Transcriptome dynamics during early embryo development
Early developing porcine embryos displayed a great adaptive capacity towards their environment, evidenced by largely similar transcriptome dynamics observed in both in vivo developed and in vitro produced embryos. In vitro produced embryos offer the opportunity to study molecular pathways of interest in a developmental-stage specific manner, as there is a higher degree of certainty regarding the time of fertilization compared to in vivo developed embryos. However, developmental rates and embryo competence of in vitro produced embryos are still lower compared to their in vivo developed counterparts [5]. A number of factors are known to contribute to embryo development. The presence of cumulus cells during maturation facilitates full oocyte maturation [19]. In pigs, the presence of cumulus cells during oocyte maturation is essential for oocyte maturation, fertilization and subsequent embryo development [20]. The discrepancy in embryo development between in vivo developed and in vitro produced embryos at early post-fertilization developmental stages might be explained by the use of a pool of non-selected oocytes of overall lower competence for in vitro maturation, compared to those selected for ovulation, and the effects of in vitro maturation on oocyte quality. A higher blastocyst rate has previously been shown after oocyte maturation under a 20% oxygen atmosphere [21]. However, blastocyst quality assessed by the expression of genes related to metabolism (GLUT1 and LDHA), antioxidant response (SOD2 and GPX1), growth factors and apoptosis (IGF2R, BCL2 and BAX), methylation (DNMT3B), and blastocyst quality (AKR1B1, POU5F1 and CDX2) were not affected [21]. In addition, the blastocyst rates of in vivo and in vitro matured rabbit oocytes did not significantly differ, while at earlier developmental stages, the in vivo embryo development rates were significantly higher than observed for embryos produced with in vitro matured oocytes [22]. Thus, while oocyte quality and competence, and subsequent embryo development are affected by the maturation conditions, only minor transcriptional differences have been reported at the hatched blastocyst stage [23]. In line with previous findings, we found more similar transcriptome profiles at later developmental stages. At the hatched blastocyst stage, only limited transcriptional differences persisted. Additionally, the developmental-stage specific differences were more pronounced than the sex-specific differences, as previously described by Zeng et al. (2019), studying the transcriptome dynamics in in vivo developed day 8, 10, and 12 porcine embryos [16].
Early porcine embryo development
The early embryo development was studied at the 4-cell, morula and hatched blastocyst stage for both in vivo developed and in vitro produced embryos. Previously, porcine embryos after EGA have been shown to display an increased abundance of transcripts involved in, among others, transcription [13]. In our in vivo developed embryos, the ERK/MAPK signaling pathway was significantly enriched during the 4-cell to morula transition, which is predicted to result in a lower rate of transcription at the 4-cell stage. This is in line with the increased number of detectable transcripts at the morula compared to the 4-cell stage, and indicates that the in vivo embryos might have been sampled at an earlier 4-cell stage, i.e., closer to EGA. In addition, the TNFR1 signaling pathway was enriched in the in vivo 4-cell to morula transition. A lower degree of apoptosis and cell survival was predicted at the 4-cell stage compared to in vivo morulae stage. An inverse pattern was observed in in vitro produced embryos, indicating a higher degree of apoptosis in early in vitro embryos. This is congruent with previous findings that in vitro embryos around EGA have an increased cytoplasmic content of apoptotic factors and the low developmental rates observed for in vitro embryo production [13]. This could indicate that in vitro produced embryos with compromised developmental competence are arrested at an early stage of development.
Both the in vivo developed and in vitro produced 4-cell to morula transition was characterized by an enrichment of oxidative phosphorylation, tRNA charging and EIF2 signaling. An increase in oxidative phosphorylation with developmental progression has previously been reported for mouse embryos [24]. Oxidative phosphorylation accounted for 60-70% of consumed oxygen in blastocysts, compared to 30% of consumed oxygen in cleavage stage embryos [24]. In addition, oxygen consumption of in vivo bovine blastocysts increased with increasing morphological quality and developmental stage [25]. Yet, in vitro produced embryos displayed a higher oxygen consumption, which was related to lower pregnancy rates [25]. Thus, after initial selection around the time of EGA, in vitro morulae seem developmentally competent, as they display increased transcription of genes related to oxidative phosphorylation, as observed for the in vivo embryos. EIF2 signaling has previously been shown to be downregulated in parthenogenetically activated expanded porcine blastocysts compared to in vivo developed embryos, evidencing a correlation between aberrant EIF2 signaling and reduced developmental competence [26]. EIF2 signaling was upregulated in morulae compared to 4-cell embryos, irrespective of embryo source, evidencing cell growth and proliferation [27].
During the morula to the hatched blastocyst transition, both in vivo developed and in vitro produced embryos displayed an enrichment of the pathways 14-3-3-mediated signaling, signaling of Rho family GTPases, and NRF2-mediated oxidative stress response. The 14-3-3 signaling plays a role in normal growth and development [28], cell polarity [29], and cell fate [30]. Signaling of Rho family GTPases in mice has recently been shown to be important in blastocoel formation and the regulation of trophectoderm-specific marker genes [31]. The upregulation of genes related to this signaling pathway indicates a physiological blastulation of embryos, with normally developing trophectoderm and expanded blastocoels. In bovine, the NRF-2 mediated oxidative stress response is enriched in competent blastocysts [32], and the functions and processes related to the NRF-2 mediated oxidative stress response and oxidative phosphorylation pathways have been suggested to be related to developmental competence [33]. Thus, our data suggests an initial natural selection of in vitro produced embryos around the EGA. Embryos that develop past the 2- to 4-cell stage display a higher developmental competence. The enrichment of the shared signaling pathways in both in vivo developed and in vitro produced embryos during further early embryo development appeared to be indicative of largely similar developmental transcriptional profiles, potentially related to embryo competence.
In vivo developed versus in vitro produced hatched blastocysts
The differences between in vivo developed and in vitro produced hatched blastocysts were investigated to understand persisting transcriptional differences and their relationship to embryo competence. Whitworth et al. (2005) previously reported DEGs in porcine blastocyst stage embryos by comparing in vivo developed and in vitro produced embryos [23]. Unlike the difference in expression of HMGB1 they reported, we did not find a difference in its expression between in vivo developed and in vitro produced hatched blastocysts. The expression of HMGB1 has been associated with the number of nuclei per embryo [23], suggesting that the stage of our hatched blastocysts is likely similar, thereby allowing the comparison between in vivo developed and in vitro produced embryos at this developmental stage. Likewise, there was no significant difference in the expression of ATP5A1 between in vivo developed and in vitro produced hatched blastocysts. The expression of ATP5A1 has previously been used to indicate differences in metabolic rates in in vivo developed and in vitro produced blastocysts [23]. In addition, 71% of genes related to cellular metabolism were reported to be upregulated in in vivo developed compared to in vitro produced porcine blastocysts [34]. The in vitro hatched blastocysts in this study displayed a significant increase in amino acid metabolism. Among the genes related to amino acid metabolism, the arginine transporter SLC7A1 has previously been reported to be significantly upregulated in in vitro produced embryos compared to in vivo developed embryos [35]. Porcine embryos deplete arginine from the culture medium at a higher rate at the expanded blastocyst stage compared to early blastocysts [36]. The arginine concentration in the embryo culture medium used in this study was at 0.1 mM [37]. It has previously been shown that adding arginine to a final concentration of 0.36 mM to the embryo culture medium decreased the SLC7A1 transcript level in in vitro produced embryos to a level comparable to the in vivo developed embryos [35]. In our study, the in vitro produced hatched blastocyst displayed a higher transcript expression of genes related to tRNA charging, cell cycle: G1/S checkpoint regulation, PEDF signaling and neuroinflammation signaling pathway. The in vivo developed embryos displayed a higher transcript expression of genes related to cyclins and cell cycle regulation. In vitro produced porcine blastocyst have previously been reported to display a higher transcript expression of genes involved in, among others, mRNA transcription, nucleotide metabolism, DNA metabolism, amino acid metabolism, and lipid metabolism [35]. The higher metabolic rate of in vitro produced embryos is evidenced in our in vitro hatched blastocysts by an enrichment in tRNA charging, the G1/S checkpoint in which DNA damage is usually repaired prior to replication, and the PEDF signaling which is related to an increased level of apoptosis. This transcriptional profile is in line with the proposed quiet embryo hypothesis, where viability is highest for embryos with a low rate of metabolism [38]. In addition, embryos with high DNA damage display an increased amino acid turnover [39, 40]. Thus, we propose that the transcriptome of in vitro produced hatched blastocysts is indicative of an increased level of DNA damage, as evidenced by the enrichment of the G1/S checkpoint regulation and the PEDF signaling, and the higher degree of amino acid metabolism. The effect of adding higher concentrations of arginine, i.e., 0.36 mM instead of 0.1 mM, to the embryo culture medium on the embryos’ amino acid metabolism and DNA damage should be evaluated. Thereby, an improvement of the currently employed in vitro fertilization pipelines can be assessed.