The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632

Abstract

Trophoblasts (TR) are specialized cells of the placenta and play an important role in embryo implantation. The in vitro culture of trophoblasts provided an important tool to investigate the mechanisms of implantation. In the present study, porcine trophoblast cells were derived from pig in vitro fertilized (IVF) and parthenogenetically activated (PA) blastocysts via culturing in medium supplemented with KnockOut serum replacement (KOSR) and basic fibroblast growth factor (bFGF) on STO feeder layers, and the effect of ROCK (Rho-associated coiled-coil protein kinases) inhibiter Y-27632 on the cell lines culture was tested. 5 PA blastocyst derived cell lines and 2 IVF blastocyst derived cell lines have been cultured more than 20 passages; one PA cell lines reached 110 passages without obvious morphological alteration. The derived trophoblast cells exhibited epithelium-like morphology, rich in lipid droplets, and had obvious defined boundaries with the feeder cells. The cells were histochemically stained positive for alkaline phosphatase. The expression of TR lineage markers, such as CDX2, KRT7, KRT18, TEAD4, ELF5 and HAND1, imprinted genes such as IGF2, PEG1 and PEG10, and telomerase activity related genes TERC and TERF2 were detected by immunofluorescence staining, reverse transcription PCR and quantitative real-time PCR analyses. Both PA and IVF blastocysts derived trophoblast cells possessed the ability to differentiate into mature trophoblast cells in vitro. The addition of Y-27632 improved the growth of both PA and IVF blastocyst derived cell lines and increased the expression of trophoblast genes. This study has provided an alternative highly efficient method to establish trophoblast for research focused on peri-implantation and placenta development in IVF and PA embryos.

The bovine endometrial epithelial cells promote the differentiation of trophoblast stem-like cells to binucleate trophoblast cells

“…Therefore, the development of in vitro models is required to study implantation processes. Bovine endometrial epithelial cells (EECs) are often used as a bovine in vitro model for studying the biological characteristics, cellular and molecular mechanisms, and responses to infection or damage of the bovine uterine epithelium, as well as for investigating the impacts of bovine embryo implantation and development of interactions between the embryo and mother (Bai et al 2014;Fortier et al 1988;Hou et al 2015;Skarzynski et al 2000;Zhang et al 2013). Coculture of bovine EECs and bovine trophoblast cells can be used to investigate the maturation of trophoblasts and the formation of binucleate cells.…”

Utilization of porcine in vitro‐produced parthenogenetic embryos for co‐transfer with vitrified and warmed embryos

“…The proportion of PA blastocysts was 20.5%, which was lower than that in previous studies (> 29.6%, Hou et al . ; Sangho & Woo‐suk ). In the present study, we did not select mature oocytes after IVM culture because we performed IVF with cumulus‐enclosed oocytes.…”

“…We consider that co-transfer of in vitro-produced embryos may be a promising avenue for achieving this goal, and two sources of embryos produced in vitro for cotransfer could be potentially utilized: (i) PA embryos and (ii) in vitro fertilization (IVF) embryos. Utilization of PA embryos has already been reported (Kawarasaki et al 2009), and we have shown that in vitro-matured (IVM)/IVF oocytes have the ability to develop to term even after in vitro culture (IVC) to the blastocyst stage (Nakamura et al 2017). In the present study, we compared the abilities of both PA and IVM/IVF oocytes to develop to the blastocyst stage, and their quality after in vitro culture.…”

“…PA embryos were produced using a modification of the method reported by Kawarasaki et al (2009). Briefly, after IVM for 48 h and denudation of cumulus cells by hyaluronidase treatment and pipetting, the oocytes were stimulated with an electrical pulse (DC at 150 kV/cm) in 0.28 mmol/L mannitol solution.…”

“…This physiological peculiarity of pigs means that in order to avoid the loss of rare and important embryos after transfer, some other viable embryos need to be co-transferred with them. One well-known report by Kawarasaki et al (2009) indicated that when a single fresh blastocyst was transferred together with 20 parthenogenetic (PA) blastocysts, seven out of nine recipient pigs went to term and produced one piglet. That report clearly demonstrated that co-transfer of variable embryos (in that study, parthenotes) can aid the implantation and development of a target embryo(s).…”