survival was significantly decreased by 10 microM endosulfan treatment. Surviving embryos showed various developmental abnormalities including tail dysplasia at 50 microM. By hampering the embryonic development endosulfan may cause the decline in the natural populations of this frog species breeding on farmland and in the surrounding aquatic environment.

Heparin-binding epidermal growth factor (HB-EGF) may improve embryonic development and implantation by increasing vitronectin receptor (integrin alphanubeta3) expression in peri-implantation mouse embryos.

PURPOSE: This study investigated the effects of HB-EGF on expression of integrin alphanubeta3 and implantation of embryos. METHODS: Two-cell embryos were recovered and cultured with or without 10 ng/mL HB-EGF for 96h. Expression of integrin alphanubeta3 in cultured embryos was examined by real time-RT-PCR and immunofluorescence analysis; embryos were cultured with or without HB-EGF, then transferred into the uteri of pseudo-pregnant female mice in order to analyze their implantation rate. RESULTS: HB-EGF improved embryonic hatching and outgrowth during extended culture, and up-regulated expression of integrin alphanubeta3 in both the preimplantation embryo and outgrowing blastocyst. Also, integrin alphanubeta3 subunits were localized at the pericellular borders and cell-cell contact areas. The number of successful implantation sites of transferred HB-EGF-treated embryos in the uterus was increased when compared to number of implantation sites with non-treated controls. CONCLUSIONS: HB-EGF may improve implantation by accelerating expression of integrin alphanubeta3 in peri-implantation mouse embryos.

Comparative characteristics of three human embryonic stem cell lines.

Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.

Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition.

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.

Available human feeder cells for the maintenance of human embryonic stem cells.

Mouse embryonic fibroblasts (MEFs) have been previously used as feeder cells to support the growth of human embryonic stem cells (hESCs). In this study, human adult uterine endometrial cells (hUECs), human adult breast parenchymal cells (hBPCs) and embryonic fibroblasts (hEFs) were tested as feeder cells for supporting the growth of hESCs to prevent the possibility of contamination from animal feeder cells. Cultured hUECs, hBPCs and hEFs were mitotically inactivated and then plated. hESCs (Miz-hES1, NIH registered) initially established on mouse feeder layers were transferred onto each human feeder layer and split every 5 days. The morphology, expression of specific markers and differentiation capacity of hESCs adapted on each human feeder layer were examined. On hUEC, hBPC and hEF feeder layers, hESCs proliferated for more than 90, 50 and 80 passages respectively. Human feeder-based hESCs were positive for stage-specific embryonic antigen (SSEA)-3 and -4, and Apase; they also showed similar differentiation capacity to MEF-based hESCs, as assessed by the formation of teratomas and expression of tissue-specific markers. However, hESCs cultured on hUEC and hEF feeders were slightly thinner and flatter than MEF- or hBPC-based hESCs. Our results suggest that, like MEF feeder layers, human feeder layers can support the proliferation of hESCs without differentiation. Human feeder cells have the advantage of supporting more passages than when MEFs are used as feeder cells, because hESCs can be uniformly maintained in the undifferentiated stage until they pass through senescence. hESCs established and/or maintained under stable xeno-free culture conditions will be helpful to cell-based therapy.

CDNA cloning and expression of ATP synthase subunit b from the fire-bellied frog Bombina orientalis (Anura: Discoglossidae).

We cloned the Bombina orientalis adenosine triphosphate (ATP) synthase subunit b gene from a B. orientalis oviduct cDNA library. The transcript was 997 bp long and encoded 250 amino acid residues. It showed high similarity to amphibian (84-85%), mammalian (56-62%) and invertebrate (46-50%) sequences. In phylogenetic analyses, it clustered with other amphibian sequences. This gene was highly expressed in brain, intestine and oviduct but not in muscle and liver. In this paper, we report the basic characteristics of B. orientalis ATP synthase subunit b gene.

Estrogen regulates the expression of the small proline-rich 2 gene family in the mouse uterus.

Small proline-rich (SPRR) proteins are structural components of the cornified cell envelope (CE), a specialized structure beneath the plasma membrane of stratified squamous epithelia. They are divided into four families, of which SPRR2 is the most complex consisting of 11 members (2a-2k) in the mouse. To assess the possible influence of estrogen on expression of the SPRR2 family in the uterus, we examined the effect of 17b-estradiol (E2) on SPRR2 mRNA levels on ovariectomized (OVX) adult mice. We employed a combination of laser capture microdissection (LCM) and semiquantitative RT-PCR to examine expression in particular uterine cell types - luminal epithelia, and stromal and muscle cells. We also used quantitative real-time PCR to measure levels of the mRNA of several SPRR2 proteins in the mouse uterus over the estrous cycle and during early pregnancy. Expression of SPRR2a, 2b, 2c, 2d, 2e, 2f and 2g mRNA was increased by estrogen treatment. SPRR2a, 2b, 2d and 2e were highly expressed on day 1 and 2 of pregnancy, but decreased markedly by days 3-6. Interestingly, several members of the SPRR2 family were preferentially up-regulated at implantation sites compared to inter-implantation sites around day 4 of pregnancy. They were abundant during proestrus and estrus but declined rapidly during metestrus. These results indicate that estrogen is a key regulator of the expression of the SPRR2 family in the mouse uterus during the estrous cycle and early pregnancy. In addition, they suggest that some members of the family play an important role in uterine processes such as the estrous cycle, early pregnancy and implantation.

Role of occludin, a tight junction protein, in blastocoel formation, and in the paracellular permeability and differentiation of trophectoderm in preimplantation mouse embryos.

Tight junctions (TJ) are critical for blastocoel formation in mammalian embryos. The present study aimed to examine the role of tight junctions in the differentiation of the trophectoderm (TE), and in the pluripotency of blastomeres, as well as in the formation and integrity of the blastocoel. We examined the effect of occludin antibody on blastocoel formation, blastocyst permeability, and expression of H19 and Oct-4, markers of TE differentiation and blastomere pluripotency, respectively. Eight-cell mouse embryos and morulae were cultured in the presence or absence of occludin antibody for 31 h. Occludin antibody inhibited blastocoel formation and increased permeability of the TE of nascent and expanding blastocysts to FITC-dextran (4 kDa), a permeability tracer. At the same time Oct-4 expression increased while expression of H19 became barely detectable. These observations indicate that occludin is involved in establishing the blastocoel, as well as in maintaining its impermeability, and that the development of tight junction is critical for TE formation in mouse embryos.

Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos.

We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus lambdaGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced approximately 7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFkappaB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 3'-UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms' c-fos genes. We propose its potential use in ecotoxicology.

Analysis of estrogen-regulated genes in mouse uterus using cDNA microarray and laser capture microdissection.

The steroid hormone, estrogen, plays an important role in various physiological events which are mediated via its nuclear estrogen receptors, ERalpha and ERbeta. However, the molecular mechanisms that are regulated by estrogen in the uterus remain largely unknown. To identify genes that are regulated by estrogen, the ovariectomized mouse uterus was exposed to 17beta-estradiol (E2) for 6 h and 12 h, and the data were analyzed by cDNA microarray. The present study confirms previous findings and identifies several genes with expressions not previously known to be influenced by estrogen. These genes include small proline-rich protein 2A, receptor-activity-modifying protein 3, inhibitor of DNA binding-1, eukaryotic translation initiation factor 2, cystatin B, decorin, secreted frizzled-related protein 2, integral membrane protein 2B and chemokine ligand 12. The expression patterns of several selected genes identified by the microarray analysis were confirmed by RT-PCR. In addition, laser capture microdissection (LCM) was conducted to determine the expression of selected genes in specific uterine cell types. Analysis of early and late responsive genes using LCM and cDNA microarray not only suggests direct and indirect effects of E2 on uterine physiological events, but also demonstrates differential regulation of E2 in specific uterine cell types. These results provide a basic background on global gene alterations or genetic pathways in the uterus during the estrous cycle and the implantation period.