Transcriptome profiling of longissimus dorsi during different prenatal stages to identify genes involved in intramuscular fat deposition in lean and obese pig breeds.

BACKGROUND: There was significant difference in muscle development between fat-type and lean-type pig breeds. METHODS AND RESULTS: In current study, transcriptome analysis and bioinformatics analysis were used to compare the difference in longissimus dorsi (LD) muscle at three time-points (38 days post coitus (dpc), 58 dpc, and 78 dpc ) between Huainan (HN) and Large white (LW) pig breeds. A total of 24500 transcripts were obtained in 18 samples, and 2319, 2799, and 3713 differently expressed genes (DEGs) were identified between these two breeds at 38 dpc, 58 dpc, and 78 dpc, respectively. And the number and foldchange of DEGs were increased, the alternative splice also increased. The cluster analysis of DEGs indicated the embryonic development progress of LD muscle between these two breeds was different. There were 539 shared DEGs between HN and LW at three stages, and the top-shared DEGs were associated with muscle development and lipid deposition, such as KLF4, NR4A1, HSP70, ZBTB16 and so on. CONCLUSIONS: The results showed DEGs between Huainan (HN) and Large white (LW) pig breeds, and contributed to the understanding the muscle development difference between HN and LW, and provided basic materials for improvement of meat quality.

Identification and characterization of circRNAs related to meat quality during embryonic development of the longissimus dorsi muscle in two pig breeds.

Meat quality, an important economic trait, is regulated by many factors, especially by genetic factors, including coding genes, miRNAs, and lncRNAs. Recent studies have elucidated that circRNAs also play a key role in muscle development and lipid deposition. However, the functions and regulatory mechanisms of circRNAs in meat quality remain mostly unknown. The circRNA expression profiles between Huainan pigs (Chinese indigenous pigs, fat-type, Huainan HN) and Large White pigs (Western commercial pigs, lean-type, LW) in the longissimus dorsi (LD) muscle at 38, 58, and 78 days post conception (dpc) were compared by sequencing. In total, 39,887 circRNAs were identified in 18 samples, and 60, 78, and 86 differentially expressed circRNAs (DECs) were found at the three stages mentioned above between these two breeds. The parent genes of DECs were enriched in myogenesis, proliferation, adipogenesis and muscle fiber-type transition. The circRNA-miRNA interaction networks included 38 DECs and 47 miRNAs, and these miRNAs were involved in muscle development and lipid metabolism. Two shared DECs (circ_0030593 and circ_0032760) of these three stages were selected, their head-to-tail junction sites were validated by Sanger sequencing, and RT‒qPCR results suggested that these two DECs might be involved in intramuscular fat deposition. These findings provide a basis for understanding the role of circRNAs in meat quality.

Analysis of differentially expressed circRNAs in longissimus muscle between castrated and intact male pigs.

Castration can reduce odor and fights in boars, but the carcass yield is reduced, and the intramuscular fat content is increased. Understanding its molecular mechanism is of great significance for production. Recent studies have shown that circular RNAs (circRNAs) play an important role(s) in the regulation of muscle development. To explore the effects of circRNAs on the development of longissimus dorsi (LD) muscle after castration, six Huainan male pigs were selected and three of which were randomly castrated. Six pigs were slaughtered when their body weight reached around 130 kg, and the LD muscle samples were collected. The differentially expressed circRNAs (DECs) were screened by high-throughput sequencing and functionally analyzed using the KEGG databases. DECs-miRNAs network was constructed, and the expression profiles of candidate circRNAs and their interactions with miRNAs were verified in porcine skeletal muscle satellite cells. The results showed that a total of 5866 circRNAs were obtained, and 370 DECs were identified in LD muscle between the castrated and intact groups (| log2Foldchange | > 1, padj <0.8). KEGG enrichment indicated that the parental genes for the DECs were mainly enriched in the pathways associated with muscle development, muscle fiber type transformation, and energy metabolism. There were 8 miRNAs and 69 circRNAs enriched in the DECs-miRNA network. circRNA_2241 and circRNA_4237 were selected for verification, which showed that these two circRNAs really existed and their expression profiles were consistent with the sequencing results. Further, preliminary analysis showed that circRNA_2241 interacted with miR-1, and testosterone promoted circRNA_2241 but inhibited miR-1 expression. These results confirmed that circRNAs might participate in the regulation of LD muscle development after castration by interacting with miRNAs, thereby providing new materials and references for analyses on the molecular mechanisms of castration on the regulation of muscle development.

Autophagy Contributes to Oxidative Stress-Induced Apoptosis in Porcine Granulosa Cells.

Oxidative stress-induced granulosa cell (GC) death is a major cause of follicular atresia. As the major types of programmed cell death, autophagy and apoptosis have been observed in response to H2O2-mediated oxidative stress and have been demonstrated to be responsible for porcine GC death. To date, however, the cellular reactions linking autophagy to the apoptosis of porcine GC under oxidative stress are still poorly understood. Porcine GC were treated with H2O2, and autophagic flux was examined by western blotting. Cell viability and cell death assays were performed after cotreatment of porcine GC with autophagy activator (rapamycin) or inhibitor (3-methyladenine, 3-MA) together with H2O2. We revealed that short exposure (1-3 h) of porcine GC to H2O2 dramatically increased autophagic flux (1.8- to 2.5-fold over that in the control), whereas 6-12 h prolonged treatment decreased autophagy but elevated the caspase-3 activity and GC apoptotic rate. Furthermore, we showed that pretreatment with rapamycin exacerbated H2O2-mediated cytotoxicity and caspase-3 activation but that 3-MA or siRNAs specific for Beclin 1 and Atg7 genes ameliorated H2O2-mediated GC apoptosis. Together, our results indicate that autophagy plays a pivotal role in H2O2-mediated porcine GC apoptosis. Importantly, we show that the early induction of autophagic flux contributes to oxidative stress-induced apoptosis in porcine GC. The results also suggest that regulating the autophagy response in porcine GC under oxidative stress might be a new strategy for abnormal follicular atresia.

LncRNA IMFlnc1 promotes porcine intramuscular adipocyte adipogenesis by sponging miR-199a-5p to up-regulate CAV-1.

BACKGROUND: Local Chinese local pig breeds have thinner muscle fiber and higher intramuscular-fat (IMF) content. But its regulation mechanism has not been discussed in-depth. Studies indicated that long non coding RNAs (lncRNAs) play important role in muscle and fat development. RESULTS: The lncRNAs expressional differences in the longissimus dorsi (LD) muscle were identified between Huainan pigs (local Chinese pigs, fat-type, HN) and Large White pigs (lean-type, LW) at 38, 58, and 78 days post conception (dpc). In total, 2131 novel lncRNAs were identified in 18 samples, and 291, 305, and 683 differentially expressed lncRNAs (DELs) were found between these two breeds at three stages, respectively. The mRNAs that co-expressed with these DELs were used for GO and KEGG analysis, and the results showed that muscle development and energy metabolism were more active at 58 dpc in HN, but at 78 dpc in LW pigs. Muscle cell differentiation and myofibril assembly might associated with earlier myogenesis and primary-muscle-fiber assembly in HN, and cell proliferation, insulin, and the MAPK pathway might be contribute to longer proliferation and elevated energy metabolism in LW pigs at 78 dpc. The PI3K/Akt and cAMP pathways were associated with higher IMF deposition in HN. Intramuscular fat deposition-associated long noncoding RNA 1 (IMFlnc1) was selected for functional verification, and results indicated that it regulated the expressional level of caveolin-1 (CAV-1) by acting as competing endogenous RNA (ceRNA) to sponge miR-199a-5p. CONCLUSIONS: Our data contributed to understanding the role of lncRNAs in porcine-muscle development and IMF deposition, and provided valuable information for improving pig-meat quality.

[Comparison and analysis of lincRNAs expression profile in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows].

The normal estrus in weaned primiparous sows has a great impact on pig production and abnormal estrus is the main reason for the elimination of primiparous sows. In this study, we studied the long intergenic noncoding RNAs (lincRNAs) in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows. These long intergenic noncoding RNAs (lincRNAs) were screened and compared through RNA-seq analysis. The expression profiles of lincRNAs were obtained and their characteristics and functions were preliminarily analyzed. There are 3519 novel lincRNAs identified in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows. Compared with estrous primiparous sows, 17 differentially expressed lincRNAs were indentified, including 12 up-regulated lincRNAs and 5 down-regulated lincRNAs (FC≥2, P<0.05). The four lincRNA transcripts obtained through selection were verified by qRT-PCR, which are consistent with the RNA-seq results. The GO, KEGG pathway, and lincRNA-mRNA co-expression network analysis of these 17 lincRNAs revealed that these lincRNAs were mainly involved in reproductive activities, such as oocyte meiosis mature, ovarian cells differentiation and granulosa cells apoptosis. The results enriched the data resources of pig lincRNAs and provided useful information for further research about the reproductive performance of primiparous sows.

miR-181a promotes porcine granulosa cell apoptosis by targeting TGFBR1 via the activin signaling pathway.

Activin/Smad3 signaling plays a pivotal role in follicle development and atresia. However, the precise mechanisms underlying this process are not yet fully understood. Herein, we identified miR-181a as a central component of activin/Smad3-mediated follicle atresia. miR-181a was strikingly upregulated in porcine atretic follicles, which induced the apoptosis of porcine granulosa cells (GCs) in vitro. Furthermore, the transforming growth factor-ß type 1 receptor (TGFBR1) was confirmed as a direct target of miR-181a by bioinformatics analysis and luciferase assays. Transfection with an miR-181a agomir repressed the TGFBR1 mRNA and protein levels. In addition, TGFBR1 overexpression repressed GC apoptosis, whereas TGFBR1 inhibition promoted GC apoptosis. miR-181a overexpression downregulated the phosphorylation of Smad3 and blocked the activation of TGF-ß signaling. Moreover, activin A downregulated miR-181a expression and upregulated the TGFBR1 and p-Smad3 protein levels. Collectively, these data suggest that miR-181a regulates porcine GC apoptosis by targeting TGFBR1 via the activin signaling pathway.

Grape Seed Procyanidin B2 Protects Porcine Ovarian Granulosa Cells against Oxidative Stress-Induced Apoptosis by Upregulating let-7a Expression.

Oxidative stress is a causal factor and key promoter of all kinds of reproductive disorders related to granulosa cell (GC) apoptosis that acts by dysregulating the expression of related genes. Various studies have suggested that grape seed procyanidin B2 (GSPB2) may protect GCs from oxidative injury, though the underlying mechanisms are not fully understood. Therefore, whether the beneficial effects of GSPB2 are associated with microRNAs, which have been suggested to play a critical role in GC apoptosis by regulating the expression of protein-coding genes, was investigated in this study. The results showed that GSPB2 treatment protected GCs from a H2O2-induced apoptosis, as detected by an MTT assay and TUNEL staining, and increased let-7a expression in GCs. Furthermore, let-7a overexpression markedly increased cell viability and inhibited H2O2-induced GC apoptosis. Furthermore, the overexpression of let-7a reduced the upregulation of Fas expression in H2O2-treated GCs at the mRNA and protein levels. Dual-luciferase reporter assay results indicated that let-7a directly targets the Fas 3'-UTR. Furthermore, the overexpression of let-7a enhanced the protective effects of GSPB2 against GC apoptosis induced by H2O2. These results indicate that GSPB2 inhibits H2O2-induced apoptosis of GCs, possibly through the upregulation of let-7a.

Critical Role of FoxO1 in Granulosa Cell Apoptosis Caused by Oxidative Stress and Protective Effects of Grape Seed Procyanidin B2.

Reactive oxygen species (ROS) are closely related to the follicular granulosa cell apoptosis. Grape seed procyanidin B2 (GSPB2) has been reported to possess potent antioxidant activity. However, the GSPB2-mediated protective effects and the underlying molecular mechanisms in granulosa cell apoptosis process remain unknown. In this study, we showed for the first time that GSPB2 treatment decreased FoxO1 protein level, improved granulosa cell viability, upregulated LC3-II protein level, and reduced granulosa cell apoptosis rate. Under a condition of oxidative stress, GSPB2 reversed FoxO1 nuclear localization and increased its level in cytoplasm. In addition, FoxO1 knockdown inhibited the protective effects of GSPB2 induced. Our findings suggest that FoxO1 plays a pivotal role in regulating autophagy in granulosa cells, GSPB2 exerts a potent and beneficial role in reducing granulosa cell apoptosis and inducing autophagy process, and targeting FoxO1 could be significant in fighting against oxidative stress-reduced female reproductive system diseases.

Chronic Exposure to Diquat Causes Reproductive Toxicity in Female Mice.

Diquat is a bipyridyl herbicide that has been widely used as a model chemical for in vivo studies of oxidative stress due to its generation of superoxide anions, and cytotoxic effects. There is little information regarding the toxic effects of diquat on the female reproductive system, particularly ovarian function. Thus, we investigated the reproductive toxic effects of diquat on female mice. Chronic exposure to diquat reduced ovary weights, induced ovarian oxidative stress, resulted in granulosa cell apoptosis, and disrupted oocyte developmental competence, as shown by reactive oxygen species (ROS) accumulation, decreased polar body extrusion rates and increased apoptosis-related genes expression. Additionally, after diquat treatment, the numbers of fetal mice and litter sizes were significantly reduced compared to those of control mice. Thus, our results indicated that chronic exposure to diquat induced reproductive toxicity in female mice by promoting the ROS production of gruanousa cells and ooctyes, impairing follicle development, inducing apoptosis, and reducing oocyte quality. In conclusion, our findings indicate that diquat can be used as a potent and efficient chemical for in vivo studies of female reproductive toxicity induced by oxidative stress. Moreover, the findings from this study will further enlarge imitative research investigating the effect of ovarian damage induced by oxidative stress on reproductive performance and possible mechanisms of action in large domestic animals.