Comparative stepwise pattern of reactive oxygen species production during in vitro development of fertilized and nuclear transferred goat embryos

Background: A unique feature of embryo metabolism is production of reactive oxygen species [ROS]. It is well established that during in vitro culture, ROS levels increase over normal ranges observed for embryos developed in vivo. This study evaluates and compares the stepwise pattern of ROS production during in vitro development of reconstructed goat embryos produced by zona-free method of somatic cell nuclear transfer [SCNT]. Furthermore, the pattern of ROS production of SCNT embryos were compared with zona free embryos derived from in vitro fertilization [IVF]

Materials and Methods: In this experimental study, zona-free oocytes, SCNT and IVF embryos at different stages of in vitro development [2, 4, 8, 16-cells, morula, and blastocyst] were used for assessment of ROS production using 2, 7-dichloro dihydroflourescein diacetate [DCHFDA] probe and the result were presented as fold increase or decrease relative zona free oocytes

Results: The relative level of ROS compared to metaphase-II [MII] oocytes insignificantly decrease during early stages post embryo reconstitution and regained its value by 8-cell and morula stage and, significantly increase compared to MII oocytes by blastocyst stage

Conclusion: The pattern of ROS change in SCNT embryos is similar to zona free IVF derived embryos, except it decrease from two cell stage and regain its value at morula stage. The sudden rise in ROS at blastocyst stage, further emphasizes the special need of IVF and SCNT derived embryos during this stage of development

Stage-specific profiling of transforming growth factor-beta, fibroblast growth factor and wingless-int signaling pathways during early embryo development in the goat

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

Expression profile of developmentally important genes in pre- and peri-implantation goat embryos produced in vitro

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

Identification of a specific pseudo attP site for phage PhiC31 integrase in bovine genome

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

Cloning, expression, and in vitro functional activity assay of phiC31 integrase cDNA in Escherichia coli

The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 [DE3]. Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] of the purified phiC31 integrase confirmed the size of protein [70 kDa]. Finally, the functionality of purified phiC31 integrase was verified. The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration

Developmental competence and pluripotency gene expression of cattle cloned embryos derived from donor cells treated with 5-aza-2'-deoxycytidine

Reconstructed embryos from terminally differentiated somatic cells have revealed high levels of genomic methylation which results in inappropriate expression patterns of imprinted and non-imprinted genes. These aberrant expressions are probably responsible for different abnormalities during the development of clones. Improvement in cloning competency may be achieved through modification of epigenetic markers in donor cells. Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine [5-aza-dc; 0-0.3 microM], a DNA methyl transferase inhibitor, improved development and expression of Oct-4. In comparison with untreated cells, 0.01 and 0.08 microM 5-aza-dc treated cells insignificantly decreased the blastocyst rate [32.1% vs. 28.6% and 27.2%, respectively] while it was significant for 0.3 microM treated cells [6.5%]. Embryo quality as measured by the total cell number [TCN] decreased in a dose-related fashion, which was significant at 0.08 and 0.3 microM 5-aza-dc treated cells when compared with 0 and 0.01 microM 5-aza-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 microM 5-aza-dc treated cells showed lower levels of DNA methylation and histone H3 acetylation, development to blastocyst stage was decreased. The epigenetic markers of embryos cloned from 0.01 microM 5-aza-dc remained unchanged. These results show that 5-aza-dc is not a suitable choice for modifying nuclear reprogramming. Finally, it was concluded that the wide genomic hypomethylation induced by 5-aza-dc deleteriously impacts the developmental competency of cloned embryo

Effect of ovarian cyclic status on in vitro embryo production in cattle

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

Effects of culture system on developmental competence, cryosurvival and DNA-fragmentation of in vitro bovine blastocysts

This study investigated the effect of two in vitro embryo culture systems [co-culture system versus cell-free sequential-media] on developmental competence, cryosurvival and DNA-fragmentation of in vitro developed bovine blastocysts. Bovine presumptive zygotes were cultured in Menezo's B2 [B2] plus vero-cells or sequential synthetic oviductal fluid [SOF] for eight days. Subsequently, half of the expanded blastocysts developed in both groups were vitrified, warmed within 30 minutes and post-warming embryos along with their corresponding non-vitrified embryos were cultured for two additional days in the same medium used before vitrification. Embryo development, cryosurvival and apoptosis were compared between the groups. For non-vitrified embryos, culture in SOF significantly promoted the potency of embryos to develop into blastocysts compared with the co-culture system. The difference in post vitrification survival rate of SOF blastocysts [83.3%] was insignificant compared with co-culture [84.3%]. However, while total cell number of warmed blastocysts in the co-culture system was significantly higher in the co-culture versus the sequential system [215.4 vs. 170.4], the quality of survived embryos in terms of hatching ability and apoptosis was adversely affected by co-culture compared with SOF [65.0% vs. 74.3%, and 13.5% vs. 10.0%, respectively; p<0.05]. Although co-culture system may increase the viability of embryos following cryopreservation, the potency and dynamics of blastocyst formation significantly increased with sequential media compared to the co-culture system which can compensate for the lower efficiency of sequential media for vitrification/warming purposes