ZGA is essential for early embryonic development with a series of physiological and biochemical reactions, including transcription factor activation, DNA methylation, protein modification, RNA binding, metabolic changes, and maternal factor degradation[22, 23]. However, there are few reports on lncRNA in pig early embryos. In this study, we used transcriptome data at different stages of early porcine embryos to identify a novel lncRNA specifically expressed at ZGA stage: XLOC-040580. We found that the expression of ZGA related genes changed after knocking down XLOC-040580, which proved that it was indispensable in this process. After knocking down XLOC-040580 in pig embryos, it was found that the blastocyst formation rate was significantly reduced, the number of TE cells were significantly reduced, and the total number of blastocysts were also significantly reduced. In addition, we found that two target genes of XLOC-040580 were also involved in the regulatory network of early embryonic development in pigs. The above results support that XLOC-040580 plays a key role in the ZGA process, and provide a reference for further study of pig ZGA-related lncRNAs.
In this study, we selected PA embryos similar to fertilization activation mechanism. PA embryos can develop to blastocyst stage, and because the offspring of PA embryos are not affected by sperm, it is more conducive to the study of single controllable variable. At the early stage of embryonic development, the transcription of embryonic genome is completely regulated by maternal mRNA and protein during embryonic development, but maternal mRNA is gradually degraded during ZGA[24, 25]. Previous studies have shown that there is no significant difference in the expression between IVF and PA during early embryonic development[26]. Similarly, eIF1A associated with differential expression can be used as a marker gene for IVF and PA in ZGA, KDM7A[26], PDHA1[27], KDM5B, KDM5C[28] and other ZGA-related transcription factors have also been shown that share the same expression pattern in IVF and PA embryos. In this study, the expression trend of XLOC-040580 was consistent with the qRT-PCR results of RNA-Seq and PA embryos. So PA embryos can be used for subsequent experiments.
In previous reports, 252 testis-related lncRNAs and 159 placenta-related lncRNAs were analyzed by sequencing of porcine placenta and testis tissues[29]. It has also been reported that lncRNAs TCONS_01729386 and TCONS_01325501 may play an important role before embryo implantation[11], providing ideas for the study of lncRNAs in early porcine embryos. We further explored early embryos and found a novel lncRNA specifically expressed in 4–8 cell stages of early embryonic development, XLOC-040580. When we knocked down XLOC-040580 by siRNA, the blastocyst formation r ate decreased significantly. More importantly, the expression levels of ZGA-related genes were affected, including eIF1a[30], Smg[31], ACADL[32] and Hsp70[33]. These genes were considered to be the marker genes in the ZGA period, which proved that XLOC-040580 played a more extensive role in the ZGA period. More interestingly, knockdown of XLOC-040580 significantly reduced the number of embryonic TE cells, suggesting that XLOC-040580 may play a role in late ZGA and affect the normal development of embryonic TE cells. This is consistent with recent studies, transcription factors generally enriched in cis-regulatory elements of ICM and TE, and the expression of transcription factors in ZGA is the highest consistent, indicating that ICM and TE programs first started in ZGA[34].
In order to better test the regulatory network of lncRNA, we focused on two XLOC-040580 target genes: TPRA1 and BCL2L1. We found that the expression abundance of TPRA1 and BCL2L1 increased from 4-cell stage to 8-cell stage, and peaked at 8-cell stage, which were consistent with ZGA expression pattern. TPRA1 is generally believed to be closely related to cell growth and embryonic differentiation. It has been reported that it mediates intracellular cAMP level, mainly affects cell growth and differentiation by regulating Hedgehog (Hh) signaling pathway, and has a positive regulatory effect on embryonic formation and tissue homeostasis[35]. BCL2 protein family is a key regulator of apoptosis and participates in a variety of signaling pathways related to cell development[36]. We knocked down these two target genes, resulting in early embryonic development blocked. Interestingly, we used single-cell transcriptome sequencing technology to detect the effect of knockdown target genes on the whole gene of porcine embryos, and found that TPRA1 was related to oocyte gene inhibitor AGO2[37], male reproductive factor TDRP[38] and WDR19[39]. BCL2L1 is related to the 2-cell cleavage and pretreatment mRNA factor SNRNP[40], the PRDXs that affect mitochondrial activity throughout embryonic developmen[41], and the apoptosis and senescence-related gene GADD45B[42]. We further drew the regulatory network diagrams of XLOC-040580, TPRA1 and BCL2L1. TPRA1 is associated with many genes in the G protein-coupled receptor (GPR) family, and GPR107 is the key gene for embryonic lethality[43]. However, it was found that AGO2 and TDRP were not directly related to TPRA1. We speculated that the knockdown of TPRA1 might affect the expression level of XLOC-040580, thereby changing the expression of TDRP and AGO2. More interestingly, we found that BCL2L1 could be associated with mediator complex subunit (MED) family, and MED family was also associated with ZGA marker genes eIF1a and HSP70.2. MED31 can promote embryonic growth and cell proliferation[44], and excessive expression of MED28 can also disturb cell cycle and lead to genomic instability[45]. We continued to carry out KEGG enrichment analysis, which found that TPRA1 and BCL2L1 jointly participate in MAPK signaling pathway. In addition, TPRA1 is involved in signaling pathways such as oocyte maturation, gonadotropin-releasing hormone signal and oocyte meiosis, and MAPK factor is also involved in these pathways. It is well known that MAPK signaling pathway plays a key role in spindle connection, microtubule organization and asymmetric division during oocyte maturation. It is well known that MAPK signaling pathway plays a key role in spindle connection, microtubule organization and asymmetric division during oocyte maturation[46], and is closely related to many intracellular events such as cell proliferation, cell differentiation and apoptosis. In addition, MAPK14 induces BCL2 phosphorylation in apoptotic germ cells[47]. The above results showed that the target genes TPRA1 and BCL2L1 of XLOC-040580 played an important role in the regulatory network of oocyte maturation and preimplantation embryo development.