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  • Development of mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media. 16876344

    This study investigated the effects of the in vitro co-culture of mouse embryos with non-polarized or polarized uterine epithelial cells, using sequential culture media, on their development to blastocysts, blastocyst quality (blastocyst diameter and cell number), apoptosis, Bcl-2 and Bax gene expression. There were three treatments, all of which used sequential culture media. The treatments were no co-culture (control), non-polarized or polarized epithelial cell monolayer co-culture in 24-well tissue culture plates. Mouse uterine epithelial cells were isolated enzymatically and were seeded either on the surface of the culture plate (non-polarized monolayer) or on a Millipore filter insert coated with extra-cellular matrix extract (polarized monolayer) that was then placed in the culture plate. Two-cell mouse embryos were cultured in G-1 ver3 medium to the eight-cell stage when they were randomly assigned to the treatments. The culture medium was G-2 ver3 during the treatment phase of the study. Significances of differences were evaluated by the one-way analysis of variance for continuous data. The epithelial cells cultured on Millipore filters became polarized and their morphology compared favorably with those cultured on the surface of the culture plate and in vivo uterine epithelial cells. After 96 h on the treatments, the polarized monolayer had supported the development of significantly more hatched blastocysts (80.0%; P<0.05) than the non-polarized monolayer (63.4%) or the control (61.4%) culture treatments. Co-culture resulted in the production of blastocysts with significantly more cells (non-polarized monolayer 56.7+/-2.1, polarized monolayer 61.9+/-2.1) than the control culture (42.8+/-2.6; P<0.05) but the diameter and shape of the blastocysts were not significantly different. The proportion of blastocysts with apoptotic blastomere was higher for the control culture (94.4%) than for the non-polarized (68.2%) or polarized (66.7%) co-culture systems (P<0.05). Moreover, the apoptotic index was significantly higher in control blastocysts (5.6+/-0.9; P<0.05) than in non-polarized (1.7+/-0.3) or polarized (1.5+/-0.3) co-culture. In the control, Bax mRNA was strongly expressed when compared to co-culture treatments (P<0.05), whereas, the relative abundance of Bcl-2 mRNA to the beta-tubulin was lower than co-culture treatments (P<0.05). It is concluded that a co-culture system involving polarized uterine epithelial cells and sequential culture media is a promising method of producing mouse embryos.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1636

  • The effects of dexamethasone on insulin release and biosynthesis are dependent on the dose and duration of treatment. 11269888

    Complex results concerning the effect of glucocorticoids on insulin secretion have been reported. The aim of this study is to clarify the direct effects of glucocorticoids on pancreatic islets and to determine whether the effect of glucocorticoids on insulin biosynthesis or release is dependent on the dose and duration of treatment with glucocorticoid. Studies on insulin secretion and biosynthesis were performed with different concentrations (0, 1, 10, 100 nmol/l) and durations (1 and 6 h) of treatment with dexamethasone (dexa) in rat pancreatic islets. (1) One nmol/l dexa had no inhibitory effect on insulin secretion and biosynthesis. Ten and 100 nmol/l had an inhibitory effect on insulin secretion, which was mainly due to suppression of the first phase of insulin secretion. (2) Insulin content was significantly increased regardless of the concentration in 1-h treated islets. However, insulin content was markedly diminished with 100 nmol/l dexa in 6-h treated islets. (3) The preproinsulin mRNA expression of 6-h treated islets was suppressed in a dose-dependent manner. Our data revealed that, in the condition of short-term and low-dose glucocorticoid exposure, insulin secretion and biosynthesis are not affected. The secretory process of insulin seems to be the initial step of the inhibitory action of glucocorticoid. Both insulin release and biosynthesis are inhibited by chronic exposure to high dose dexamethasone. It can be concluded that glucocorticoid might be involved in the multisteps of insulin release and biosynthesis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    RI-13K
    Nombre del producto:
    Rat Insulin RIA

  • Epithelial to mesenchymal transition in gingival overgrowth. 20489142

    Epithelial to mesenchymal transition (EMT) occurs normally in development. In pathology, EMT drives cancer and fibrosis. Medication with phenytoin, nifedipine, and cyclosporine-A often causes gingival overgrowth. Based partly on the histopathology of gingival overgrowth, the present study investigates the hypothesis that EMT could contribute to its development. We found that phenytoin-induced human gingival overgrowth tissues, the most fibrotic drug-induced variety, contain diminished epithelial E-cadherin expression, whereas fibroblast-specific protein-1 (FSP-1) and alphavbeta6 integrin levels are up-regulated. In connective tissue stroma, fibronectin and alternatively spliced fibronectin extra type III domain A (FN-ED-A) levels are increased in overgrowth lesions. Transforming growth factor (TGF)-beta1 treatment of primary human gingival epithelial cells cultured in transwell plates resulted in inhibited barrier function as determined by reduced electrical resistance, paracellular permeability assays, and cell surface E-cadherin expression. Moreover, TGF-beta1 altered the expression of other markers of EMT determined at the mRNA and protein levels: E-cadherin decreased, whereas SLUG, fibronectin, matrix metalloproteinase (MMP)2, MMP9, and MMP13 increased. Nifedipine- and cyclosporine A-induced gingival overgrowth tissues similarly contain diminished E-cadherin and elevated levels of FSP-1 and fibronectin, but normal levels of alphavbeta6 integrin. In summary, data in vitro support that human gingival epithelial cells undergo functional and gene expression changes consistent with EMT in response to TGF-beta1, and in vivo studies show that important EMT markers occur in clinical gingival overgrowth tissues. These findings support the hypothesis that EMT likely occurs in drug-induced gingival overgrowth.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB2074Z
    Nombre del producto:
    Anti-Integrin αVβ6 Antibody, clone E7P6, azide free

  • Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. 19429786

    Mammalian oocytes are arrested in meiotic prophase by an inhibitory signal from the surrounding somatic cells in the ovarian follicle. In response to luteinizing hormone (LH), which binds to receptors on the somatic cells, the oocyte proceeds to second metaphase, where it can be fertilized. Here we investigate how the somatic cells regulate the prophase-to-metaphase transition in the oocyte, and show that the inhibitory signal from the somatic cells is cGMP. Using FRET-based cyclic nucleotide sensors in follicle-enclosed mouse oocytes, we find that cGMP passes through gap junctions into the oocyte, where it inhibits the hydrolysis of cAMP by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells (from approximately 2 microM to approximately 80 nM at 1 hour after LH stimulation) and by closing gap junctions between the somatic cells. The resulting decrease in oocyte cGMP (from approximately 1 microM to approximately 40 nM) relieves the inhibition of PDE3A, increasing its activity by approximately 5-fold. This causes a decrease in oocyte cAMP (from approximately 700 nM to approximately 140 nM), leading to the resumption of meiosis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    70-500