Differences in the growth performance between the IUGR and normal body weight (NBW) piglets
In this study, the body weight of all piglets was summarized in Fig. 1a. The initial body weight of the IUGR neonates was significantly lower than that of the NBW on Day 1 as expected (P < 0.01). However, the body weight of the IUGR piglets was consistently lower than that of the NBW on Day 7 and Day 28 (P < 0.01). By calculating the relative body weight of the IUGR piglets to NBW piglets, the results showed that the body weight ratios were 45, 44, and 66% on Days 1, 7, and 28, respectively (Fig. 1b). It was noteworthy that the gaps in body weight between the IUGR and NBW piglets was reduced on Day 28 compared with that on Day 1 and Day7, which implies a catch-up growth compensation in IUGR piglets.
Furthermore, in line with the decreased body weight difference between the IUGR and NBW piglets, growth compensation was also supported by the increasing ADG ratio of the IUGR piglets throughout the postnatal period (Fig. 1c and d). In addition, no significant sexual-dimorphic effects on the growth performance of the body weight and ADG were observed between the IUGR and NBW piglets at each time point.
General profiling of DEGs between the IUGR and NBW piglets
Transcriptome sequencing was performed using a total of 42 liver samples from the IUGR and NBW piglets on Days 1, 7, and 28, respectively [Day 1: IUGR n = 8 (4 females and 4 males) vs NBW n = 8 (4 females and 4 males); Day 7: IUGR n = 7 (4 females and 3 males) vs NBW n = 7 (4 females and 3 males); Day 28: IUGR n = 6 (3 females and 3 males) vs NBW n = 6 (3 females and 3 males)]. Approximately 20,000 transcripts were detected in each sample. Compared with NBW, the liver of IUGR piglets contained 516 differentially expressed genes (DEGs) on Day 1 (P < 0.05; FC > 2 or < 0.5). Of these, 292 were up-regulated and 224 were down-regulated. On Day 7, 173 DEGs were screened out, 105 of which were upregulated and 68 were downregulated. Notably, the number of DEGs decreased along with the postnatal period, and only 84 DEGs were screened out on Day 28. At each time point, the mildly altered DEGs (4 > FC > 2 or 0.5 > FC > 0.25) accounted for the largest proportion of DEGs (Fig. 2a and b; Supplementary file: Table S1-S3). These results suggested that the altered gene expression profiles in the IUGR piglet livers could be attenuated with postnatal development.
In addition, a Venn diagram was used to screen the consistently dysregulated DEGs during the postnatal stage. The results showed that an extremely small number of DEGs were consistently regulated between each time point. Only one DEG was consistently dysregulated throughout the entire postnatal period in the IUGR piglets. There were 24 DEGs that were consistently dysregulated from Days 1 to 7, 3 DEGs were consistently dysregulated from Days 7 to 28. There were 484, 145, and 73 DEGs specifically dysregulated on Days 1, 7, and 28, respectively. The large proportion of stage-specific DEGs at each time point suggested that disordered liver functions or development are highly dynamic in IUGR piglets. Despite this finding, 12 and 10 DEGs were consistently up- and down-regulated from postnatal Days 1 to 7 (Fig. 2c). These DEGs were involved in multiple cellular processes, including inflammatory immunity (SCUBE1 and CD200R1), nutrient transport (SLC38A5, SLC51B, and MCT7), and cellular proliferation and migration (CCDC38, ARMC12, and CDH16) (Fig. 2d). Five of these DEGs (SLC38A5, SLC51B, DMRTA1, ADAD1, and CD200R1) that were involved in important biological processes and functions, were further detected using real-time qPCR to validate the reliability of the RNA-Seq analysis (Fig. 2e).
Detailed functional profiles of the DEGs between the IUGR and NBW piglets
The following functional analyses were based on Gene Ontology (GO) for the dynamically altered DEGs between the IUGR and NBW piglets to explore the potential physiological changes in the IUGR liver. GO classification of the biological processes (BP) showed that the dysregulated DEGs were most significantly enriched in the hepatic immune response on Day 1, including ‘lymphocyte migration’, ‘leukocyte cell-cell adhesion’, ‘regulation of chemotaxis’, and ‘regulation of leukocyte activation’ (Fig. 3a). These findings suggest that the liver of IUGR piglets may suffer from immune-related stress. DEGs were also clustered in items, such as ‘response to glucocorticoid’ and ‘response to steroid hormone’, which may imply a disordered steroid hormone metabolism and response. It is important to note that most of the DEGs related to immune regulation were down-regulated, whereas those related to sterol hormone regulation were up-regulated through GOCircle plot analysis (Fig. 3b). We further focused on these DEGs, and the GOChord plot was performed to select the DEGs, which were assigned to at least three BP terms (Fig. 3c). Among these, GPR183, STAP1, HAVCR2, CCR7, TNF, CCL4, WNT5A, and CCL2 were all involved in the innate and adaptive immune response and homeostasis, whereas IGF1, IGFBP2, RORA, AGTR2, NTRK3, and HSPH1 were related to cellular growth, differentiation, and developmental regulation (Fig. 3d). To further investigate the functional relationship among the DEGs on Day 1, the protein-protein interaction (PPI) was constructed using the STRING database. The interconnected DEGs were also clustered in the subnetwork of steroid hormone biosynthesis and regulation, fatty acid metabolism, and immune response (Fig. 3e). Next, the node genes of the DEG network were ranked by the CytoHubba, and the top 10 hub genes and related functions were presented. These genes contained TNF, chemokines (CCL4), and their receptors (CCR7 and CCR8), which can cause inflammation. It also contained genes from the G protein-coupled receptor family (GPR183, GRM4, GALR1, and AGTR2), which regulated G protein activity in the liver (Fig. 3d). Some of the screened DEGs were overlapping in the GOChord and CytoHubbar analysis, implying the importance of these genes in determining the phenotype of IUGR piglets.
Next, we performed a detailed analysis of the DEGs on Day 7. The majority of the DEGs were enriched in the regulation of actin filament depolymerization and polymerization processes. DEGs in these terms were primarily involved in the assembly of the actin filament network and maintenance of the actin skeleton (ADD2, KIAA1211, and SPTB). Moreover, the DEGs were also concentrated in the muscle tissue growth (DKK1, EGR1, EGR2, FOS, KEL, and SHOX2), as well as hormone biosynthesis and metabolism processes (ADM and EGR1) (Fig. 3f). These indicate that the dysregulated DEGs may affect the cytoskeleton reorganization in the IUGR liver tissue on Day 7. The DEGs on Day 28 were analyzed in the same manner, which were primarily enriched in the ‘cellular transition metal ion homeostasis’ process, including ATP6V1G1, HAMP, SLC30A4, and TFRC. Of these, both HAMP and TFRC regulated the maintenance of ion homeostasis, and SLC30A4 exerted zinc transmembrane transporter activity. Dysregulation of transition metal ion homeostasis may be the molecular basis for the abnormal physiological characteristics of IUGR piglets. At the same time, these DEGs contained CD209, TLR8, and UBE2D2, which were clustered in inflammatory entries (e.g., ‘positive regulation of T cell proliferation’, ‘innate immune response-activating signal transduction’, and ‘type I interferon biosynthetic process’). All of these entries may be suggestive of an abnormal state of immune stress in IUGR piglets (Fig. 3g).
Finally, a KEGG analysis was performed to determine the pathways that participate in the disordered functions exhibited in the livers of the IUGR piglets. The PI3K-AKT signaling pathway, glycerolipid metabolism, and the HIF-1 signaling pathway were significantly enriched consistently during the postnatal period. Moreover, the cAMP signaling pathway, cytokine-cytokine receptor interaction, phagosome, MAPK signaling pathway, and steroid hormone biosynthesis were also enriched (Fig. 3h). These enrichment pathways fully revealed the pathophysiological status of the IUGR piglets. Moreover, the number and significance of the enriched pathways also supported the concept that disordered state of IUGR appeared to be alleviated during postnatal development.
Analysis of serum biochemical parameters and liver histology between the IUGR and NBW piglets
Given that the DEGs between IUGR and NBW piglets were related to the abnormal immune response, we next compared the liver function index between the IUGR and NBW piglets to assess the potential impact of immune stress on the liver damage in IUGR piglets. The liver function indexes in the IUGR piglets changed significantly, as the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity in the IUGR piglets was significantly higher than that in the NBW piglets at all of the time points. Moreover, the total protein (TP) content, a biomarker of the inflammatory status in the liver, was found to be significantly lower in the IUGR piglets than that in the NBW piglets (Fig. 4a), which predicted the inflammatory status in the livers of the IUGR piglets.
We subsequently detected the hepatic pathological sections in IUGR piglets. Compared with the NBW piglets, the IUGR piglets displayed marked inflammatory lymphocytic infiltration in the hepatic lobules at different time points. Additionally, apparent vacuolar and severe structural damage appeared in the IUGR hepatocytes on Day 28 (Fig. 4b). These results further confirm the existence of liver injury in IUGR piglets.
In addition, a comparison of the ultrastructural morphology of the liver between IUGR and NBW piglets was evaluated using transmission electron microscopy (TEM). In the present study, ultrastructural pathological lesions were observed in the hepatocytes of IUGR piglets. Striking structural alterations were identified in the IUGR piglets, including vacuolar dilatation of the cytoplasm, loss of cytoplasmic material and degeneration of hepatocyte organelles, especially in the mitochondria and endoplasmic reticulum. These observations indicated that the mitochondria were swollen, round-shaped, and the mitochondrial cristae were disrupted. Furthermore, discontinuous endoplasmic reticulum cisternae were also observed among the hepatocytes in IUGR piglets at each time point. Whereas a normal histological appearance with well-organized organelles was observed in the liver sections of the NBW piglets (Fig. 4c). These results further support that ultrastructural cytoskeleton is disrupted in hepatocytes of IUGR piglets.
Sexual-dimorphic effects on the liver expression patterns between the IUGR and NBW piglets
Given the sex-biased growth phenotypes that we observed, it was hypothesized that the transcriptomic changes also exhibited sexual dimorphic patterns in the IUGR piglet livers. Transcriptional information was analyzed between the IUGR and NBW groups within the male and female piglets (Supplementary file: Table S4-S9). Sex-specific profiling of the DGEs during postnatal development revealed different dynamics between the male and female IGUR piglets. In female IUGR piglets, the number of DGEs decreased as early as Day 7, whereas the number of DGEs decreased until Day 28 in the male IUGR piglets (Fig. 5a and b). The different patterns of gene expression raise the possibility that female IUGR piglets may have a greater potential to compensate for postnatal growth.
Secondly, we filtered sex-specific DEGs at each time point using a Venn diagram of DEGs from both female and male IUGR piglets (Fig. 5c). On Day 1, 909 DGEs were specifically dysregulated among the female IUGR piglets, whereas 544 DGEs were specifically regulated in the male IUGR piglets, and only 72 DGEs were common to both the male and female IUGR piglets. On Day 7, there were 87 and 636 DGEs specifically dysregulated in both female and male IUGR piglets, respectively, with only 2 shared DEGs between the males and females. On Day 28, 127 and 68 DGEs were specifically dysregulated in female and male IUGR piglets, with only 2 shared DEGs between the males and females. Given that the great majority of the dysregulated DEGs exhibited sexual dimorphism, we propose that the mechanisms underlying the IUGR-associated liver disorders may differ between male and female piglets.
Next, to explore the possible differential mechanisms, DEGs specific to males and females were analyzed. On Day 1, the GO classification showed that the DEGs in the female IUGR were most enriched during the process of cell cycle regulation (Fig. 6a). The GOCircle plot analysis showed that most DEGs enriched in cell cycle regulation were down-regulated (Fig. 6b). With the same setting parameters as described above, the candidate DEGs were focused through the GOChord plot analysis and PPI analysis (Fig. 6c and d), including serine/threonine kinase subfamily members (CDK1, AURKB, and CHEK1), kinesin family members (KIF2C and KIF18A), chromosome replication, repairment-related genes (RPA2, RPA3, CDT1, CDC6, DSCC1, BRCA1, GEN1, and FBXO5), and cell cycle regulation-related genes (CCNA1, CCNB2, NUF2, CENPE, SGOL1, SKA1, and CDDA5). The detailed functions of these genes are presented in Fig. 6e. Similarly, the DEGs on Day 7 were also associated with the regulation of the cell cycle (e.g., ‘synaptonemal complex assembly’, ‘meiosis I cell cycle process’, and ‘meiotic nuclear division’). The candidate genes included in these terms were STAG3, C11orf80, and SYCP2. In addition, some physiological metabolic processes, including the regulation of transcription (EGLN3 and MT3), immune response (GNLY, LYZ, and PGC), and apoptosis-related processes (EPO, GZMB, IL20RA, and MMP9) were also significantly enriched (Fig. 6f). On Day 28, the DEGs specific to female IUGR piglets were functionally associated with GO terms, including a response to a toxic substance (CBL, HP, and FOS), cold-induced thermogenesis (ADRB1, NPR3, and PEMT), epithelial cell apoptotic process (ANGPT1, CCL2, KRT18, and KRT8), as well as phosphatidylinositol 3-kinase signaling (ANGPT1, IER3, and PRR5) (Fig. 6g).
Unlike the female IUGR group, the GO classification of DEGs in male IUGR piglets on Day 1 were enriched in factors relevant to carboxylic acid and organic anion transport, monosaccharide metabolic processes, ribonucleotide biosynthetic processes, ribose phosphate biosynthetic processes, and hexose metabolism (Fig. 7a). Using a GOCircle plot analysis, we found that most DEGs associated with transport-related processes were up-regulated, whereas the DEGs in metabolism-related processes were down-regulated (Fig. 7b). We further obtained the following candidate genes through a GOChord plot analysis, including transporters (SLC26A2, SLC35B4, and ABCC2) and regulatory factors involved in both glucose and lipid metabolism (RORA, RORC, PDK4, PPARA, ACSL1, ACSL3, and HK2) (Fig. 7c). In addition, CytoHubba revealed that most of the top 10 hub genes were cytokines (CCL4, CCR5, CCR7, CCR8, CCR2, and GPR183) (Fig. 7d and e), which were involved in inflammatory regulation, indicating an immune response disorder in male IUGR piglets. Next, we analyzed the GO cluster on Day 7, DEGs of the male IUGRs were enriched regarding immune cell differentiation (SOCS1, NFKBIZ, and LAG3) and hematopoiesis (GATA1, MYB, TAL1, TRIM58, HLX, DLL1, and MAFB) (Fig. 7f). In the end, the GO items in the male IUGR on Day 28 included thyroid hormone metabolic processes (DIO2, PAX8, EGR1, and AFP) and lipoprotein transport (APOA4, MFSD2A, LIPG, SLC25A33, and SLC44A5). (Fig. 7g).
Serum lipid metabolites between the IUGR and NBW piglets
To explore the metabolic status of IUGR livers, we next tested the serum lipid profiles, highlighting the cholesterol (CHOL) and triglycerides (TG) that were commonly used as the clinical indexes to reflect the physiological or pathological state of the liver. As the results showed in Fig. 8, the level of CHOL and TG were both significantly increased in the IUGR piglets compared with the NBW groups for both the female and male groups. Interestingly, the TG level of male IUGR piglets was consistently higher than that of the female IUGR piglets from Days 1 to 7, and the CHOL level of male IUGR piglets was also significantly higher than that of female IUGR piglets on Day 1, which also exhibited sexual dimorphic patterns.