IMF content and serum lipid parameters
The amount of IMF was estimated by Oil Red O staining the neutral lipids in breast muscle with the aim to visualize the differences in IMF deposition of male and female chickens. Our results revealed that the density and size of lipid droplets in the female chicken group were higher as compared to those of the male chicken group. The IMF content reflected a sex-dependent pattern with significantly higher levels in 150-day female chickens (P < 0.05) (Fig. 1A). Besides, the estimated serum lipid parameters (TG, TC, HDL-C, LDL-C, and VLDL-C) highlighted significantly higher serum TG, LDL-C, and VLDL-C (P < 0.05), whereas significantly lower HDL-C (P < 0.01) in the female chickens than the male chickens (Fig. 1B, D, E, F, G). However, there was no statistical difference in TC between 150-day males and females (Fig. 1C).
Differentially expressed genes analysis
RNA-Seq experiment derived overall 83.14 Gb clean data and 597.82 million reads. Aligned with the reference genome, the mapping frequencies were found to vary from 82.38% to 93.53% for each sample. Among the mapped reads, an average of 82.66% of the total mapped reads was mapped to exons, 7.38% mapped to introns, and 9.96% mapped to the intergenic regions.
The DESeq2 program detected a total of 1281 transcripts as DEGs in the breast muscle tissue between male chickens and female chickens, of which 219 transcripts were upregulated, and 1062 transcripts were down-regulated in female chickens (Fig. 2A). In the liver tissue, a total of 598 DEGs were identified, of which 360 transcripts were upregulated and the remaining 238 transcripts were down-regulated in female chickens (Fig. 2B). The lipid metabolism-related DEGs in the breast muscle and liver were summarized in Table S1 and Table S2, respectively. Among the male and female chickens, 58 DEGs were common in the breast muscle and liver. Among these common DEGs, 10 genes, including ETNK2, PLIN2, OSBPL10, MFSD2A, SLC51B, GCNT2, ALDH7A1, CPT1A, LSS, and MSMO1, were relevant for lipid transport and metabolism. Among the DEGs, lipid biosynthetic process and lipid storage-related genes in pectoralis muscle tissue include ACSL1, MSMO1, HACD4, SLC27A1, LSS, LPIN1, DGAT2, and APOB that were upregulated in female chickens, indicating a significant role in lipid deposition of female chickens. As compared to the male chicken group, the expression of PLCZ1, LPIN2, and DDHD2, associated with lipid or fatty acid catabolic process, was significantly lower in the female chickens. Moreover, significant down-regulation of NR4A3 and PPARA, which participate in positive regulation of fatty acid oxidation, was witnessed in female chickens. Furthermore, some genes involved in long-chain fatty acid transport (THBS1, PLIN2), phospholipid transport (PLTP, MFSD2A, OSBPL5), cholesterol transport (LIPG, ABCA1), were significantly down-regulated in female chickens. These findings suggested the probable association of these genes with the higher IMF content in the female chickens compared with that in the male chickens. In terms of DEGs in the liver, up-regulation was evident in the genes belonging to the PPAR signaling pathway, such as ACOX1, ACOX3, ACAA1, CPT1A, FABP1, and FABP3, in the female chickens. Additionally, significantly higher expression of the fatty acid biosynthetic process-related genes, including HADHA, ELOVL2, ABHD2, ABHD3, ABDH6, and ABCD3, was observed than that in the female chicken group. However, the female chicken group manifested down-regulation of the genes involved in the steroid biosynthetic process (DHCR24, SQLE, NSDHL, CYP51A1, LSS, MVD, CYP17A1, HSD17B7, DHCR7, MSMO1). KEGG pathway analysis of the DEGs
The significantly enriched pathways obtained from the KEGG pathway analysis, conducted based on the known DEGs, are illustrated in Fig. 3. We identified 238 DEGs annotated into 174 pathways in FBM vs. MBM (Fig. 3A) and 197 DEGs annotated into 160 pathways in FL vs. ML (Fig. 3B). Since the homeostasis between synthesis, transport, and degradation of lipids regulates the IMF content (Zhang et al. 2017), we focused mainly on the pathways directly involved in lipid metabolism. The breast muscle and liver of female and male chickens witnessed five common significantly enriched pathways (metabolic pathways, fatty acid degradation, fatty acid biosynthesis, glycolysis/gluconeogenesis, and PPAR signaling pathway) (P<0.05). Moreover, the DEGs in the liver tissue were also found to be significantly enriched in steroid biosynthesis, steroid hormone biosynthesis, fatty acid metabolism, citrate cycle, and five amino acid-related metabolism pathways (P<0.05).
3.4 Gene expression validation of DEGs by qRT-PCR
The results of the RNA-Seq analysis were further confirmed by validating the expression data of randomly selected ten DEGs. Results revealed expression of five DEGs (APOB, SLC27A1, DGAT2, PLTP, LIPG) in the muscle, whereas five DEGs (CPT1A, ACAA1, FABP3, DHCR24, MSMO1) were expressed in the liver. Consistency of the qRT-PCR result with the RNA-Seq data with a correlation coefficient of 0.986 authenticated the accuracy of RNA-Seq (Fig. 4).
3.5 PLIN2 promoted intramuscular preadipocyte proliferation, differentiation, and inhibited apoptosis
PLIN2, a common DSG in the breast muscle and liver, was studied to further explore the biological significance of candidate genes. The tissue expression patterns of PLIN2 were determined by analyzing 13 tissues of female chickens using qRT-PCR. High expression of PLIN2 in the liver, subcutaneous fat, and abdominal fat indicated its potential role in the lipid metabolism of chicken (Fig. 5). Transfection with siRNA knocked down the PLIN2 expression, which helped to elucidate the regulatory role of PLIN2 in chicken intramuscular preadipocyte proliferation, apoptosis and differentiation. Compared with the negative control (NC) group, transfection with siRNA significantly downregulated the mRNA expression level of PLIN2 (Fig. 6A).
To explore the role of PLIN2 on chicken intramuscular preadipocyte proliferation, we detected the mRNA expression of cell-proliferation-related genes, including Cyclin D1 (CCND1), Cyclin D2 (CCND2), cyclin dependent kinase 2(CDK2), proliferating cell nuclear antigen (PCNA) and a marker of proliferation Ki-67 (Ki67). Results showed that the mRNA expression levels of CCND1, CCND2, CDK2, PCNA and Ki67 were all significantly decreased after transfected with si-PLIN2, compared with the negative control (NC) group (Fig. 6B). The protein expression level of CDK2 was also detected by western blotting and the result was consistent with the qRT-PCR results (Fig. 6C). Furthermore, CCK-8 results showed that the cell vitality of the si-PLIN2 transfected group was significantly lower than that of the NC group after 12, 24, 36, and 48h of transfection (Fig. 6D). EdU assay results showed that the percentage of EdU positive cells were significantly reduced in si-PLIN2 transfected group (Fig. 6E and F).
To investigated the role of PLIN2 in chicken intramuscular preadipocyte differentiation, we examined the mRNA expression levels of the adipogenic transcripts PPARγ, FABP4, C/EBPα and C/EBPβ. Results showed that mRNA expression levels of these genes were all downregulated after transfected with si-PLIN2 (Fig. 7A). The protein expression level of C/EBPβ was consistent with the qRT-PCR results (Fig. 7B). Besides, oil red O staining corroborated a decrease in the number of intracellular lipid droplets in the siRNA-PLIN2 group (Fig. 7C), and the OD510 values of the siRNA-PLIN2 group were also significantly lower as compared to that of the NC group (Fig. 7D). Cumulatively, these results indicated PLIN2 promoted intramuscular preadipocyte proliferation, differentiation, and inhibited apoptosis.
To further investigated whether PLIN2 could also modulate preadipocyte apoptosis, the mRNA expression levels of crucial mediators of apoptosis, including caspase-3, caspase-8 and Bcl-2 were examined. We found the mRNA expression levels of caspase-3, caspase-8, caspase-9 were upregulated and Bcl-2 mRNA and protein expression levels were downregulated after transfected with si-PLIN2 (Fig. 8A and B). The Apoptosis and Necrosis Assay results suggested that the interfere with PLIN2 significantly increased preadipocyte apoptosis when compared with the NC group (Fig. 8C and D).