Anti-Human KRT8 (Phospho-Ser74) polyclonal antibody

Trophectoderm cells are the foremost embryonic cells to differentiate with prospective stem-cell properties. In the current study, we aimed at improving the current approach for trophoblast culture by using granulosa cells as feeders. Porcine granulosa cells (PGCs) compared to the conventional mouse embryonic fibroblasts (MEFs) were used to grow trophectoderm cells from hatched bovine blastocysts. Isolated trophectoderm cells were monitored and displayed characteristic epithelial/cuboidal morphology. The isolated trophectoderm cells expressed mRNA of homeobox protein (CDX2), cytokeratin-8 (KRT8), and interferon tau (IFNT). The expression level was higher on PGCs compared to MEFs throughout the study. In addition, primary trophectoderm cell colonies grew faster on PGCs, with a doubling time of approximately 48 hrs, compared to MEFs. PGCs feeders produced a fair amount of 17 beta-estradiol and progesterone. We speculated that the supplementation of sex steroids and still-unknown factors during the trophoblasts coculture on PGCs have helped to have better trophectoderm cell's growth than on MEFs. This is the first time to use PGCs as feeders to culture trophectoderm cells and it proved superior to MEFs. We propose PGCs as alternative feeders for long-term culture of bovine trophectoderm cells. This model will potentially benefit studies on the early trophoblast and embryonic development in bovines.

Studies on the effects of various factors, including xenobiotics, on the maternal-fetal connections in the placenta are restricted by the lack of a simple and inexpensive research model. We used placentomes collected at a slaughterhouse to in vitro study the bovine sections contained integral maternal-fetal connections. The placentomes from cows (n = 4/experiment, 120-150 days post coitum) were cut using a razor blade into 60-80 mg sections and incubated in either DMEM/Ham's F-12 or M-199 supplemented with FCS (2%, 5% or 10%), amniotic fluid (AF or inactive AF, 10% or 20%) or both. The sections (n = 4/supplement) were incubated for 24 or 48 h in a water bath at 37.5 degrees C in an atmosphere of 5% CO2 and 95% O-2. The structure and secretory activity of placentome sections were maintained when incubated in DMEM/Ham's F-12 with 2% FCS and 10% AF. M-199 was less acidified than DMEM/Ham's F-12 during incubation, and thus, this medium was better able to maintain the integrity of the placenta and the secretion of estradiol, progesterone and oxytocin for 48 h. Moreover, we detected a decrease in the expression of placenta-specific 1 (PLAC1) mRNA (an indicator of trophoblast proliferation) and an increase in the levels of keratin 8 (KRT8; a marker of normal placental barrier function) and hypoxia induced factor in (HIF1e; a marker of hypoxia) mRNA. These results indicate the presence of adaptation and repair mechanisms and confirm the biological activity of the placentome sections. We propose the use of placentome sections as an in vitro model to study maternal-fetal connections in cows. (C) 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved.