Somatic cell nuclear transfer (SCNT) is a proven technique with immense potential in the production of transgenic animals and embryonic stem (ES) cells, conservation of endangered species, multiplication of elite animals and employing farm animals as models for human diseases and xenotransplantation. Though SCNT has been successfully used to clone almost 20 mammalian species, still the success rate is very low (< 5%) of reconstructed embryos develop into viable offspring (Young, 2003; Campbell et. al.2007). Additional indicators like poor development, lower number of cells per blastocyst, and higher incidence of apoptosis (Vajta and Gjerris, 2006) also reflect towards its poor efficiency. World over, researchers have been trying to enhance the efficiency of the SCNT technique so as to harness its potential as a cloning technique for the benefit of both livestock as well as biomedical sectors.
Incomplete reprogramming of the donor nucleus and epigenetic defects are probably the two major known factors that causes most of the developmental problems of SCNT produced embryos and thus reduce the efficiency of the SCNT technique (Ng and Gurdon, 2005; Vajta and Gjerris, 2006). Incomplete epigenetic reprogramming of the donor cell nuclei associated with aberrant gene expression during embryo development is probably the most crucial cause of the low efficiency of SCNT technique (Dean et. al.2001). Also, the success rate of cloned embryos is reported to be significantly lower than that of IVF embryos in terms of live birth (Bauersachs et. al.2009). However, no concrete mechanistic viewpoints have been proposed for the differential outcome of two types of embryos at different developmental stages. Therefore, to address some of the above issues, the present study was carried out to identified the DEGs and molecular mechanisms of the SCNT embryos with respect to IVF embryos at different developmental stages in riverine buffalo, an important dairy species in India.
In the present study, RNA-seq data analysis led to valuable information about the DEGs in both types of embryos at different developmental stages. The large number of significantly (p < 0.05) expressed genes were detected in the present study pointed towards the fact that these might have important functional and regulatory roles at different developmental stages of the embryos. 148, 497 and 26 unique significant (p < 0.05) DEGs were identified at the 2 cells, 8 cells and blastocyst stage respectively, however, 47 significant DEGs were common throughout the all-development stage from the 2 cells to the blastocyst stage (Supplementary Fig. 1 and Supplementary Table 10). Some of the common genes, such as TNNC1, HSF5, GDF1, GPX8, TRAM1L1, SPATC1L, PPEF1, KBTBD11, MN1, COL4A5, JSRP1 were significantly upregulated, while XIST, DNAH7, TAGLN, ZEB1, TEX12, IGSF22 were significantly downregulated in SCNT as compared to the IVF embryos at all developmental stages. The expression profile of these genes suggested the embryos undergo the dynamic changes in pre-implantation embryo development, that might be responsible for the low success rate of the SCNT. Similar to our study, Sood et al. (2019), identified higher expression of the SPATC1L gene in SCNT embryos as compared to IVF embryos. In addition, ZEB1 plays crucial role in the development of the embryo and necessary for the maturation and migration of embryonic cells (Poonaki et. al.2022; Yamakoshi et. al.2012). However, in our study, the expression of ZEB1 was downregulated in the SCNT embryos than IVF embryos at all developmental stages. Rankin et al. (2000), reported basal level expression of the GDF1 for the proper development of the embryos in mouse. Although, GDF1 (-7.986-fold) was downregulated in SCNT relative to IVF counterparts at different developmental stage in the current study. Apart from this, higher expression of HSF1 and GPX8 might be associated with the high level of the oxidative stress in SCNT embryos as compared to IVF embryos at different developmental stages.
Moreover, several genes such as TNNC (23.140-fold), GPR50 (21.207-fold), ITGA4 (20.943-fold), AGMAT (10.544-fold), PCGF5 (9.351-fold), KBTBD11 (7.796-fold) were upregulated, whereas, PDE3B (-9.506-fold), VIM (-9.427-fold), IL17RB (-8.818-fold), S100A5 (-8.801-fold), TAGLN (-7.592-fold), ZEB1 (-7.9862-fold), HSD3B1 (-5.495-fold) were downregulated in SCNT blastocyst as compared to IVF blastocyst. Upregulated gene agmatinase (AGMAT), associated with the development of the embryos through the AGMAT pathway in sheep (Hussain et al. 2017). In addition, several genes such as ITGA4, PCGF5, GPR50, ILDR2 were involved in the development of the embryos in bovine, human, yak and goat (Yamakoshi et. al.2012; Yao et. al.2018; Chen et. al.2022; Liu et. al.2020). Liang et al. (2019) identified higher expression of the TAGLN associated with embryo implantation by promoting the actin filament in mice. Similarly, upregulation of the S100A5 protein responsible for the implantation of embryos in human (Tong et. al.2010). Another study, Gupta et al. (2017), reported that the HSD3B1 gene associated with lipid peroxidation and reactive oxygen species (ROS) in bovine blastocysts. In addition, VIM, CXCL12, DNAH7 and COL5A1 genes were associated with the cell structure and differentiation (Gupta et. al.2017; Ling et. al.2018; Hu et. al.2021). Therefore, upregulation and down regulation of these genes compromised the development of the preimplantation of the embryos. Interestingly, TEX12, EPHA10, C11H2ORF61, RESP18, SPATC1L genes were associated with testis function (Sood et. al.2019; Aasheim et. al.2005; Schiller et. al.1996). However, the function of these genes was unknown in embryos at different developmental stages (2 cells, 8 cells and blastocyst stage). Additionally, Sood et al. (2019), reported higher expression of several genes related to pluripotency, trophectoderm formation and epigenetic modification in the blastocyst stage, which was responsible for the low cloning efficiency in buffalo.
Furthermore, GO identified several biological processes (cellular process, metabolic process, developmental process, biosynthetic process, cellular metabolic process, single organism process, response to stimulus, biological regulation, immune system processes, signaling etc.), molecular functions (binding, catalytic activity, transporter, structure molecule activity etc.) and cellular components (cell part, membrane, cell, cell junction, synapse etc.) in our data set which might be associated with the development of the SCNT embryos at different developmental stages. The identified putative target genes could lead to a better understanding of DEGs in SCNT and IVF blastocysts. Therefore, target genes for each of the DEGs were predicted for their roles in regulating various cellular and developmental processes. Moreover, in our study, 110 pathways were reported in the 2 cells and 8 cells stages and 89 pathways were identified in the blastocyst stage. However, the 87 pathways were common in all developmental stages of embryos (Supplementary Fig. 2 and Supplementary Table 11). The major pathways (Wnt signalling pathway, PDGF signalling pathway, Apoptosis signalling pathway, Ras signalling pathway, integrin signalling pathway, TGF- signalling pathway, FGF signalling pathway, p53 signalling pathway, EGF receptor signalling, and cadherin signalling pathway) were affected the embryonic development of the SCNT embryos relative to their IVF counterparts. Similarly, Goel et al. (2022) identified the Wnt signalling pathway, TGF- signalling pathway, apoptosis signalling pathway, the FGF signalling pathway the p53 pathway was the major pathway associated with the development of the SCNT embryos relative to their IVF counterparts in buffalo. Likewise, Integrin signalling pathway, Wnt signalling pathway, TGF- signalling pathway, CCKR signalling map, apoptosis signalling pathway, gonadotropin-releasing hormone receptor pathway, angiogenesis, chemokine & cytokine signalling pathway were the most significant pathways related to development of the SCNT embryos relative to IVF counterparts at different developmental stages in buffalo (Malpotra et. al.2022). Several studies were suggested that Wnt signalling pathways and TGF- signalling pathway (also reported in our study) play a significant role to control a wide range of cellular processes, including cell fate, cell division, cell-cell adhesion, cellular polarity and pluripotency (Nance, 2014; Sonderegger et. al.2010; Chen et. al.2009; Shyam et. al.2020). De (2011), identified canonical Wnt pathway has been associated with regulation of cell fate while non-canonical Wnt pathways involved in regulation of polarity, asymmetric cell divisions, and cell movements during gastrulation. However, Denicol et al. (2013), reported that the canonical wnt pathway reduces the competence of the In-vitro produced embryo. Apart from this, TGF- signalling pathway associated with nodal signalling and involved in the development of blastocyst through proper specification and patterning of the cells (Zinski, et. al.2018). Apoptosis signalling pathway affected the development of the blastocyst and early-stage embryos in SCNT as compared to the IVF counterparts. Therefore, this may be a major cause of lower live birth rate obtained with cloned embryos. Some of the studies reported, apoptosis related genes showed the higher expression in early stage of embryos as compared to IVF counterparts (Fahrudin et. al.2002). These identified molecular pathways might be affected the development of the SCNT embryos as compared to the IVF counterparts.