Molecular Regulation of Porcine Skeletal Muscle Development: Insights from Research on CDC23 Expression and Function.

Cell division cycle 23 (CDC23) is a component of the tetratricopeptide repeat (TPR) subunit in the anaphase-promoting complex or cyclosome (APC/C) complex, which participates in the regulation of mitosis in eukaryotes. However, the regulatory model and mechanism by which the CDC23 gene regulates muscle production in pigs are largely unknown. In this study, we investigated the expression of CDC23 in pigs, and the results indicated that CDC23 is widely expressed in various tissues and organs. In vitro cell experiments have demonstrated that CDC23 promotes the proliferation of myoblasts, as well as significantly positively regulating the differentiation of skeletal muscle satellite cells. In addition, Gene Set Enrichment Analysis (GSEA) revealed a significant downregulation of the cell cycle pathway during the differentiation process of skeletal muscle satellite cells. The protein-protein interaction (PPI) network showed a high degree of interaction between genes related to the cell cycle pathway and CDC23. Subsequently, in differentiated myocytes induced after overexpression of CDC23, the level of CDC23 exhibited a significant negative correlation with the expression of key factors in the cell cycle pathway, suggesting that CDC23 may be involved in the inhibition of the cell cycle signaling pathway in order to promote the differentiation process. In summary, we preliminarily determined the function of CDC23 with the aim of providing new insights into molecular regulation during porcine skeletal muscle development.

Genetic analysis of milk citrate predicted by milk mid-infrared spectra of Holstein cows in early lactation.

Milk citrate is regarded as an early biomarker of negative energy balance in dairy cows during early lactation and serves as a suitable candidate phenotype for genomic selection due to its wide availability across a large number of cows through milk mid-infrared spectra prediction. However, its genetic background is not well known. Therefore, the objectives of this study were to (1) analyze the genetic parameters of milk citrate; (2) identify genomic regions associated with milk citrate; and (3) analyze the functional annotation of candidate genes and quantitative trait loci (QTL) related to milk citrate in Walloon Holstein cows. In total, 134,517 test-day milk-citrate phenotypes (mmol/L) collected within the first 50 d in milk on 52,198 Holstein cows were used. These milk-citrate phenotypes, predicted by milk mid-infrared spectra, were divided into 3 traits according to the first (citrate1), second (citrate2), and third to fifth parity (citrate3+). Genomic information for 566,170 SNPs was available for 4,479 animals. A multiple-trait repeatability model was used to estimate genetic parameters. A single-step GWAS was used to identify candidate genes for citrate and post-GWAS analysis was done to investigate the relationship and function of the identified candidate genes. The heritabilities estimated for citrate1, citrate2, and citrate3+ were 0.40, 0.37, and 0.35, respectively. The genetic correlations among the 3 traits ranged from 0.98 to 0.99. The genomic correlations among the 3 traits were also close to 1.00 across the genomic regions (1 Mb) in the whole genome, which means that citrate can be considered as a single trait in the first 5 parities. In total, 603 significant SNPs located on 3 genomic regions (chromosome 7, 68.569-68.575 Mb; chromosome 14, 0.15-1.90 Mb; and chromosome 20, 54.00-64.28 Mb), were identified to be associated with milk citrate. We identified 89 candidate genes including GPT, ANKH, PPP1R16A, and 32 QTL reported in the literature related to the identified significant SNPs. These identified QTL were mainly reported associated with milk fatty acids and metabolic diseases in dairy cows. This study suggests that milk citrate in Holstein cows is highly heritable and has the potential to be used as an early proxy for the negative energy balance of Holstein cows in a breeding objective.

Identification of miR-192 target genes in porcine endometrial epithelial cells based on miRNA pull-down.

INTRODUCTION: MicroRNAs (miRNAs)-a class of small endogenous non-coding RNAs-are widely involved in post-transcriptional gene regulation of numerous physiological processes. High-throughput sequencing revealed that the miR-192 expression level appeared to be significantly higher in the blood exosomes of sows at early gestation than that in non-pregnant sows. Furthermore, miR-192 was hypothesized to have a regulatory role in embryo implantation; however, the target genes involved in exerting the regulatory function of miR-192 required further elucidation. METHODS: In the present study, potential target genes of miR-192 in porcine endometrial epithelial cells (PEECs) were identified through biotin-labeled miRNA pull-down; functional and pathway enrichment analysis was performed via gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Bioinformatic analyses were concurrently used to predict the potential target genes associated with sow embryo implantation. In addition, double luciferase reporter vectors, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and Western blot were performed to verify the targeting and regulatory roles of the abovementioned target genes. RESULTS: A total of 1688 differentially expressed mRNAs were identified via miRNA pull-down. Through RT-qPCR, the accuracy of the sequencing data was verified. In the bioinformatics analysis, potential target genes of miR-192 appeared to form a dense inter-regulatory network and regulated multiple signaling pathways, such as metabolic pathways and the PI3K-Akt, MAPKs, and mTOR signaling pathways, that are relevant to the mammalian embryo implantation process. In addition, CSK (C-terminal Src kinase) and YY1 (Yin-Yang-1) were predicted to be potential candidates, and we validated that miR-192 directly targets and suppresses the expression of the CSK and YY1 genes. CONCLUSION: We screened 1688 potential target genes of miR-192 were screened, and CSK and YY1 were identified as miR-192 target genes. The outcomes of the present study provide novel insights into the regulatory mechanism of porcine embryo implantation and the identification of miRNA target genes.

Genome-wide association for milk urea concentration in Dual-Purpose Belgian Blue cows.

The objectives of this study were to estimate genetic parameters and identify genomic regions associated with milk urea concentration (MU) in Dual-Purpose Belgian Blue (DPBB) cows. The data were 29,693 test-day records of milk yield (MY), fat yield (FY), protein yield (PY), fat percentage (FP), protein percentage (PP) and MU collected between 2014 and 2020 on 2498 first parity cows (16,935 test-day records) and 1939 second-parity cows (12,758 test-day records) from 49 herds in the Walloon Region of Belgium. Data of 28,266 single nucleotide polymorphisms (SNP), located on 29 Bos taurus autosomes (BTA), on 1699 animals (639 males and 1060 females) were used. Random regression test-day models were used to estimate genetic parameters through the Bayesian Gibbs sampling method using a single chain of 100,000 iterations after a burn-in period of 20,000. SNP solutions were estimated using a single-step genomic best linear unbiased prediction approach. The proportion of genetic variance explained by windows of 25 consecutive SNPs (with an average size of ~2 Mb) was calculated, and regions accounting for at least 1.0% of the total additive genetic variance were used to search for candidate genes. The mean (SD) of MU was 22.89 (10.07) and 22.35 (10.07) mg/dl for first and second parity, respectively. The mean (SD) heritability estimates for daily MU were 0.18 (0.01) and 0.22 (0.02), for first and second parity, respectively. The mean (SD) genetic correlations between daily MU and MY, FY, PY, FP and PP were -0.05 (0.09), -0.07 (0.11), -0.03 (0.13), -0.05 (0.08) and -0.03 (0.11) for first parity, respectively. The corresponding values estimated for second parity were 0.02 (0.10), -0.02 (0.09), 0.02 (0.08), -0.08 (0.06) and -0.05 (0.05). The genome-wide association analyses identified three genomic regions (BTA2, BTA3 and BTA13) associated with MU. The identified regions showed contrasting results between parities and among different stages within each parity. This suggests that different groups of candidate genes underlie the phenotypic expression of MU between parities and among different lactation stages within a parity. The results of this study can be used for future implementation and use of genomic evaluation to reduce MU in DPBB cows.

Identification of circular RNAs in the ovarian follicles of Meishan and Duroc sows during the follicular phase.

Circular RNAs (circRNAs) are a newly discovered class of endogenous non-coding RNAs that play an important role in growth and development by regulating gene expression and participating in a variety of biological processes. However, the role of circRNAs in porcine follicles remains unclear. Therefore, this study examined middle-sized ovarian follicles obtained from Meishan and Duroc sows at day 4 of the follicular phase. High-throughput RNA sequencing (RNA-seq) was utilized to construct circRNAs, and differential expression was identified. The findings were validated using reverse transcription PCR (RT-PCR) and DNA sequencing, GO and KEGG analyses were performed, and potential miRNA targets were identified. The RNA-seq identified a total of 15,866 circRNAs, with 244 differentially expressed in the Meishan relative to the Duroc (111 up-regulated and 133 down-regulated). The RT-PCR finding confirmed the RNA-seq results, and quantitative real-time PCR (qPCR) analysis examining a subset of the circRNAs showed that they are resistant to RNase R digestion. Bioinformatics analysis (GO and KEGG) showed that the host genes associated with the differentially expressed circRNAs are involved in reproduction and follicular development signaling pathways. Furthermore, many of the circRNAs were found to interact with miRNAs that are associated with follicular development. This study presents a new perspective for studying circRNAs and provides a valuable resource for further examination into the potential roles of circRNAs in porcine follicular development.

Molecular phylogenetics and evolutionary history of the endemic land snail genus Everettia in northern Borneo.

Borneo has gone through dramatic changes in geology and topography from the early Eocene until the early Pliocene and experienced climatic cycling during the Pleistocene. However, how these changes have shaped the present-day patterns of high diversity and complex distribution are still poorly understood. In this study, we use integrative approaches by estimating phylogenetic relationships, divergence time, and current and past niche suitability for the Bornean endemic land snail genus Everettia to provide additional insight into the evolutionary history of this genus in northern Borneo in the light of the geological vicariance events and climatic fluctuations in the Pleistocene. Our results show that northern Borneo Everettia species belong to two deeply divergent lineages: one contains the species that inhabit high elevation at the central mountain range, while the other contains lowland species. Species diversification in these lineages has taken place before the Pliocene. Climate changes during the Pleistocene did not play a significant role in species diversification but could have shaped contemporary species distribution patterns. Our results also show that the species-rich highland habitats have acted as interglacial refugia for highland species. This study of a relatively sedentary invertebrate supports and enhances the growing understanding of the evolutionary history of Borneo. Species diversification in Everettia is caused by geological vicariance events between the early Miocene and the Pliocene, and the distribution patterns were subsequently determined by climatic fluctuations in the Pleistocene.