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Keiichiro Kizaki, Yoshiaki Tanaka, Yasunori Suzuki, Tadashi Furusawa, Koichi Ushizawa, Misa Hosoe, Toru Takahashi, Kazuyoshi Hashizume, Isolation of Binucleate Trophoblastic Cells from Bovine Placenta., Biology of Reproduction, Volume 81, Issue Suppl_1, 1 July 2009, Page 417, https://doi.org/10.1093/biolreprod/81.s1.417
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The differentiation of the trophoblast lineage in mammals is recognized from the appearance of phenotypic cells distinct in morphology and function. Bovine trophoblastic binucleate cells (BNCs) are large cells with two nuclei, differentiate from the mononuclear trophoblastic cells (MNCs) at the beginning of implantation and throughout pregnancy, and represent about 20% of trophoblastic cells. The BNCs produce hormones such as placental lactogens (CSH1), prolactin-related proteins (PRPs) and pregnancy-associated glycoproteins (PAGs), and play an important role in fetal development and maternal adaptation to pregnancy. Although BNCs are known to produce these specific molecules, the characteristics and differentiation mechanisms remain to be investigated. Therefore, we try to isolate the BNCs and MNCs from bovine placenta, and characterize the both cell types. The placentomes were collected from cows which were ranged from middle to late gestation. In brief, fetal cotyledonary tissue was separated manually from placentomes, washed and cut into small pieces. The pieces of tissue were treated with collagenase, and the digested tissue was filtered through stainless steel meshes to remove undigested tissue. The filtrate was washed with the medium and layered on discontinuous Percoll gradients with isopycnic densities at 1.03–1.07 g/ml, and centrifuged. The interface between 1.04 and 1.06 g/ml were collected, and the cell fractions were incubated with Hoechist 33342 and propidium iodide. Further cell fractionation was performed using high performance cell-sorting system EPICS ALTRA MultiCOMP. The nuclear phase, cell size and the inner complexity of the cells were used as separation indicator. In flow cytometric analysis, the analyzed trophoblastic cells exhibit approximately 80% diploid and 20% tetraploid (or hexaploid) cells, and two different sizes of tetraploid cells in the fraction were observed. These cells were sorted and the mRNA expression of trophoblastic cell specific molecules were analyzed by quantitative RT-PCR. The mRNA of CSH1, PRP1 and PAG1 were highly expressed in tetraploid and hexaploid cells compared with diploid cells. However, the transcriptional factors including CDX2, ETS2 and POU5F1 mRNA levels were not different in each cell types. The present results imply that BNCs and multinucleated throphoblastic cells were highly contained in the tetraploid and hexaploid cell fractions. These results further suggest that the methodology using high performance cell-sorting system following discontinuous density gradients centrifugation in the present study may supply an appropriate technique for isolation of BNCs from bovine placenta, and allow the investigation of trophoblastic cell differentiation and function. Supported by a Research Project for Utilizing Advanced Technologies (05-1770) grant from the MAFF of Japan; grants (Kiban-kenkyu B 20380159, Kiban-kenkyu C 19580335) from Ministry of Education, Culture, Sport, Science.
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