Validation of primary epitheloid cell cultures isolated from bovine placental caruncles and cotyledons

Bridger, P.; Haupt, S.; Klisch, Karl; Leiser, Rudolf; Tinneberg, H.-R.; Pfarrer, Christiane

doi:10.1016/j.theriogenology.2007.05.046

Cited by 27 publications

(29 citation statements)

References 39 publications

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“…However, cotyledon samples are mostly contaminated with caruncles [57]. We also found similar cross contaminations in our samples and were not able to include them in our study.…”

Section: Discussionmentioning

confidence: 55%

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

et al. 2018

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Genomic imprinting is an epigenetic phenomenon of differential allelic expression based on parental origin. To date, 263 imprinted genes have been identified among all investigated mammalian species. However, only 21 have been described in sheep, of which 11 are annotated in the current ovine genome. Here, we aim to i) use DNA/RNA high throughput sequencing to identify new monoallelically expressed and imprinted genes in day 135 ovine fetuses and ii) determine whether maternal diet (100%, 60%, or 140% of National Research Council Total Digestible Nutrients) influences expression of imprinted genes. We also reported strategies to solve technical challenges in the data analysis pipeline. We identified 80 monoallelically expressed, 13 new putative imprinted genes, and five known imprinted genes in sheep using the 263 genes stated above as a guide. Sanger sequencing confirmed allelic expression of seven genes, CASD1, COPG2, DIRAS3, INPP5F, PLAGL1, PPP1R9A, and SLC22A18. Among the 13 putative imprinted genes, five were localized in the known sheep imprinting domains of MEST on chromosome 4, DLK1/GTL2 on chromosome 18 and KCNQ1 on chromosome 21, and three were in a novel sheep imprinted cluster on chromosome 4, known in other species as PEG10/SGCE. The expression of DIRAS3, IGF2, PHLDA2, and SLC22A18 was altered by maternal diet, albeit without allelic expression reversal. Together, our results expanded the list of sheep imprinted genes to 34 and demonstrated that while the expression levels of four imprinted genes were changed by maternal diet, the allelic expression patterns were un-changed for all imprinted genes studied.

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“…However, cotyledon samples are mostly contaminated with caruncles [57]. We also found similar cross contaminations in our samples and were not able to include them in our study.…”

Section: Discussionmentioning

confidence: 55%

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

et al. 2018

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“…F3 spheroid formation did not lead to the downregulation of vimentin in the spheroidal F3 cells either. However, the emergence and maintenance of vimentin expression has also been described in bovine caruncular epithelial cells in vitro [Bridger et al, 2007].…”

Section: Discussionmentioning

confidence: 98%

Formation of Bovine Placental Trophoblast Spheroids

Haeger¹,

Hambruch²,

Dilly³

et al. 2010

Cells Tissues Organs

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Introduction: In this study, we aimed to form spheroids with the bovine placental trophoblast cell line F3. Spheroids are 3-dimensional culture models which can be used to conduct versatile in vitro and in vivo experiments. Materials and Methods: The spheroids were generated using the hanging drop technique, 25% methocel and matrigel. The F3 spheroids were characterized morphologically by light microscopy and transmission (TEM) and scanning electron microscopy (SEM) and immunohistochemistry (ezrin, vimentin, cytokeratin, placental lactogen). The fluorescent dyes calcein and ethidium homodimer were used to determine the viability of the spheroidal F3 cells by immunofluorescence microscopy. Results: The cell line F3 only formed spheroids by the hanging drop technique when matrigel was added. The trophoblast spheroids were delimited and fully covered by extracellular matrix (light microscopy/TEM/SEM). Cells contributing to spheroids could not be discriminated from each other (light microscopy). The outer spheroidal layer consisted of cells which possessed an apical pole with microvilli that were directed to the outside (light microscopy/TEM). All of the spheroidal F3 cells expressed ezrin, vimentin and cytokeratin, but not placental lactogen. The spheroid core contained degenerating cells whilst the F3 cells of the outer rim were viable (TEM/immunofluorescence microscopy). Discussion: We have established a 3-dimensional spheroid model for the bovine placental trophoblast cell line F3. The developed culture model might prove valuable for future in vitro studies on the differentiation of bovine trophoblast cells.

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“…Low-passage (nontransformed) cell cultures were generated and cryopreserved as described previously [13,14]. Briefly, prior to tissue collection 50-ml tubes containing 200 U/ml collagenase I, 10% Hanks Balanced Salt Solution (HBSS) with Ca 2þ and Mg 2þ (Biochrom, Berlin, Germany), and 90% medium composed of Dulbecco modified Eagle medium (DMEM)/Ham F12 (PAA, Cölbe, Germany) containing 10% fetal calf serum (FCS) and 1% penicillin/ streptomycin (10 000 IU/10 000 lg/ml; Biochrom) in a 10-ml solution were prepared for disaggregation.…”

Section: Cell Culturementioning

confidence: 99%

“…If primary antibodies were derived from different hosts, double labeling was performed. Epithelial cells were characterized using antibodies directed against cytokeratin, tight junctional zonula occludens-1, vimentin, desmin, and smooth muscle actin as described previously [14]. Specificity of primary antibody binding was determined via Western blot analysis as described above.…”

Section: Immunofluorescence Microscopymentioning

confidence: 99%

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Integrin Activation in Bovine Placentomes and in Caruncular Epithelial Cells Isolated from Pregnant Cows

Bridger

Haupt

Leiser

et al. 2008

Biology of Reproduction

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In the bovine synepitheliochorial placenta, restricted trophoblast invasion requires complex interactions of integrin receptors with proteins of the extracellular matrix (ECM) and integrin receptors of neighboring cells. Activated integrins assemble to focal adhesions and are linked to the actin cytoskeleton via signaling molecules including alpha-actinin (ACTN), focal adhesion kinase (PTK2 or FAK), phosphotyrosine, and talin (TLN1). Aims of this study were to assess integrin activation and focal adhesion assembly within epithelial cells of bovine placentomes and low-passage (not transformed) placentomal caruncular epithelial cells cultured on dishes coated with ECM proteins. Immunofluorescence analysis was performed to colocalize the signaling molecules ACTN, PTK2, phosphotyrosine, and TLN1 with each other and with beta(1)-integrin (ITGB1) in placentomal cryosections throughout pregnancy and in caruncular epithelial cells in vitro. Antibody specificity was confirmed by Western blot. Cells were cultured on uncoated dishes, and the dishes were coated with fibronectin (FN), laminin (LAMA), and collagen type IV (COL4), thereby statistically assessing cell number and qualitatively assessing the expression pattern of ITGB1, phosphotyrosine, and TLN1. Results demonstrated integrin activation and focal adhesion assembly in the placentome and that low-passage caruncular epithelial cells maintain integrin-associated properties observed in vivo. Expression and/or colocalization of signaling molecules with ITGB1 confirmed, for the first time, integrin activation and participation in "outside-in" and "inside-out" signaling pathways. The prominent role of ECM, and FN in particular, in integrin signaling is supported by the in vitro enhancement of proliferation and focal adhesion expression. Thus, this in vitro model provides excellent potential for further mechanistic studies designed to elucidate feto-maternal interactions in the bovine placentome.

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Validation of primary epitheloid cell cultures isolated from bovine placental caruncles and cotyledons

Cited by 27 publications

References 39 publications

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

Formation of Bovine Placental Trophoblast Spheroids

Integrin Activation in Bovine Placentomes and in Caruncular Epithelial Cells Isolated from Pregnant Cows

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