Effects of retroviral oncogenes on myogenesis (original) (raw)
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.
Endocrine Journal, 1998
The transforming growth factor f3 (TGFI3) family is known to control cell migration, growth, differentiation, function and regulation of extracellular matrix, all of which are required for the process of implantation. Expression of TGF/3 by the conceptus and endometrium was studied during the period of implantation in the ewe. A total of thirty-four ewes were hysterectomized on day 12, or 20 of pregnancy (day 0 = day of estrus). Conceptus (200 mg wet weight) and endometrial (300 mg wet weight) tissues were cultured in vitro in 7 and 10 ml Eagle's minimal essential medium, respectively. The culture media were subjected to a bioassay to determine concentrations of TGF/3. Conceptus culture media (CCM) were also analyzed for contents of ovine interferon-tau (oIFNi), low molecular weight acidic protein, produced by the trophectoderm between days 8 and 21 of pregnancy. Whole uteri including conceptus(es) and conceptuses (day 16) only were fixed and subjected to immunohistochemical and in situ hybridization studies. Levels of oIFNr produced by conceptuses were the highest on day 16 at 4.4 µg/ml. Concentrations of TGFJI in day 12, and 20 CCM were 38 ± 19, 102 ± 56, 862 ± 152, 728 ± 191 and 336 ± 106 pg/ml, respectively, and approximately 90% of TGFI3 activity in CCM was due to TGFfJ1 whereas less than 10% was due to TGFI33 based on neutralization with TGFI3 subtype-specific antibodies. Immunohistochemical studies revealed that day 16 conceptuses displayed major staining for TGFfI1, no f32 staining and minor staining for f3. In situ hybridization studies also revealed that day 16 trophectoderm possessed most TGF/31 mRNA while day 14 trophectoderm and day 20 chorion/amnion displayed weaker staining for TGFI31 mRNA. TGFI3 in day 12, and day 20 endometrial culture media was 156 ± 37,129 ± 33, 49 ± 22, 62 ± 23 and 179 ± 40 pg/ml, respectively, and approximately 65% and 35% of the activities were due to TGFfI1 and f2, respectively. These results indicate that TGFf3 production by the conceptus coincides with the time when oIFNr production starts to decline. These observations support the postulate that TGF/3 may play an important role in implantation in the ovine species.
236 Localization of Interferon-Tau in Bovine Embryos and Cumulus Cells by Confocal Microscopy
Reproduction Fertility and Development, 2003
The oviduct epithelium undergoes marked morphological and functional changes during the estrous cycle. It has been shown that a dramatic change in the frequencies of ciliated and non-ciliated cells occurs during the estrous cycle. At estrus the epithelium consists of secretory and ciliated cells and at diestrus mainly of ciliated cells. The oviduct provides the microenvironment for sperm capacitation, fertilization, and early cleavage-stage embryonic development. At the molecular level, only a few genes or proteins are known that change during the estrous cycle and which may be important for fertility, so as the bovine oviduct-specific glycoprotein, the major secretory protein in the oviduct. Therefore, we studied systematically the changes in gene expression in bovine ipsilateral oviduct epithelial cells at estrus and diestrus. To identify differentially expressed genes, a combination of subtracted cDNA libraries and cDNA array hybridization was used. Two subtracted libraries were produced to enrich cDNAs of upregulated genes at estrus and at diestrus. A total of 1536 cDNA clones of each library were analyzed with radioactively (33-P) labeled probes generated from the oviduct epithelial cells of six Simmental heifers, three of them slaughtered at Day 0 (estrus) and three at Day 12 after standing heat (diestrus). After normalization of the raw data and statistical analysis, all cDNAs showing significant differences in their expression levels at estrus compared to diestrus were sequenced. Sequencing revealed 84 different cDNAs; 42 of them matched bovine genes or their human/mouse homologs with known functions, and 42 matched genes without a known function. Half of the genes (n = 42) were expressed at a higher level at estrus; for the other (n = 42) expression levels were higher at diestrus. The regulated genes or their products represented a variety of functional classes, such as genes of the secretory pathway, genes involved in transcription regulation, cell-surface proteins, cell-cell interaction proteins, secreted proteins, members of signal transduction pathways, immune-related proteins, and some enzymes. The identification of genes differentially regulated in ipsilateral oviduct epithelial cells at estrus v. diestrus is the first step of a systematic analysis of differential gene expression during the estrous cycle. Further studies will follow, focusing on different compartments of the bovine oviduct and additional times of the estrous cycle.
Journal of Virology, 2010
The sheep genome contains multiple copies of endogenous betaretroviruses highly related to the exogenous and oncogenic jaagsiekte sheep retrovirus (JSRV). The endogenous JSRVs (enJSRVs) are abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus trophectoderm and are essential for conceptus elongation and trophectoderm growth and development. Of note, enJSRVs are present in sheep and goats but not cattle. At least 5 of the 27 enJSRV loci cloned to date possess an intact genomic organization and are able to produce viral particles in vitro. In this study, we found that enJSRVs form viral particles that are released into the uterine lumen of sheep. In order to test the infectious potential of enJSRV particles in the uterus, we transferred bovine blastocysts into synchronized ovine recipients and allowed them to develop for 13 days. Analysis of microdissected trophectoderm of the bovine conceptuses revealed the presence of enJSRV RNA and, in some cases, DNA. Interestingly, we found that RNAs belonging to only the most recently integrated enJSRV loci were packaged into viral particles and transmitted to the trophectoderm. Collectively, these results support the hypothesis that intact enJSRV loci expressed in the uterine endometrial epithelia are shed into the uterine lumen and could potentially transduce the conceptus trophectoderm. The essential role played by enJSRVs in sheep reproductive biology could also be played by endometrium-derived viral particles that influence development and differentiation of the trophectoderm.
Biology of Reproduction, 1999
Trophoblast-derived interferon tau (IFN) acts on the endometrium to increase secretion of several proteins during the pregnancy recognition period in ruminants. One of these is a 70-kDa acidic protein that has not been identified. Our hypothesis was that the 70-kDa acidic protein is osteopontin (OPN). OPN is an acidic glycoprotein that fragments upon freezing and thawing or treatment with proteases including thrombin. OPN contains a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins to promote cell-cell attachment and cell spreading. Using antisera to recombinant human OPN, both 70-kDa and 45-kDa proteins were identified in uterine flushings from pregnant ewes by Western blotting. A clone containing the entire ovine OPN cDNA coding sequence was isolated by screening a Day 15 pregnant ovine endometrial cDNA library with a partial ovine OPN cDNA. In pregnant ewes, steady-state levels of OPN endometrial mRNA increased (P Ͻ 0.01) after Day 17. In both cyclic and pregnant ewes, in situ hybridization analysis showed that OPN mRNA was localized on unidentified immune cells within the stratum compactum of the endometrium. In pregnant ewes, OPN mRNA was also expressed by the glandular epithelium. Results suggest that progesterone and/or IFN induce expression and secretion of OPN by uterine glands during the periimplantation period and that OPN may induce adhesion between luminal epithelium and trophectoderm to facilitate superficial implantation.