Expression Profile of Developmentally Important Genes
in preand peri-Implantation Goat Embryos
Produced In Vitro
The first two authors equally contributed to this manuscript.
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
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.
The gradual pattern of reduction in the maternally stored transcripts without renewal thereafter (cluster-1:
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.
Mammalian preimplantation embryonic development encompasses the period from fertilization to implantation. During this period, the embryonic stages and critical developmental events assessed are transition from germinal vesicle stage (GV) to metaphase-II (MII) oocyte, maternal-to-embryonic transition (MET), and the first lineage differentiation to the inner cell mass (ICM) and trophectoderm (TE) during blastocyst formation (1). Notably, implantation in ungulates, unlike human and mice, occurs with a delay of around 7 days. During this “peri-implantation” period, the rapid development of TE dramatically alters the blastocyst morphology from a sphere to a day 14 hatched blastocyst (2).
An improved understanding of gene activity that regulates preimplantation development is crucially important for assisted reproduction techniques and for derivation of embryonic stem cells (3). This goal has been largely achieved in mouse and human (4, 5). For example, it has been shown that embryonic developmental program is regulated by intricate cooperation of several important genes in the context of cell-signaling pathways. It was initially presumed that the developmental genes regulating early embryonic events are conserved across all mammalian species. Researchers attempted to extrapolate mice and human knowledge databases to the embryonic development of other species as well. However, further comparative studies revealed that species-specific differences exist between gene regulatory networks regulating embryo development in mammals (3, 5-7), which will provide a roadmap for differentiating definitive species-specific differences.
The goat is a valuable livestock animal with
promising importance in agriculture, biomedicine
and transgenesis (8, 9). However, the molecular basis of goat early embryonic development is
poorly understood. Yan et al. (10) for the first time
demonstrated that the expression of Oct4 and Nanog proteins were not restricted to the ICM of goat
blastocysts. To date, four studies have reported
derivation of goat “putative” embryonic stem cells
(ESCs) from embryos produced either
An improved understanding of expression pro-
files of developmentally important genes in pre- and peri-implantation goat embryos would improve current attempts to establish ESC in this
valuable farm species. Therefore, this study for
the time 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
Materials and Methods
In this experimental study , unless otherwise stated, all chemicals and media were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and Gibco (Grand Island, NY, USA), respectively.
Selection of genes set
Nineteen candidate genes for the investigation
were selected from the human and mouse data
bases if i. They were only present in ESCs
and either in the oocyte or blastocyst and ii.
Their gene ontology applications indicated a critical role in transcription regulation, pluripotency
and differentiation. This gene set included
In vitro production of goat embryos
The procedure used for
For real time-quantitative PCR (RT-qPCR) experiments, oocytes and embryos at five different
stages of goat
in vitro D14 embryos
RNA extraction and real time-quantitative polymerase chaine reaction
The procedure for RT-qPCR was as described previously (18). In brief, total RNA of oocytes and embryos was extracted suing RNeasy Micro kit (Qiagen, Mississauga, ON, Canada) followed by treatment with DNase I (Ambion, Streetsville, ON, Canada) according to the manufacturer’s protocol. The RNA quality and quantity was determined using WPA Biowave spectrophotometer (Cambridge, United Kingdom). For reverse transcription, 10 µl of total RNA was used in a final volume of 20 µl reaction containing 1 µl of Random Hexamer, 4 μl RT buffer (10 x), 2 µl of dNTP, 1 μl of RNase inhibitor (20 IU), and 1μl of reverse transcriptase (Fermentas, Glen Burnie, Ontario, Canada). Reverse transcription was carried out at 25°C for 10 minutes, 42°C for 1 hour and 70°C for 10 minutes.
The selection of appropriate reference gene is of crucial importance in the accuracy and fidelity of the data of RT-qPCR results. Accordingly, we searched the literature to find the best suitable candidate reference gene for the goat. We observed that in almost infield studies, ACTB has been used as the choice reference gene in several similar studies in the goat (19), bovine (3, 9, 20). Moreover, ACTB was selected as a suitable internal control for study of gene expression in cryopreserved egg and embryo because it efficiently withstands cryoshocks and oocyte manipulation (9, 17, 21). After ascertaining that the expression of ACTB was stable among different development stages of embryos (data not shown), relative quantification of the target genes was undertaken with ACTB as the reference gene. For RT-qPCR, total RNAs of oocytes and embryos were extracted and used for cDNA synthesis. RT-qPCR was carried out using 1 µl of cDNA (50 ng), 5 μl of the SYBR Green/0.2 μl ROX qPCR Master Mix (2X) (Fermentas, Germany) and 1 µl of forward and reverse primers (5 pM) adjusted to a total volume of 10 µl using water nuclease-free. Three technical replicates of RT-qPCR were conducted for each primer. CT samples of each target gene were normalized to the CT of ACTB and represented as 2-ΔΔCT (22). The primer sequences, annealing temperatures and the size of amplified products are shown in Table 1.
|Gene||Primer sequences (5ˊ-3ˊ)||Length of PCR product||TM|
PCR; Polymerase chain reaction and TM; Melting temperature.
Statistical analysis were carried out using SPSS software. For the analysis of developmental data and real-time PCR data a two-tailed Students t test with equal variance was used to determine significance data. Statistical significance was accepted at P<0.05.
All the 19 genes examined were expressed throughout embryo development, from MII-oocyte to D14 developing blastocysts (23, 24). Even though, the levels of expression of all genes varied during different developmental stages as no gene was found to be stably expressed throughout embryo development. Moreover, different genes had different levels of expression with respect to a certain stage of development. Stage profiling revealed robust and dynamic patterns of stage-specific gene activity that fall into four major clusters depending on the respective mRNA profile (Figes.2,,,,-6,).
The first gene cluster (Figes.2,, 3,) exhibited highest levels of mRNA in MII-oocyte which gradually and consistently decreased during subsequent
stages of embryo development. This cluster encompassed 11 genes including
|- Profiles of expression of genes categorized in the first class based on Figure 2. a, b, and c; Significant difference at P<0.05%.|
The second gene cluster (Figes. 2,, 4,) showed low
levels of the transcripts in MII-oocytes, reached
their highest relative mRNA levels in D3 embryos
and significantly decreased thereafter with no sign
of regain in transcription in D7 and D14 blastocysts. This gene set encompassed 4 genes including
|- Profiles of expression of genes categorized in the second class based on Figure 2. a, b, and c; Significant difference at P<0.05%.|
In the third gene cluster (Figes.2,, 5,), the original levels of transcripts of MII-oocyte gradually decreased in developing embryos with a significant reduction in D7 blastocysts. However,
these genes initiated transcription from D7 onwards which resulted in a peak of expression in
D14 blastocysts. This gene set composed of 2
|- Profiles of expression of genes categorized in the third class based on Figure 2. a, b, and c; Significant difference at P<0.05%.|
The fourth gene cluster (Figes.2,, 6,), showed
low levels of the transcripts in MII-oocytes,
reached their first the peak of expression in D3
embryos and followed by a significant decrease
in transcription in D7 blastocysts. However,
this group of genes reinitiated transcription
from D7 onwards which resulted in the second
peak of expression in D14 blastocysts. This
gene set encompassed 2 genes including
This study demonstrated that all developmental
genes assessed are present throughout the pre and
peri implantation stages of goat
The first set of genes, which importantly comprised two-third of the examined genes, revealed a consistent trend of gradual mRNA reduction as the highest and lowest levels of transcripts were observed in MII-oocyte and D14 blastocysts, respectively. This may suggest that the transcripts of these set of genes have been transcribed and accumulated during oocyte growth phase. Because MII-oocyte and early embryo are considered transcriptionally silent (25) and since there is no evidence of active transcription during meiosis resumption, these maternal transcripts should be produced during earlier stages of oocyte growth preceding germinal vesicle breakdown stage.
Theoretically, the steady state of mRNA reduction without renewal (gene cluster 1) may suggest the potential importance of these mRNA for production of proteins that are required during early stages of embryo development, especially to support maternal embryonic transition (MET), when the goat embryos are self-reliant in their transcription. Mechanistically, the distinctive processes have been associated with the declines of maternal mRNA in the eukaryotes including deadenylation, degradation, and protein translation or synthesis (26). Moreover, it has been suggested that the oocyte unlikely would keep useless products (27). Therefore, the quick reduction in relative mRNA abundance could be associated with the protein production.
The second group of genes revealed a significant elevation in their transcripts at D3 compared to MII-oocyte. But, this elevation in the transcripts did not continue and gradually declined and reached the lowest levels in D14 blastocysts . The potential importance of D3 burst-in-the-transcription is indicative of their crucial importance at the MET stage. Accordingly, the lower abundances of these transcripts during post-MET period may not play an important role in the regulation of blastocyst formation and further stages.
The third group of genes revealed a gradual decrease in the maternal mRNA abundances similar to the first group, but their transcripts increased from D7 and reached their highest levels at D14. This would imply that this set of genes is of critical importance during pre- and post- MET phases and underscores the facts that: i. The exact time windows that the second and third sets of genes are in demand for the embryo development are different, and ii. The maternal stockpiles of the third, but not second, set of genes are quite enough to support MET without any need for the additional source of embryo-specific transcripts.
The fourth group of genes was those showing two peaks in embryo-specific mRNA transcription at two distinctive time points of MET and day 14 blastocyts. This may suggest that these transcripts are crucially needed during both stages. This group can be considered as the combined model of the second and third groups, and correspondingly, the genes in this final group may be of theoretical alternative capacity to cover the duties of genes in both second and third groups.
One of the shortcomings of this study was usage of
The results obtained through this work highlight the fact that transcription factors involved in the regulation of early events of pluripotency and differentiation are present through pre- and periimplantation in the goat embryos. Since the blastocysts of ungulates, unlike human and mice, implant with a delay of around 7 days, the obtained results in D7 and 14 blastocysts may provide useful information to figure out the expression profiles of developmentally important genes between these two stages. The profiles obtained may be useful for derivation of ESC in this valuable farm species.
This study was performed by the grant from Royan Institute. Authors would like to thank Dr. Farnoosh Jafarpour and Nima TanhaieVash for technical helps. There is no conflict of interest in this study.