Effects of retroviral oncogenes on myogenesis (original) (raw)
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Paracrine activities of porcine trophoblastic interferons
Reproduction, 1994
Interferon-\g=g\IFN-\g=g\ and a type I IFN (spl IFN) are transiently coexpressed by trophoblastic cells of pig conceptuses at implantation between day 12 and day 20 of gestation. The local effects of these trophoblastic IFNs were examined on endometrial cells and on trophoblast by measuring antiviral activity and the induction of (2\m=' \,5\m=' \)-oligoadenylatesynthetase activity. Trophoblastic vesicles were shown to be susceptible to infection by vesicular stomatitis virus and transmissible gastroenteritis virus. Vesicular stomatitis virus multiplied by about 1000 times in trophoblastic vesicles, and endogenous trophoblastic IFNs or exogenous recombinant IFN-\g=g\ or spl IFN had no effect on virus production. No (2\m=' \,5\m=' \)-oligoadenylatesynthetase activity could be measured on the trophoblast, even after treatment with IFN-\g=g\ or spl IFN. These results clearly show that trophoblastic IFNs cannot induce antiviral resistance or (2\m=' \,5\m=' \)-oligoadenylatesynthetase activity in the trophoblast, suggesting that these IFNs have no autocrine function. Endometrial epithelial and stromal cells in primary cultures displayed distinct sensitivity to the antiviral effect of IFN-\g=g\ and spl IFN. Stromal fibroblasts were highly sensitive to spl IFN but weakly sensitive to IFN-\g=g\; epithelial cells were sensitive to both IFNs. The same sensitivity pattern was obtained when measuring the (2\m=' \,5\m=' \)-oligoadenylatesynthetase activity. Flushing fluid, containing IFN-\g=g\ and type I IFN, was a potent inducer of antiviral effect and (2\m=' \,5\m=' \)-oligoadenylatesynthetase activity. It is therefore postulated that the endometrial epithelium is the most likely target of trophoblastic IFNs. It is possible that these IFNs play a role in the viral protection of conceptuses.
Placenta, 1993
led to a high interferon (IFN) production. The magnitude of the production was dependent on the gestational age of the trophoblast, type of inducer and the stage of differentiation of the trophoblast. The data obtained indicated that the first trimester trophoblast cultures produced five to sixfold more IFN than the third trimester trophoblast on per cell basis whereas syncytiotrophoblast at term produced twice as much IFN than the mononuclear term trophoblast when stimulated with the viruses.
Expression of the protooncogene fos (c-fos) by preimplantation blastocysts of the pig
Blastocyst material was obtained from 25 pigs during the period 10 to 33 days post coitum, and fixed thin sections of tissue were hybridized in situ to sense and antisense fos RNA probes synthesized using the expression vector Bluescribe M13 + . Indirect immunofluorescence using antisera to a synthetic peptide fragment of c-fos was used to confirm the tissue distribution of oncogene-encoded proteins, which were shown by immunoprecipitation to have MrS of 55 000 and 40 000, which are the known MrS of the fos gene product and an associated nucleoprotein, respectively. Northern and slot blots were used to assess the distribution of c-fos mRNA and the size of the fos transcript was found to be 2-3kbases.
Expression of proto-oncogenes in bovine preimplantation blastocysts
Anatomy and Embryology, 2000
Proto-oncogenes are involved in the regulation of gene expression, for example after ligand binding to growth factor receptors. Expression of the proto-oncogenes c-fos, c-jun, c-ha-ras and c-myc was studied in in vivo grown and in vitro cultured bovine preimplantation blastocysts employing RT-PCR, ribonuclease protection assay and immunohistochemistry. Thirteen-and 14-dayold preimplantation blastocysts, i.e. stages before and during trophoblast elongation, were used. In in vivogrown blastocysts c-fos, c-jun and c-ha-ras transcripts as well as c-Fos, c-Jun and c-Myc proteins were detected in all stages studied. Cultured blastocysts were treated with 10 nM epidermal growth factor and 10 nM transforming growth factor-alpha simultaneously. Epidermal growth factor and transforming growth factor-alpha treatment induced c-fos mRNA and c-Myc protein expression. The induction of downstream targets of the epidermal growth factor receptor by epidermal growth factor and transforming growth factor-alpha indicates a functional epidermal growth factor signal transduction pathway in elongating bovine blastocysts.
Secretion of biologically active interferon tau by in vitro-derived bovine trophoblastic tissue
Biology of Reproduction, 1995
Secretion of interferon tau (IFNt) by trophoblastic tissue has been shown to be the first embryonic signal for pregnancy recognition. Therefore we tried to derive biologically active trophoblastic tissue by in vitro techniques. Since conventional in vitro conditions for bovine embryo development were not sufficient for long-term culture, we tested more complex culture conditions, including M6nezo B2 or Buffalo rat liver (BRL) cell-conditioned medium, for their ability to support proliferation and IFNt secretion by in vitro-derived trophoblastic tissue. IFNt activity was determined by using a biological assay based on the inhibition of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney cells. When cultures of individual hatched blastocysts were started in 60-pl drops of BRL cell-conditioned medium, mean IFNt secretion (antiviral units/ml/48 h) corresponded to 1200 on Day 11 and to 5000 on Day 13 (p < 0.01). To characterize trophoblast cell-specific secretions, the inner cell mass was removed from all embryos by microsurgery on Day 13. IFNT secretion by trophoblastic tissue increased to mean levels of > 105 antiviral units/ml/48 h on Day 23, stayed high for about 1 wk, and then slowly declined to levels below 10 3 antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFNc was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFNt. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFNt.
Molecular Reproduction and Development, 2008
The expression of interferon-tau (IFN-t) is essential for bovine embryo survival in the uterus. An evaluation of IFN-t production from somatic cell nuclear transfer (NT)-embryo-derived primary trophectoderm cultures in comparison to trophectoderm cultured from parthenogenote (P) and in vitro matured, fertilized, and cultured (IVP) bovine embryos was performed. In Experiment 1, the success/ failure ratio for primary trophectoderm colony formation was similar for IVP and NT blastocysts [IVP ¼ 155/ 29 (84%); NT 104/25 (81%)], but was decreased (P ¼ .05) for P blastocysts [54/43 (56%)]. Most trophectoderm colonies reached diameters of at least 1 cm within 3-4 weeks, and at this time, 72 hr conditioned cell culture medium was measured for IFN-t concentration by antiviral activity assay. The amount of IFN-t produced by IVP-outgrowths [4311 IU/mL (n ¼ 155)] was greater (P < .05) than that from NT-[626 IU/mL (n ¼ 104)] and P -[1595 IU/ mL (n ¼ 54)] derived trophectoderm. Differential expression of IFN-t was confirmed by immunoblotting. In Experiment 2, colony formation was again similar for IVP and NT blastocysts [IVP ¼ 70/5 (93%); NT 67/ 1 (99%)] and less (P < .05) for P blastocysts [65/ 27 (70%)]. Analysis of trophectoderm colony size after 23 days in culture showed a similar relationship with P-derived colonies being significantly smaller in comparison to IVP and NT colonies. A differential expression of IFN-t was also observed again, but this time as measured over time in culture. Maximal IFN-t production was found at day-14 of primary culture and diminished to a minimum by the 23rd day. Mol.
Endocrinology, 2006
Uterine-derived factors are essential for conceptus development and secretion of the maternal recognition-of-pregnancy factor, interferon-(IFNT), in ruminant species. The objectives of this study were to determine whether fibroblast growth factor-2 (FGF-2) is expressed in the bovine uterus during early pregnancy in cattle and to determine whether FGF-2 supplementation affects IFNT mRNA and protein abundance in bovine trophectoderm. FGF-2 mRNA was present in endometrium throughout the estrous cycle and was localized to the luminal and glandular endometrial epithelium at d 17-18 after estrus in pregnant and nonpregnant cows. Immunoreactive FGF-2 protein was detected within the endometrium and in the uterine lumen at d 17-18 after estrus, and concentrations did not differ based on pregnancy status. In a bovine trophectoderm cell line, CT-1, supplementation of medium with at least 1 ng/ml FGF-2 increased the incorporation of [ 3 H]thymidine into DNA. Similarly, IFNT secretion from CT-1 cells increased after FGF-2 supplementation (1-100 ng/ml) for 72 h. Abundance of IFNT mRNA in CT-1 cells increased after 24 h exposure to 1, 10, or 100 ng/ml FGF-2. In bovine blastocysts, FGF-2 supplementation did not affect cell number after 72 h of culture but did stimulate IFNT protein concentrations in conditioned medium. In summary, FGF-2 is present in the uterine lumen during early pregnancy and increases IFNT mRNA and protein abundance in trophectoderm. The magnitude by which FGF-2 stimulates IFNT expression suggests that this uterine-derived factor plays an active role in regulating the establishment and maintenance of pregnancy in ruminants. (Endocrinology 147: 3571-3579, 2006)
Polarized Porcine Trophoblastic Cell Lines Spontaneously Secrete Interferon-Gamma
Placenta, 2002
Following the demonstration of high levels of interferon-gamma (IFN-) synthesis by the pig trophectoderm around implantation, an adequate experimental system was established so as to study the regulation of endogenous IFN-gene. Several stable cell lines have been isolated from Day 14 and 15 pig trophoblast, that could withstand indefinite growth when cultured on collagen-coated supports. Since no feeder cells were used for culture, and cell lines could be successfully cloned, these lines represent the first pure porcine trophoblastic (TB) cell lines isolated so far. These cells were shown to exhibit most differentiation markers of epithelial cells and in addition to express the porcine trophectoderm-specific antigen SN1-38. The TB cell line A (TBA) was characterized in more details upon culture on microporous filters, where a high apico-basal polarity could be obtained. In those conditions, a transient and acute interferon-gamma secretion was detected only in the apical medium. Moreover, the two trophoblast-specific mRNA of 1.3 and 1.4 kb that have been described in the blastocyst collected in vivo were shown to be synchronously transcribed. This polarized synthesis and secretion of IFN-was correlated with the acquisition of maximal levels of electric resistance of TBA monolayers on filters, and was not observed on the same cells cultured on plastic. This differentiation was maintained over 30 passages, demonstrating that the induction of IFN-secretion by pig conceptus is not maternally controlled. This cellular model will be of prime importance for studies on the developmental regulation of the IFN-gene, and more generally for studies on relationship between secretion and polarity in transporting epithelia.