ABSTRACT
Objective: This research intends to unravel the temporal expression profiles of genes involved in three developmentally important signaling pathways [transforming growth factor-beta [TGF-beta], fibroblast growth factor [FGF] and wingless/int [WNT]] during pre- and peri-implantation goat embryo development
Materials and Methods: In this experimental study, we examined the transcripts that encoded the ligand, receptor, intracellular signal transducer and modifier, and the downstream effector, for each signaling pathway. In vitro mature MII oocytes and embryos at three distinctive stages [8-16 cell stage, day-7 [D7] blastocysts and day-14 [D14] blastocysts] were separately prepared in triplicate for comparative real-time reverse transcriptase polymerase chain reaction [RT-PCR] using the selected gene sets
Results: Most components of the three signaling pathways were present at more or less stable levels throughout the assessed oocyte and embryo developmental stages. The transcripts for TGF-beta, FGF and WNT signaling pathways were all induced in unfertilized MII-oocytes. However, developing embryos showed gradual patterns of decrease in the activities of TGF-beta, FGF and WNT components with renewal thereafter
Conclusion: The results suggested that TGF-beta, FGF and WNT are maternally active signaling pathways required during earlier, rather than later, stages of pre- and peri-implantation goat embryo development
ABSTRACT
Background: Little is understood about the regulation of gene expression during early goat embryo development. This study investigated the expression profile of 19 genes, known to be critical for early embryo development in mouse and human, at five different stages of goat in vitro embryo development [oocyte, 8-16 cell, morula, day-7 blastocyst, and day 14 blastocyst]
Materials and Methods: In this experimental study, stage-specific profiling using real time-quantitative polymerase chain reaction [RT-qPCR] revealed robust and dynamic patterns of stage-specific gene activity that fall into four major clusters depending on their respective mRNA profiles
Results: The gradual pattern of reduction in the maternally stored transcripts without renewal thereafter [cluster-1: Lifr1, Bmpr1, Alk4, Id3, Ctnnb, Akt, Oct4, Rex1, Erk1, Smad1 and 5] implies that their protein products are essential during early cleavages when the goat embryo is silent and reliant to the maternal legacy of mRNA. The potential importance of transcription augment at day-3 [cluster-2: Fzd, c-Myc, Cdc25a, Sox2] or day-14 [cluster-3: Fgfr4, Nanog] suggests that they are nascent embryonic mRNAs which intimately involved in the overriding of MET or regulation of blastocyst formation, respectively. The observation of two expression peaks at both day-3 and day-14 [cluster-4: Gata4, Cdx2] would imply their potential importance during these two critical stages of pre-and peri-implantation development
Conclusion: Evolutionary comparison revealed that the selected subset of genes has been rewired in goat and human/goat similarity is greater than the mouse/goat or bovine/goat similarities. The developed profiles provide a resource for comprehensive understanding of goat preimplantation development and pluripotent stem cell engineering as well
ABSTRACT
PhiC31 integrase system provides a new platform in various felid of research, mainly in gene therapy and creation of transgenic animals. This system enables integration of exogenous DNA into preferred locations in mammalian genomes, which results in robust, long-term expression of the integrated transgene. Identification of a novel pseudo attP site. Genomic DNA was extracted from primary bovine fetal fibroblast cells, which were stably trans-fected with EGFP and phiC31 integrase cDNAs carrying vectors. An inverse PCR was carried out for production of mini-circle DNAs and followed by sequencing. A new specific pseudo attP site termed BF5 was identified in bovine genome. This site is located in an intergenic AT rich region on chromosome 5 with similar features of other mammalian attP pseudo sites. Furthermore, direct sequencing of generated attL site confirmed that site-specific transgene recombination was occurred at this site. This finding confirmed that phiC31 integrase could be feasible for production of transgenic animals for biotechnological applications
ABSTRACT
The relationship between cyclic status of cattle ovaries on in vitro embryo development up to the blastocyst stage was investigated. Cattle ovaries were collected immediately after slaughter and divided into three categories based on their cyclic status, which included: 1. the presence of a large follicle [LF], 2. the presence of a corpus luteum [CL] and 3. ovaries without LF or CL [WLCF]. Oocytes of these ovaries were obtained and used for in vitro maturation and fertilization. Presumptive zygotes were then cultured up to the blastocyst stage in synthetic oviductal fluid culture medium. There were no significant differences between cleavage rates of the three groups. The rate of embryos in the compact morula stage for the CL group was 48.2% which was significantly higher than the related rate of the LF group [36.6%], but non-significantly higher than that of the ST group [45.7%]. The highest blastocyst rate belonged to the CL group [54.6%] which was significantly greater than the WLCF group [32.9%] and non-significantly higher than the LF group [52.4%]. There was no significant difference in blastocyst rates in the CL and LF groups. Preselection of oocyte donor ovaries containing a CL or LF can be used as a feasible and noninvasive criterion to obtain the most competent oocytes capable of development to the blastocyst stage