Mammalian single somatic cell RNA has a concentration range of 10-30 pg, of which rRNA accounts for approximately 85% of the total RNA content. Approximately 15-20% of the rRNA content is as low as 1-5%. The basic requirements for the total RNA used in general transcriptome sequencing are as follows: concentration of RNA samples ≥ 400 ng/µl, total amount ≥ 20 µg, purity of A260/280 between 1.8-2.4, A260/230 > 1.8, optimal integrity (RIN 7.0-8.0) and RNA28S/8S > 1.0mm. However, the total RNA concentration of each embryo from the mammalian 2-cell to the blastocyst stage is only 200-2,000 pg. Therefore, the trace RNA cannot meet the basic requirements of the construction of the transcriptome sequencing library and of high-throughput sequencing. With the continuous development of a new generation of high-throughput sequencing technology, RNA-Seq can be used to sequence the total transcriptional activity of any biological growth and developmental stage without designing probes in advance. Moreover, it can accurately detect gene expression under various conditions and discover several unknown molecular regulatory mechanisms. In the present study, RNA-Seq technology was used for the first time to reveal the mechanism of sheep early embryo development from the perspective of single embryo transcriptome, which provides a new method for improving sheep embryo production in vitro. It also provides a theoretical basis for further improving sheep gene structure information and identification of new genes related to embryonic development. Therefore, Smart-Seq2 amplification technology was used to enrich the samples and construct a sequencing library.The Illumina Hi SeqXten high-throughput sequencing technology was used for transcriptome sequencing. The results of sequencing quality evaluation and data analysis indicated that the sequencing quality and library construction quality were high and that the sequencing data were accurate and reliable.
Following filtration of the data of the original sequence, the filtered sequence of the sheep embryos at the four developmental stages of the 8-cell, 16-cell, morula and blastocysts embryos exhibited the following range: 441698590-48957974. Using the TopHat software to compare the obtained Clean reads with the reference genome, the results indicated a range of 93.71-95.29% clean reads alignment on the sheep reference gene in each stage. The sequence ratio of (multi map rate) multiple locations of the genome was 5.79-15.62%, which satisfied the requirements. The correlation between biological repeats was 90.86% and the repeatability between the samples was optimal. In the present study, a large number of alternative splicing in sheep 8-cell, 16-cell, morula and blastocysts,Alternative splicing of transcripts is prevalent in mammalian cells and could make major differences for maintenance of cell identity and function[24, 25]. Certain genes are expressed at specific stages of embryonic development and play key regulatory roles at specific stages. It was found that the large number of novel transcripts was due to the lack of the Y chromosome sequence in the reference genome of the sheep.
Embryonic development is the result of the expression of genetic information according to a certain time, space and order. This result (genetic program) is derived from the genetic program of development. The development of early embryos belongs to the process of maternal regulation, indicating that it is regulated by a large number of mRNAs and proteins synthesized during oogenesis and maturation. Maternal mRNA plays an important physiological role in the early stage of embryonic development. Following developmental progression, maternal mRNA and protein are gradually degraded, while embryonic genome activation (embryonic genome activation, EGA) is initiated and developmental transition from maternal regulation to embryonic regulation (maternal to-embryonic transition, MET) is facilitated. Different species exert different stages of MET. Mouse MET occurs in the 2-cell stage, human and pig occur in the 4-cell to the 8-cell stage and the bovine embryo genome mainly transcribes from the 8-cell to the 16-cell stage.
KIT, STAT3, ZP4, BMP15, GDF9, ZP3, Pms2, Hsf1-2 and Zar1 were confirmed to be maternal genes[26, 27]. The expression of these maternal genes from the 16-cell to the morula sheep embryos did not change significantly. The expression levels of the zygotic genome-related genes were not high. However, the marker gene NANOG of EGA was initially expressed in the morula stage. Therefore, it is concluded that sheep EGA may occur from the 16-cell to the morula stage. Methylation-related genes are stably and highly expressed, suggesting that the methylation of embryos is being reconstructed, which is consistent with previous studies.
The number of DEGs in 840 in E16vsE8, 6631 in E32vsE16, 810 in BlavsE32. And the number of DEGs at different stage was summarized in Fig. 5. indicating significant temporal differences in the developmental regulatory mechanism of sheep embryos at different developmental stages.There was 6631 differential genes from 16-cell development to the morula period, mainly due to the substantial activation of zygotic genes during the ZGA period of sheep embryos, as a result of substantial consumption of maternal material.We identified 30 key genes, and the analysis found that these genes were functional enriched in the cell cycle, RNA, DNA synthesis regulation, and verified the developmental characteristics of energy metabolism during this period.We identified 30 key genes, and the analysis found that these genes were functional enriched in the cell cycle, RNA, DNA synthesis regulation, and verified the developmental characteristics of energy metabolism during this period.But its functionality will need to be further explored.
GO analysis indicated that No significant difference was found at E16 vs E8.At E32 vs E16,Cellular components contained 127 significance terms (P<0.05). And biological processes involved 338 significance terms.At Bla vs E32,Cellular components contained 7 significance terms.The GO and KEGG enrichment analyses found that the main differences were concentrated in the 16-cell to morula period, and the difference types were mainly cell metabolism and compound synthesis, indicating that after zygotic genome activation, the zygotic genome began to regulate embryonic development.In bovine, comparative transcriptome analysis demonstrated that lineage differentiation at the blastocyst stage is associated with marked differences in expression of genes involved in various biological processes, such as metabolism, endocytosis, paracrine signaling, and cellular architecture.