Embryonic Development and Implantation Related Gene Expression of Oocyte Reconstructed with Bovine Trophoblast Cells

Abstract

Abstract. The temporal progressive increase of interferon tau (IFNτ) secretion from the bovine trophoblast is a major embryonic signal of establishing pregnancy. Here, we cultured and isolated bovine trophoblast cells (BTs) from IVM/IVF oocytes and in vitro produced blastocysts, used them, for the first time, as donor cells for nuclear transfer and compared them with adult fibroblasts (AFs) as donor cells. BTs were reprogrammed in enucleated oocytes to blastocysts with similar efficiency to AFs (14.5% and 15.6% respectively, P≤0.05). The levels of IFNτ, CDX2 and OCT4 expression in IVF-, BTand AF-derived blastocysts were analyzed using reverse transcription polymerase chain reaction and reverse transcription quantitative polymerase chain reaction (RT-PCR and RT-qPCR). IVF-produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid, expanded and hatching blastocysts. RT-PCR and RT-qPCR studies showed that IFNτ expression was higher in BT-derived blastocysts than IVF-and AF-derived blastocysts. Both IVF-and BT-derived blastocysts showed a progressive increase in IFNτ expression as blastocyst development advanced when it compared with AF-derived blastocysts. OCT4 was inversely related with IFNτ expression, while CDX2 was found to be directly related with IFNτ temporal expression. Persistence of high expression of IFNτ and CDX2 was found to be higher in BT-derived embryos than in IVF-or AF-derived embryos. In conclusion, using BTs expressing IFNτ as donor cells for bovine NT could be a useful tool for understanding the IFNτ genetics and epigenetics. Key words: Bovine trophoblast, Interferon tau, Nuclear transfer, Real-time PCR, Reprogramming (J. Reprod. Dev. 58: [425][426][427][428][429][430][431] 2012) S ince the first cloned lamb was born, nuclear transfer (NT) has been challenging in several species and has produced many cloned offspring [1][2][3]. To produce cloned offspring, fetal fibroblasts have been chosen as a preferential donor cell line for NT so far because they have high proliferative potentials [4][5][6][7]. In cattle, fetal and adult fibroblasts have been dominantly used for NT to produce cloned calves. Additionally, several types of cells, like granulosa, cumulus, oviduct epithelial cells, skin, tongue and other cells, have been used for NT [8,9].After fertilization of an egg with a sperm, the one-cell stage embryo grow up through several mitosis and reaches the preimplantation stage, becoming a blastocyst, which consists of an inner cell mass (ICM) that is capable of differentiation into all embryo organs and trophoblasts, which are the first differentiated cells from the embryo, and contributes formation of the placenta and fetal membranes but does not participate the formation of the fetus proper [7]. Some reports have demonstrated trophoblast isolation and its function in vitro in cattle [10][11][12]. In mice, living pups were born by nuclear transfer of trophectoderm cells from the expanded blastocysts into enucleated oocytes [13] as a trial to show...

Presence of Transcription Factor OCT4 Limits Interferon-tau Expression during the Pre-attachment Period in Sheep

“…The presence of OCT4 mRNA in bovine and ovine trophoblasts detected in the present study and by others (Kurosaka et al, 2004; Degrelle et al, 2005; Berg et al, 2011; Saadeldin et al, 2012) contrasts with data from mouse trophoblasts, in which OCT4 expression is restricted to the ICM (Niwa et al, 2005). Berg et al (2011) showed that the upstream conserved region 4 (CR4) of OCT4 genes differs between bovine and murine species, and that bovine CR4 is effective for OCT4 expression in mouse TE.…”

“…According to Ezashi et al (2001), OCT4 expression subsides long before the initiation of conceptus elongation, when ETS2 begins to activate bovine IFNT gene transcription. In situ hybridization results suggested that OCT4 mRNA was restricted to the ICM in d 7 bovine blastocysts (Kurosaka et al, 2004; Saadeldin et al, 2012). However, further studies revealed that OCT4 expression remained until d 12 and 13, just prior to conceptus elongation (Degrelle et al, 2005), or even longer period (Berg et al, 2011).…”

Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells

“…This protocol followed our previously published protocol [11]. Motile spermatozoa were obtained by purification using Percoll gradient method as previously described [13].…”

“…Two different feeders were used: the conventional method using mouse embryonic fibroblasts (MEFs) as we described before [11] and new feeder porcine granulosa cells (PGCs). PGCs were obtained through aspiration of follicular fluid of 4–6 mm porcine ovarian follicles with an 18-gauge needle.…”

“…Consequently, maintenance of trophoblastic cell lines using feeder cells comes as a more convenient and feasible approach. In this regard, recently, some trophoblastic cell lines were established, on mouse embryonic fibroblasts as feeders, in several species as porcine [8], murine [9], and bovine [10, 11]. Henceforward, improving the culture system is of particular interest and advantage, to facilitate the in vitro investigation of the trophoblastic cells.…”