The present study describes a global metabolic perspective of the effects of different Se sources associated with B6 on PEB after maternal supplementation, and generates some hypothesis based on these new findings. Although the same microarray database was used by Dalto et al. [3] and the present study, those authors used the pig genome map draft 9.0 for genes annotation and a more restrictive statistical approach in order to evaluate highly affected processes, functions, and components related with each treatment. In the present study re-annotation was made using the updated pig genome map version 10.2 and parameters related with statistics and genes selection were chosen in order to provide a global overview of the effects of Se in PEB.
Considering that the present results were observed in self-regulated organisms (expanded blastocysts) that are genetically dissimilar (half allogenic) from those directly receiving the treatments (dams), such effects would require major metabolic alterations. In fact, Dalto et al. (2015; original database) reported that maternal blood Se was clearly modulated by dietary Se. Therefore, non-Se fractions of dietary OSe (mainly yeast) or MSe (Na + ion) are unlikely involved in such major systemic metabolic changes.
Both sources of Se (MSe and OSe) are used as dietary supplements for pigs but, depending on the Se status of the animal, they follow different metabolic pathways [4] before producing a common metabolite (selenide) required for the synthesis of selenoenzymes [5]. According to Burk et al. [14], in mouse, MSe does not take part of the maternal methionine pool but is mainly incorporated in selenoproteins whereas OSe is mostly deposited in the methionine pool with limited synthesis of selenoproteins. Considering that the uterine flushing of 5 days gestation gilts is a negligible route of Se transfer for the embryo [3], the Se content of pre-ovulatory oocytes is likely to be the main source of this mineral for pre-implantation porcine embryos. Therefore, whatever the source of maternal dietary Se, the embryo Se content would derive from an organic metabolite, either the oocyte SeMet pool and/or oocyte selenoproteins.
In this sense, we hypothesize that for both MSe and OSe supplemented gilts, maternal (oocyte) selenoproteins serve, after proteolysis, as a common source of SeCys for either embryo protein deposition or selenoproteins synthesis, whereas oocyte SeMet (OSe) might be directly deposited into embryo proteins and/or undergo transmethylation, because the transsulfuration pathway is not fully functional in embryos [1].
This hypothesis involving a common source of embryonic Se between OSe and MSe is indirectly supported by the present and previous studies. In fact, all MSeB610 vs CONT GO terms were also found in OSeB610 vs CONT comparison, suggesting that both sources of Se share a common metabolism but also that OSe may have additional metabolic pathways. Fortier et al. [15] and Dalto et al. [16] reported that even after placentation (30 days of gestation) porcine embryos from MSe or OSe supplemented gilts have similar Se-dependent glutathione peroxidase (Se-GPX) activities, indicating that independently of maternal Se source, embryos may obtain their Se content from a common intermediary Se metabolite, which is subjected to similar regulations for the synthesis of Se-GPX. Nevertheless, in both studies, the Se content of embryo from OSe supplemented gilts was greater than in those from MSe supplemented gilts, indicating the presence of different and/or additional Se metabolic pathways in OSe embryos.
DNA repair
DNA damage triggers repair pathways, but also signalling pathways that stimulate cell cycle checkpoints, apoptosis, transcription, and chromatin remodelling [17]. It has been proposed that, among the mechanisms for monitoring DNA integrity, cells may detect stalled RNA polymerases or abortive transcripts, activating DNA damage signaling [18]. The present results for OSeB610 x MSeB610 indicate that Se acts in one of these signaling pathways, the nucleotide excision repair (NER). More specifically, it affects the transcription-coupled NER (TC-NER), a specialized sub-pathway of NER responsible to repair DNA lesions in transcribed strands and coupled to active transcription [19, 20]. The high similarity between GO terms for OSeB610 vs CONT and OSeB610 vs MSeB610 and the up-regulation of CDK7 (gene that links transcription initiation, DNA repair, and cell cycle) in OSeB610 vs CONT suggests a major influence of OSe in this process.
SeMet (main OSe form) have been reported to impact protein p53 activity [21], a protein that under conditions of permanent blockage of transcription may stimulate p53-dependent apoptosis [22]. Therefore, the fast removal of transcription blocking lesions is crucial to avoid the detrimental effects of transcription inhibition. Studies have proposed a direct role of p53 in the OSe-induced activation of the NER pathway through its interaction with NER-associated proteins [23,24,25]. However, in the present data, none of the genes proposed to link p53 and NER [growth arrest and DNA damage inducible alpha (GADD45A), proliferating cell nuclear antigen (PCNA) and apurinic/apyrimidinic endodeoxyribonuclease 1(APE1)] [26] were expressed. Moreover, GADD45GIP1, a gene induced by p53 and that inhibits G1 to S phase of cell cycle progression was, in fact, down-regulated. Therefore, the present study suggests the possible role of alternative mechanisms for OSe in maintaining genomic stability in PEB through TC-NER. The present data suggests two likely possibilities, the activation of caspases by C-terminal-modified TXNR or ubiquitylation of damage-arrested RNA polymerase.
C-terminal-modified TXNR is unable to reduce TXN, but may replace the p53 apoptotic function via activation of caspases [27]. Interesting, Dalto et al. [3] reported a down-regulation of TXN in OSeB610 x CONT and OSeB610 x MSeB610, whereas in the present study CASP3, 6, and 7 were up-regulated in these comparisons. For damage-arrested RNA polymerase, it might be released from the template by a mechanism that leads to its ubiquitylation and degradation [22], in which genes ERCC excision repair (ERCC) and cullin (CUL) play a main role. In OSeB610 x CONT, genes encoding proteins that are important in ubiquitination of proteins involved in cell cycle progression, signal transduction and transcription were up-regulated [ANAPC1, CUL1, CUL2, DDB1 and CUL4 associated factor 7 (DCAF7) and ERCC8]. Globally, these results on DNA repair are consistent with the numerically greater amount of viable embryos collected from OSeB610 gilts by Dalto et al. [3], and this, in absence of degenerated embryos.
mRNA processing
After the formation of the precursor mRNa (pre-mRNA) in the nucleus, it has to be converted to mature mRNA by splicing [28] in order to be translated. Pre-mRNA splicing occurs in the spliceosome, a large ribonucleoprotein complex composed of five U-type small nuclear ribonucleoprotein particles (snRNPs) and splicing factors [29].
The present data for OSeB610 x CONT indicates that OSe acts on the ribonucleoprotein complex assembly. Although SeMet may have a structural function by replacing methionine in proteins, in PEB the most important effect of OSe appears to be on splicing factors activity. In fact, many dead-box proteins, pre-mRNA processing factors, snRNPs polypeptides, and other splicing factors (among them serine/arginine-rich splicing factors, poly (A) polymerases, and cleavage stimulation factors 3′ pre RNA) were impacted by maternal OSe supplementation.
During the second catalytic step of splicing, the exon junction complex (EJC) is formed on the pre-mRNA strand at the exon-exon junction [30, 31]. In the present data for OSeB610 x CONT, two [eukaryotic translation initiation factor 4A3 (EIF4A3) and mago homolog, exon junction complex core component (MAGOH)] of the three core proteins of the EJC were expressed. This protein complex has major influences on translation, localization of the spliced mRNA, and mRNA surveillance. According to Brogna & Wen [32], in mammalian cells, EJC mediates the link between splicing and nonsense-mediated decay pathway (NMD), a surveillance translation-coupled mechanism that eliminates mRNAs containing premature translation-termination codons [33].
The NMD pathway is assumed to affect selenoproteins expression because they contain multiple SeCys residues, which are encoded by the UGA codon that normally signals translation termination. In selenoproteins, however, SeCys insertion sequence (SECIS) recognizes UGA as a SeCys codon rather than as a stop signal. Under Se deficiency, Seyedali & Berry [34] showed that NMD may act on selenoproteins mRNA. In the present data, SECIS binding protein 2 (SECISBP2) was up-regulated in OSeB610 x MSeB610, suggesting that selenoproteins synthesis was active. In fact, selenoprotein K was up-regulated in both OSeB610 x CONT and OSeB610 x MSeB610. For the most known selenoproteins (glutathione peroxidases, iodothyronine deiodinases, and thioredoxin reductases), none of them was differentially expressed. This, however, does not imply that their synthesis was impaired but that maternal Se supplementation did not further stimulate their synthesis compared to the control diet or between Se sources.
mRNA translation
The present data indicates that besides the function of OSe as a structural component of the ribosome, both MSe and OSe play roles in translation, stimulating RNA binding factors and influencing translational initiation. A deeper evaluation of individual genes in these GO terms revealed that, under the present experimental conditions, Se is likely to influence specifically the Cap-dependent initiation. This process involves the interaction of the eukaryotic translation initiation factor (eIF) complex and the 5′ cap as well as with the 5′ untranslated region [35]. Additionally, the transport of the initiator tRNA, which encodes the amino acid methionine in eukaryotes, to the P-site of the small ribosomal subunit is performed by eIF2. This protein is also responsible for signaling the dissociation of several factors from the small ribosomal subunit, among them eIF3 that avoids the premature binding of the large ribosomal subunit, leading to the association of the large subunit and translation elongation [36]. Except for eIF5 and eIF6 that were not expressed by MSeB610 x CONT, all three comparisons have expressed many subunits of important eIFs.
Intracellular ceramides synthesis
mRNA translation was expectedly related with peptide biosynthesis, which in turn was related with amide biosynthesis. Although the association between amides and peptides may be due to the presence of amide bonds in the forming protein, a deeper evaluation of individual genes revealed that the GO term amide biosynthetic process had many genes related with two out of the three pathways of ceramides biosynthesis.
The de novo pathway begins with the formation of 3-keto-dihydro-sphingosine from palmitoyl-CoA and serine by serine palmitoyl-transferase (SPTLC2) in the endoplasmic reticulum. Further, 3-keto-dihydro-sphingosine is reduced by 3-keto-dihydro-sphingosine reductase (KDSR) to form dihydro-sphingosine followed by an acylation by ceramide synthase (CERS1, CERS5, and CERS6) to form dihydro-ceramide, which is desaturated by delta 4-desaturase (DEGS) to form ceramide. Ceramide is subsequently transported to the Golgi apparatus where it is further metabolized and the outcomes transported to the plasma membrane [37].
Breakdown pathways allow the reversion of sphingolipids back to ceramide. One of the most biologically important reactions is the breakdown of sphingomyelin in the cell membrane releasing ceramide. Considering that sphingomyelin is the most abundant complex sphingolipid in human cells, its coordinated breakdown is an essential part of membrane homeostasis [38]. Besides the breakdown of sphingosine by ceramide synthases, the present results support that OSe plays a role in the breakdown of sphingomyelin by sphingomyelin phosphodiesterase (SMPD4 and SMPDL3A) and in the synthesis of sphingosine by N-acylsphingosine amidohydrolase 1 (ASAH1) and/or alkaline ceramidase 3 (ACER3). Sphingosine may enter the cell or it is metabolized by sphingosine kinase 1 (SPHK1), a protein regulated by SPHK1-interacting protein (SPHKAP), to form sphingosine-1-phosphate. The latter may be dephosphorylated by phospholipid phosphatase 1 (PPAP2A) to resynthesize sphingosine. In the cell, sphingosine can either go directly to the endoplasmic reticulum or enter the mitochondria where it will be metabolized back to ceramide and released into the endoplasmic reticulum [37]. Among all genes mentioned in de novo and breakdown pathways, only KDSR and SPHK1 were not expressed in OSeB610 x CONT and/or OSeB610 x MSeB610.
Simple sphingolipids have significant signaling and regulatory roles within cells, with serious consequences for mammalian physiology [37]. Ceramide and sphingosine-1-phosphate have been shown to be important mediators in the signaling cascades involved in apoptosis, proliferation, differentiation, cell growth arrest, inflammation, cell migration and adhesion. In this sense, many factors known to promote the synthesis of sphingosine-1-phosphate [39] were expressed in the present data [platelet-derived growth factor subunit A (PDGFRA), insulin-like growth factor 1 receptor (IGF1R), vascular endothelial growth factor β (VEGFB), tumor necrosis factor-related genes (TNFRSF21, TNFAIP8L3, TNFSF4, and TNFRSF9), and low-density lipoprotein receptors (LRP2 and LRP6). Globally, these results are once again consistent with the numerically greater amount of advanced-stage embryos collected from OSeB610 gilts by Dalto et al. [3].
Intracellular peptides trafficking
Besides the effects on peptides and lipid-related metabolites biosynthesis, OSeB610 x CONT and OSeB610 x MSeB610 comparisons also showed an impact on their transport via the signal recognition particle (SRP)-dependent cotranslational pathway of protein targeting to membrane. The cotranslational pathway uses SRP to deliver secretory proteins to a membrane-bound protein-conducting channel (translocon), which is present in the endoplasmic reticulum membrane, concomitantly with their synthesis in ribosomes [40]. In mammals, SRP consists of six proteins (encoded by SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72) and a 7S RNA, most of which had genes expressed in the above mentioned comparisons. This process begins with the recognition of the signal peptide of the protein by SRP during the protein synthesis in the ribosome [41], with further insertion of the nascent protein into the translocon [42]. The translocon complex consists of oligosaccharyl transferase complex, the translocon-associated protein (TRAP) complex, and the translocating chain-associating membrane protein (TRAM), besides the central element Sec61 [43]. For the present data, many genes related with the translocon complex (3 SEC61 subunits, STT3A, STT3B, and 7 TRAPPC subunits) were expressed in OSeB610 x MSeB610 and/or OSeB610 x CONT. Once the nascent polypeptide has been translocated into the endoplasmic reticulum membrane, the signal sequence is cleaved by signal peptidases (GTP hydrolysis) [44, 45], some of which were expressed (SPCS1, SPCS2, and SPCS3) in OSeB610 x MSeB610 and/or OSeB610 x CONT.
Signal peptidases are also found in the mitochondria import machinery [46]. The pre-protein containing peptide signals targeting the mitochondria is bound by translocases of outer membrane (TOM) and transported through the intermembrane space by translocases of inner membrane (TIM) [47]. Among the three mitochondrial TOMs, those responsible for binding pre-sequences and internal targeting peptides (TOM20 and TOM22) were expressed in OSeB610 x MSeB610 and OSeB610 x CONT. Additionally, TIM23, which acts as a translocator of pre-proteins for the mitochondrial matrix, the inner membrane, and the intermembrane space, was affected in these same comparisons.
Exclusively for the comparison OSeB610 x CONT, the present data show an effect on mitochondrial respiratory chain complex assembly, more specifically the NADH dehydrogenase (ubiquinone) activity (21 NADH dehydrogenase and 5 cytochrome c genes stimulated). The evaluation of individual genes in these GO terms showed that Se acts mainly as a structural component, contrary to its expected antioxidant action against reactive oxygen species in the electron transport chain.
Epigenetics
It is already known that maternal diet may play a crucial role in epigenetic programming of conceptus development [48] and that methyl dietary supplements (such as SeMet) can alter the methylation of specific imprinted genes [49]. For this reason, an individual search for epigenetic-related genes was performed. Many important genes related with epigenetic events were expressed in OSeB610 x MSeB610 and/or OSeB610 x CONT (DNMT1, DNMT3B and 19 other methyltransferases as well as MBD3, MBD4, MBD3L5, HAT1, HDAC1, HDAC9, KDM5C, and WDR5) whereas only a few in MSeB610 x CONT (HDAC1, HDAC9, KDM5C, and 6 methyltransferases).
Epigenetic processes are dynamic during embryogenesis partially because of the significant amount of DNA synthesis that occurs in this period [50]. During the pre-implantation period, embryos’ epigenome is particularly susceptible to environmentally induced modifications. At this period, de/methylation of DNA and histones modification occurs. Effects of supplemental Se on global and gene-specific DNA methylation have been reported [51, 52], but never in PEB after maternal Se supplementation. Transfer of methyl groups from SAM to the 5-carbon position of cytosine by DNA methyltransferases (DNMTs), results in 5-methylcytosine (5 mC) [53]. In contrast, DNA demethylation is not catalyzed directly but results from either DNA replication-coupled dilution or replacement of 5 mC. For histones, the interference of nutrients occurs mainly through modulation of histone modifying enzymes and via interference with substrate availability. According to Narayan et al. [54], selenoprotein biosynthesis is crucial for selenite-induced modulation of histone H4 acetylation, supporting the conclusion of Dalto et al. [3] that, although not influenced by Se sources with B6, PEB are potentially capable of synthesizing selenoproteins.
A tempting interpretation for the higher impact of OSe (SeMet) compared to MSe (selenite) on epigenetic events would be to link it to the synthesis of SAM during the demethylation of SeMet to Se-homocysteine. However, as the metabolic ratio between methionine and SeMet is generally considered to be above 3000/1 [55], it appears likely that the contribution of SeMet to SAM (a sulfurized metabolite) synthesis is negligible. Nevertheless, this is dependent upon the specific biosynthetic substitution of methionine by SeMet in PEB cells which has never been determined in pigs. Using recombinant human annexin V expressed in E. coli as a model, Budisa et al. [56] showed that, independently of experimental SeMet concentration (ranged from 0.3 to 0.8 mM), methionine was fully replace by SeMet. However, it has to be stated that the biosynthetic substitution of methionine by SeMet may vary considerably between species.
If the above mentioned ratio of 3000/1 between methionine and SeMet also applies to PEB, it has to be assumed that it is reflected on intermediary metabolites of the transmethylation pathway, such as Se-homocysteine. Therefore, the demethylation of SeMet to Se-homocysteine would also be quantitatively negligible within the total (sulfur + Se) homocysteine levels. However, some studies showed an inverse correlation between Se status and total (sulfur + Se) homocysteine levels [57, 58] indicating the important impact of Se on intermediary metabolites of the transmethylation pathway. Consequently, the question remains as whether selenized molecules would be more active than sulfurized ones within the transmethylation pathway or whether alternative Se-dependent metabolic pathways would be present in PEB to influence epigenetic events.