家畜遗传育种Livestock genetic & breeding

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The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome

XIANG Guang-ming, ZHANG Xiu-ling, XU Chang-jiang, FAN Zi-yao, XU Kui, WANG Nan, WANG Yue, CHE Jing-jing, XU Song-song, MU Yu-lian, LI Kui, LIU Zhi-guo

2023, 22 (1): 202-213. DOI: 10.1016/j.jia.2022.08.105

Abstract （307） PDF in ScienceDirect

Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research. We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus. The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene. Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting. RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e–16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes. Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P_24h=0.31, P_48h=0.96, P_72h=0.24, P_96h=0.17, and P_120h=0.38) on cell proliferation of PK15 cells. These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment

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Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

ZHANG Xue-min, HUANG Xiang-hua, WANG Jing, XING Ying, LIU Fang, XIANG Jin-zhu, WANG Han-ning, YUE Yong-li, LI Xue-ling

2023, 22 (4): 1142-1158. DOI: 10.1016/j.jia.2022.08.111

Abstract （255） PDF in ScienceDirect

Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development. In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment. At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated. From 0.1 to 10 μmol L^–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal. When the LPA concentration was 15 μmol L^–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst. Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2

and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol

L^–1. The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L^–1 to LPA-10 μmol L^–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L^–1 LPA-treated embryos compared with LPA-10 μmol L^–1 group (P<0.05). The inner cell mass in 15 μmol L^–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal. Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells. The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells. They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L^–1, while 15 μmol L^–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells. The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L^–1 group and LPA-10 μmol L^–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L^–1 group compared with LPA-10 μmol L^–1 group (P<0.05). Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L^–1 concentration. This study references the IVF embryo production and embryonic stem cell research of domestic animals.

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MicroRNA transcriptome of skeletal muscle during yak development reveals miR-652 regulates myoblasts differentiation and survival by targeting ISL1

ZHOU Xue-lan, GUO Xian, LIANG Chun-nian, CHU Min, WU Xiao-yun, YAN Ping

2023, 22 (5): 1502-1513. DOI: 10.1016/j.jia.2022.08.116

Abstract （373） PDF in ScienceDirect

The growth and development of skeletal muscle also determine the meat production of yak, ultimately affecting the economic benefits. Hence, improving growth performance is a top priority in the yak industry. Skeletal muscle development is a complex process involving the regulation of several genes, including microRNAs (miRNAs). However, the transcription of miRNAs in yak skeletal muscle during prenatal to postnatal stages is unknown. We used small RNA sequencing (small RNA-Seq) to determine the global miRNAs of longissimus dorsi muscle from yak (the samples were collected from three fetuses and three adults). Totally 264 differently expressed miRNAs (|log₂(fold change)|>1 and P-value≤0.05) were detected between the two groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differently expressed miRNAs-targeted genes participated in pathways associated with muscle development, such as MAPK, PI3K-Akt, and Hippo signaling pathways, etc. MiR-652, which was up-regulated in the fetal group, was transfected into C2C12 myoblasts to examine its role. miR-652 promoted (P≤0.05) proliferation and differentiation, but inhibited (P≤0.001) apoptosis at early period. Furthermore, miR-652 reduced (P≤0.001) the proportion of C2C12 myoblasts in the G1 phase while increasing (P≤0.01) the proportion of cells in the S and G2 phases. Dual-luciferase reporter assays indicated that ISL1 served as a target of miR-652. In general, these findings expand our understanding of yak skeletal muscle miRNAs, and suggested that miR-652 probably regulated myogenesis by regulating ISL1.

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Identification of novel antisense long non-coding RNA APMAP-AS that modulates porcine adipogenic differentiation and inflammatory responses

ZHANG Lin-zhen, HE Li, WANG Ning, AN Jia-hua, ZHANG Gen, CHAI Jin, WU Yu-jie, DAI Chang-jiu, LI Xiao-han, LIAN Ting, LI Ming-zhou, JIN Long

2023, 22 (8): 2483-2499. DOI: 10.1016/j.jia.2022.11.005

Abstract （221） PDF in ScienceDirect

Long non-coding RNAs (lncRNAs) are emerging as powerful regulators of adipocyte differentiation, fat metabolism and gene expression. However, the functional roles and mechanisms of lncRNAs in these processes remain unclear. Here, we identified a novel antisense transcript, named APMAP-AS, transcribed from adipocyte membrane-associated protein (APMAP) in the pig genome. APMAP-AS and APMAP were highly expressed in retroperitoneal adipose of obese pigs, compared with that in control pigs. Using a bone mesenchymal stem cells (BMSCs) adipogenic differentiation model, we found that APMAP-AS positively regulated adipogenic differentiation. APMAP-AS had the potential to form an RNA–RNA duplex with APMAP, and increased the stability of APMAP mRNA. Additionally, APMAP-AS promoted lipid metabolism and inhibited the expression of inflammatory factors. These findings of a natural antisense transcript for a regulatory gene associated with lipid synthesis might further our understanding of lncRNAs in driving adaptive adipose tissue remodeling and preserving metabolic health.

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Alternative polyadenylation events in epithelial cells sense endometritis progression in dairy cows

Meagan J. STOTTS, Yangzi ZHANG, Shuwen ZHANG, Jennifer J. MICHAL, Juan VELEZ, Bothe HANS, Martin MAQUIVAR, Zhihua JIANG

2023, 22 (6): 1820-1832. DOI: 10.1016/j.jia.2022.11.009

Abstract （161） PDF in ScienceDirect

Endometritis (inflammation of the endometrial lining) is one of the most devastating reproductive diseases in dairy cattle, resulting in substantial production loss and causing more than $650 million in lost revenue annually in the USA. We hypothesize that alternative polyadenylation (APA) sites serve as decisive sensors for endometrium health and disease in dairy cows. Endometrial cells collected from 18 cows with purulent vaginal discharge scored 0 to 2 were used for APA profiling with our whole transcriptome termini site sequencing (WTTS-seq) method. Overall, pathogens trigger hosts to use more differentially expressed APA (DE-APA), more intronic DE-APA, more DE-APA sites per gene and more DE-genes associated with inflammation. Host CD59 molecule (CD59), Fc fragment of IgG receptor IIa (FCGR2A), lymphocyte antigen 75 (LY75) and plasminogen (PLG) may serve as initial contacts or combats with pathogens on cell surface, followed by activation of nuclear receptor subfamily 1 group H member 4 (NR1H4) to regulate AXL receptor tyrosine kinase (AXL), FGR proto-oncogene, Src family tyrosine kinase (FGR), HCK proto-oncogene, Src family tyrosine kinase (HCK) and integrin subunit beta 2 (ITGB2) for anti-inflammation. This study is the first to show significance of cilium pathways in endometrium health and animal reproduction. MIR21 and MIR30A would be perfect antagonistic biomarkers for diagnosis of either inflammation or anti-inflammation. These novel findings will set precedent for future genomic studies to aid the dairy industry develop new strategies to reduce endometritis incidence and improve fertility.

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Signatures of positive selection for local adaptation of African native cattle populations: A review

Wondossen AYALEW, WU Xiao-yun, Getinet Mekuriaw TAREKEGN, CHU Min, LIANG Chun-nian, Tesfaye SISAY TESSEMA, YAN Ping

2023, 22 (7): 1967-1984. DOI: 10.1016/j.jia.2023.01.004

Abstract （169） PDF in ScienceDirect

Cattle are central to the lives and diverse cultures of African people. It has played a crucial role in providing valuable protein for billions of households and sources of income and employment for producers and other actors in the livestock value chains. The long-term natural selection of African cattle typically signals signatures in the genome, contributes to high genetic differentiations across breeds. This has enabled them to develop unique adaptive traits to cope with inadequate feed supply, high temperatures, high internal and external parasites, and diseases. However, these unique cattle genetic resources are threatened by indiscriminate cross-breeding, breed replacements with exotic cosmopolitan breeds, and climate change pressures. Although there are no functional genomics studies, recent advancements in genotyping and sequencing technologies have identified and annotated limited functional genes and causal variants associated with unique adaptive and economical traits of African cattle populations. These genome-wide variants serve as candidates for breed improvement and support conservation efforts for endangered cattle breeds against future climate changes. Therefore, this review plans to collate comprehensive information on the identified selection footprints to support genomic studies in African cattle to confirm the validity of the results and provide a framework for further genetic association and QTL fine mapping studies.

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MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes

JI Kai-yuan, WEN Ru-jun, WANG Zheng-zhou, TIAN Qian-qian, ZHANG Wei, ZHANG Yun-hai

2023, 22 (4): 1131-1141. DOI: 10.1016/j.jia.2023.02.018

Abstract （241） PDF in ScienceDirect

In mammals, microRNAs (miRNAs) play key roles in multiple biological processes by regulating the expression of target genes. Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors; however, its function remains unclear. In this study, we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation (P<0.01), tyrosinase activity (P=0.001) and significantly reduced (P<0.001) melanin production. Functional prediction revealed that the 3´-untranslated region (UTR) of MAP3K8 has a putative miR-370-5p binding site, and the interaction between these two molecules was confirmed using luciferase reporter assays. In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes. The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits (P<0.01) MAP3K8 expression via direct targeting of its 3´ UTR. Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition (P<0.01) of melanocyte proliferation and significant reduction (P<0.001) in melanin production, which is consistent with our observations for miR-370-5p. Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA (containing sites for the targeted binding to miR-370-5p) was significantly rescued (P≤0.001), which subsequently promoted significant increases in cell proliferation (P<0.001) and melanin production (P<0.01). Collectively, these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.

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Genome-wide association study identifies 12 new genetic loci associated with growth traits in pigs

Mu Zeng, Binhu Wang, Lei Liu, Yalan Yang, Zhonglin Tang

2024, 23 (1): 217-227. DOI: 10.1016/j.jia.2023.02.040

Abstract （391） PDF in ScienceDirect

Growth traits are among the most important economic traits in pigs and are regulated by polygenes with complex regulatory mechanisms. As the major indicators of growth performance, the backfat thickness (BFT), loin eye area (LEA), and days to 100 kg (D100) traits are commonly used to the genetics improvement in pigs. However, the available genetic markers for these traits are limited. To uncover novel loci and candidate genes associated with growth performance, we collected the phenotypic information of BFT, LEA, and D100 in 1,186 pigs and genotyped all these individuals using the Neogen GGP porcine 80K BeadChip. We performed a genome-wide association study (GWAS) using 4 statistical models, including mixed linear models (MLM), fixed and random model circulating probability unification (FarmCPU), settlement of MLM under progressively exclusive relationships (SUPER), Bayesian-information and linkage-disequilibrium Iteratively nested keyway (Blink), and identified 5, 3, and 6 high-confidence single nucleotide polymorphisms (SNPs) associated with BFT, LEA, and D100, respectively. Variant annotation and quantitative trait locus (QTL) mapping analysis suggested that 6 genes (SKAP2, SATB1, PDE7B, PPP1R16B, WNT3, and WNT9B) were potentially associated with growth performance in pigs. Transcriptome analysis suggested that the expression of Src Kinase Associated Phosphoprotein 2 (SKAP2) was higher in prenatal muscles than in postnatal muscles, and the expression of Phosphodiesterase 7B (PDE7B) continuously increased during the prenatal stages and gradually decreased after birth, implying their potential roles in prenatal skeletal muscle development. Overall, this study provides new candidate loci and genes for the genetic improvement of pigs.

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3D genome organization and its study in livestock breeding

Jie Cheng, Xiukai Cao, Shengxuan Wang, Jiaqiang Zhang, Binglin Yue, Xiaoyan Zhang, Yongzhen Huang, Xianyong Lan, Gang Ren, Hong Chen

2024, 23 (1): 39-58. DOI: 10.1016/j.jia.2023.04.007

Abstract （254） PDF in ScienceDirect

^{Eukaryotic genomes are hierarchically packaged into
cell nucleus, affecting gene regulation.
The genome is organized into multiscale structural units, including
chromosome territories, compartments, topologically associating domains (TADs),
and DNA loops. The identification of
these hierarchical structures has benefited from the development of
experimental approaches, such as 3C-based methods (Hi-C, ChIA-PET, etc.),
imaging tools (2D-FISH, 3D-FISH, Cryo-FISH, etc.) and ligation-free methods
(GAM, SPRITE, etc.). In recent two
decades, numerous studies have shown that the 3D organization of genome plays
essential roles in multiple cellular processes via various mechanisms,
such as regulating enhancer activity and promoter-enhancer interactions. However, there are relatively few studies
about the 3D genome in livestock species.
Therefore, studies for exploring the function of 3D genomes in livestock
are urgently needed to provide a more comprehensive understanding of potential
relationships between the genome and production traits. In this review, we summarize the recent
advances of 3D genomics and its biological functions in human and mouse
studies, drawing inspiration to explore the 3D genomics of livestock species. We then mainly focus on the biological
functions of 3D genome organization in muscle development and its implications
in animal breeding.}

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miR-24-3p promotes proliferation and inhibits apoptosis of porcine granulosa cells by targeting P27

Shengjie Shi, Lutong Zhang, Liguang Wang, Huan Yuan, Haowei Sun, Mielie Madaniyati, Chuanjiang Cai, Weijun Pang, Lei Gao, Guiyan Chu

2024, 23 (4): 1315-1328. DOI: 10.1016/j.jia.2023.04.008

Abstract （184） PDF in ScienceDirect

Ovarian follicle development is associated with the physiological functions of granulosa cells (GCs), including proliferation and apoptosis. The level of miR-24-3p in ovarian tissue of high-yielding Yorkshire×Landrace sows was significantly higher than that of low-yielding sows. However, the functions of miR-24-3p on GCs are unclear. In this study, using flow cytometry, 5-ethynyl-2´-de-oxyuridine (EdU) staining, and cell count, we showed that miR-24-3p promoted the proliferation of GCs increasing the proportion of cells in the S phase and upregulating the expression of cell cycle genes, moreover, miR-24-3p inhibited GC apoptosis. Mechanistically, on-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target gene of miR-24-3p in proliferation and apoptosis is cyclin-dependent kinase inhibitor 1B (P27/CDKN1B). Furthermore, the effect of miR-24-3p on GC proliferation and apoptosis was attenuated by P27 overexpression. These findings suggest that miR-24-3p regulates the physiological functions of GCs.

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Dynamic transcriptome profiles and novel markers in bovine spermatogenesis revealed by single-cell sequencing

Yuan Gao, Fuxia Bai, Qi Zhang, Xiaoya An, Zhaofei Wang, Chuzhao Lei, Ruihua Dang

2024, 23 (7): 2362-2378. DOI: 10.1016/j.jia.2023.04.036

Abstract （239） PDF in ScienceDirect

Testicular development is an important biological process in male and requires interaction between the male germ cells and somatic cells. However, the mechanisms of testicular development in livestock, particularly in cattle, are poorly understood. Furthermore, cellular heterogeneity hinders the profiling of different cell types at different developmental stages. In this study, we first performed a single-cell transcriptomic study of the bovine testis development during puberty by using 10^× genomics single-cell RNA sequencing (scRNA-seq). By collecting the scRNA-seq data from 11,083 cells from prepubertal and pubertal bovine testes, a high-resolution scRNA-seq atlas was described, identifying 9 somatic and 13 spermatogenic clusters. We also distinguished several stage-specific marker genes for bovine germ cells and somatic cells, such as GRAF2 and MORC1 for SSC (spermatogonial stem cells), HJURP and TCF19 for differentiating spermatogonia, ARSE for immature Sertoli, CLEC12B for mature Sertoli, LOC112441470 for Leydig. In conclusion, we have examined the transcription levels and constructed the single-cell developmental maps of germ cells and somatic cells during testicular development in Angus cattle. The datasets provided new insights into spermatogenesis and testicular somatic cell development in cattle.

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Effects of carcass weight, sex and breed composition on meat cuts and carcass trait in finishing pigs

XIE Lei, QIN Jiang-tao, RAO Lin, CUI Deng-shuai, TANG Xi, XIAO Shi-jun, ZHANG Zhi-yan, HUANG Lu-sheng

2023, 22 (5): 1489-1501. DOI: 10.1016/j.jia.2022.08.122

Abstract （284） PDF in ScienceDirect

Pork cutting is a very important processing in promoting economic appreciation across the swine business chain. The goal of this research is to determine the proportion and weight of meat cuts, as well as to analyze the effects of carcass weight, sex and breed composition on meat cuts. Simultaneously, we investigate the correlation between meat cuts, carcass traits and meat quality traits. To assess 17 meat cut traits, 12 carcass traits and 6 meat quality traits, we sample 2 012 pigs from four breeds, including Landrace (LD), Yorkshire (YK), Landrace Yorkshire (LY), and Duroc Landrace Yorkshire (DLY). The results showed that carcass weight, sex and breed composition have significant effects on the weight and proportion of most meat cuts. The proportion of cuts for muscle and bone decrease as carcass weight grows, whereas the proportion of cuts for fat increases. Moreover, the thickness of four-point backfat was significantly increasing (P<0.001) with increase of carcass weights, indicating that large amount of intaking energy in the late finishing stage was used for fat deposition. Besides, the proportion of Shoulder cut (SC) and Back fat (BF) in barrows was significantly higher (P<0.001) than that in sows, whereas the Leg cut (LC) showed the opposite trend. The Loin (LO) proportion and Loin muscle area (LMA) of barrows were significantly lower (P<0.001), but the proportion of fat areas in the image (PFAI) and visual marbling score (VMS) were significantly higher (P<0.001) than those of sows, respectively. In terms of breeds, LD had the longest straight carcass length, significantly longer (P<0.001) than the other three breeds, which partially explains why LD had the largest proportion of the Middle cut (MC). Moreover, the proportion of SC in DLY was the highest. Last but not least, the correlations between the proportions of most meat cuts, and also between meat cuts and meat quality or carcass traits were low or not significant (P>0.05). The effects of carcass weight, sex and breed composition on the meat cuts, meat quality and carcass traits are breed and growth stage dependent. It also reflects the asynchrony of the growth curve between different sexes. Our results laid an important foundation for breeding pig carcass cuts and composition.

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High serum reproductive hormone levels at mid-pregnancy support Meishan pig prolificacy

ZHOU Rong, YANG Yalan, LIU Ying, CHEN Jie, YANG Bing, TANG Zhong-lin

2023, 22 (11): 3489-3499. DOI: 10.1016/j.jia.2023.05.014

Abstract （296） PDF in ScienceDirect

Increasing prolificacy is an important aim in the pig industry. Regions associated with litter size have been revealed, but detailed molecular mechanisms are unclear. The Meishan pig is one of the most prolific breeds, with higher prolificacy than the Yorkshire pig, which exhibits high feeding efficiency and lean meat yield. The ovary is the key organ determining reproductive traits during pregnancy by synthesizing and secreting reproductive hormones essential for conceptus maintenance. In this comparative multi-omics study of the ovary transcriptome, proteome, and metabolome on day 49 of pregnancy, we aimed to identify genomic, proteomic, and metabolomic differences between the ovaries of Meishan and Yorkshire pigs to reveal potential molecular mechanisms conferring high prolificacy. Meishan pigs demonstrated general downregulation of steroid biosynthesis and butanoate metabolism in the ovary during mid-pregnancy at both transcriptome and proteome levels but exhibited higher serum cholesterol, estradiol, and progesterone levels than Yorkshire pigs. We also identified several single-nucleotide polymorphisms in the genes of the steroid hormone pathway associated with litter number, average birth weight, and total litter weight. Lower biosynthesis rates but elevated serum levels of reproductive hormones during mid- and late pregnancy are essential for the greater prolificacy of Meishan pigs.

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circRNA3669 promotes goat endometrial epithelial cells proliferation via miR-26a/RCN2 to activate PI3K/AKT-mTOR and MAPK pathways

Xiaorui Liu, Jiuzeng Cui, Mengyao Wei, Xiaofei Wang, Yuexia Liu, Zhongshi Zhu, Min Zhou, Gui Ba, Langda Suo, Yuxuan Song, Lei Zhang

2024, 23 (3): 960-974. DOI: 10.1016/j.jia.2023.05.029

Abstract （243） PDF in ScienceDirect

The development of receptive endometrium (RE) from pre-receptive endometrium (PE) for successful embryo implantation is a complex dynamic process in which the morphology and physiological states of the endometrial epithelium undergo a series of significant changes, including cell proliferation and apoptosis. However, the molecular mechanisms are not yet fully understood. In this study, a higher circRNA3669 level was observed in PE than in RE of goats. Functional assays revealed that this overexpression promoted the proliferation of goat endometrial epithelial cells (GEECs) by activating the expression of genes related to the PI3K/AKT-mTOR and MAPK pathways, thereby inhibiting apoptosis in vitro. Furthermore, circRNA3669 functioned as a competing endogenous RNA (ceRNA) to upregulate Reticulocalbin-2 (RCN2) expression at the post-transcriptional level by interacting with and downregulating miR-26a in GEECs. In addition, RCN2, which is highly expressed in the PE of goats, was found to be regulated by β-estradiol (E2) and progesterone (P4). Our results demonstrated that RCN2 also affected the key proteins PI3K, AKT, mTOR, JNK, and P38 in the PI3K/AKT-mTOR and MAPK pathways, thereby facilitating GEECs proliferation and suppressing their apoptosis in vitro. Collectively, we constructed a new circRNA3669-miR-26a-RCN2 regulatory network in GEECs, which further provides strong evidence that circRNA could potentially play a crucial regulatory role in the development of RE in goats.

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CRISPR/Cas9-mediated knockout of SLC15A4 gene involved in the immune response in bovine rumen epithelial cells

JIANG Mao-cheng, HU Zi-xuan, WANG Ke-xin, YANG Tian-yu, LIN Miao, ZHAN Kang, ZHAO Guo-qi

2023, 22 (10): 3148-3158. DOI: 10.1016/j.jia.2023.06.016

Abstract （295） PDF in ScienceDirect

The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide (MDP)-mediated inflammatory response of bovine rumen epithelial cells (BRECs). First, changes in the mRNA expression of pro-inflammatory factor genes in BRECs following 10 μg mL^–1 MDP treatments were examined. RT-qPCR results showed that the expression of pro-inflammatory factor (IL-1β, IL-6, and TNF-α) mRNAs were significantly increased under MDP stimulation (P<0.001). Moreover, SLC15A4-Knockout (SLC15A4-KO) cells were obtained through lentivirus packaging, transfection, screening, and cell monoclonal culture. In order to gain further insight into the potential function of SLC15A4, we utilized transcriptome data, which revealed a change in the genes between WT-BRECs and SLC15A4-KO. Five down-regulated pro-inflammatory genes and 13 down-regulated chemokine genes related to the inflammatory response were identified. Meanwhile, the down-regulated genes were mostly enriched in the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. The results of RT-qPCR also verified these detected changes. To further determine the mechanism of how WT and SLC15A4-KO BRECs are involved in inflammatory responses, we investigated the inflammatory responses of cells exposed to MDP. WT-BRECs and SLC15A4-KO were treated with a culture medium containing 10 μg mL^–1 MDP, in comparison to a control without MDP. Our results show that SLC15A4-KO BRECs had reduced the expression of genes (IL-6, TNF-α, CXCL2, CXCL3, CXCL9, and CCL2) and proteins (p-p65 and p-p44/42) from the MDP-mediated inflammatory response compared to WT-BRECs (P<0.05). In this experiment, CRISPR-Cas9 was used to KO the di/tripeptide transporter SLC15A4, and its role was confirmed via the MDP-induced inflammatory response in BRECs. This work will provide a theoretical basis for studying the pro-inflammatory mechanism of MDP and its application in the prevention and treatment of subacute rumen acidosis in dairy cows.

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Long non-coding RNA FPFSC promotes immature porcine Sertoli cell growth through modulating the miR-326/EHMT2 axis

Dan Chu, Bin Chen, Bo Weng, Saina Yan, Yanfei Yin, Xiangwei Tang, Maoliang Ran

2024, 23 (11): 3830-3842. DOI: 10.1016/j.jia.2023.06.026

Abstract （100） PDF in ScienceDirect

Sertoli cells are indispensable for guaranteeing normal spermatogenesis and male fertility. Although a huge number of long non-coding RNAs (lncRNAs) are identified from developing porcine testicular tissues and have been predicted with crucial regulatory roles in spermatogenesis, their functions and regulatory mechanisms are still in infancy. Herein, we mainly explored the regulatory and functional roles of lncFPFSC in proliferation and apoptosis of immature porcine Sertoli cells. The results demonstrated that lncFPFSC was predominantly located in the cytoplasm of immature porcine Sertoli cells. lncFPFSC overexpression promoted cell cycle progression and cell proliferation, as well as inhibited cell apoptosis, whereas siRNA-induced lncFPFSC knockdown resulted in the opposite effects. Mechanistically, lncFPFSC acted as a sponge for miR-326. Overexpression of miR-326 inhibited cell proliferation and induced cell apoptosis, which further abolished the effects of lncFPFSC overexpression. The euchromatic histone-lysine N-methyltransferase 2 (EHMT2) gene was directly targeted by miR-326, and its mRNA and protein expressions were both negatively regulated by miR-326 in immature porcine Sertoli cells. Then, siRNA-induced EHMT2 knockdown resulted a similar effect of miR-326 inhibition. Collectively, lncFPFSC promoted proliferation and inhibited apoptosis in immature porcine Sertoli cells through modulating the miR-326/EHMT2 axis. This study expanded our understanding of non-coding RNAs in participating porcine spermatogenesis through deciding the fate of Sertoli cells, and the competing endogenous RNA (ceRNA) network, and provided new molecular markers to treat Sertoli cell disorder inducing male infertility.

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Identification of transition factors in myotube formation from proteome and transcriptome analyses

ZHENG Qi, HU Rong-cui, ZHU Cui-yun, JING Jing, LOU Meng-yu, ZHANG Si-huan, LI Shuang, CAO Hong-guo, ZHANG Xiao-rong, LING Ying-hui

2023, 22 (10): 3135-3147. DOI: 10.1016/j.jia.2023.08.001

Abstract （304） PDF in ScienceDirect

Muscle fibers are the main component of skeletal muscle and undergo maturation through the formation of myotubes. During early development, a population of skeletal muscle satellite cells (SSCs) proliferate into myoblasts. The myoblasts then undergo further differentiation and fusion events, leading to the development of myotubes. However, the mechanisms involved in the transition from SSCs to myotube formation remain unclear. In this study, we characterized changes in the proteomic and transcriptomic expression profiles of SSCs, myoblasts (differentiation for 2 d) and myotubes (differentiation for 10 d). Proteomic analysis identified SLMAP and STOM as potentially associated with myotube formation. In addition, some different changes in MyoD, MyoG, Myosin7 and Desmin occurred after silencing SLMAP and STOM, suggesting that they may affect changes in the myogenic marker. GO analysis indicated that the differentiation and migration factors SVIL, ENSCHIG00000026624 (AQP1) and SERPINE1 enhanced the transition from SSCs to myoblasts, accompanied by changes in the apoptotic balance. In the myoblast vs. myotube group, candidates related to cell adhesion and signal transduction were highly expressed in the myotubes. Additionally, CCN2, TGFB1, MYL2 and MYL4 were identified as hub-candidates in this group. These data enhance our existing understanding of myotube formation during early development and repair.

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Biology of Hippo signaling pathway: Skeletal muscle development and beyond

Shuqi Qin, Chaocheng Li, Haiyan Lu, Yulong Feng, Tao Guo, Yusong Han, Yongsheng Zhang, Zhonglin Tang

2024, 23 (6): 1825-1838. DOI: 10.1016/j.jia.2023.09.031

Abstract （138） PDF in ScienceDirect

Global demand for farm animals and their meat products i.e., pork, chicken and other livestock meat, is steadily incresing. With the ongoing life science research and the rapid development of biotechnology, it is a great opportunity to develop advanced molecular breeding markers to efficiently improve animal meat production traits. Hippo is an important study subject because of its crucial role in the regulation of organ size. In recent years, with the increase of research on Hippo signaling pathway, the integrative application of multi-omics technologies such as genomics, transcriptomics, proteomics, and metabolomics can help promote the in-depth involvement of Hippo signaling pathway in skeletal muscle development research. The Hippo signaling pathway plays a key role in many biological events, including cell division, cell migration, cell proliferation, cell differentiation, cell apoptosis, as well as cell adhesion, cell polarity, homeostasis, maintenance of the face of mechanical overload, etc. Its influence on the development of skeletal muscle has important research value for enhancing the efficiency of animal husbandry production. In this study, we traced the origin of the Hippo pathway, comprehensively sorted out all the functional factors found in the pathway, deeply analyzed the molecular mechanism of its function, and classified it from a novel perspective based on its main functional domain and mode of action. Our aim is to systematically explore its regulatory role throughout skeletal muscle development. We specifically focus on the Hippo signaling pathway in embryonic stem cell development, muscle satellite cell fate determination, myogenesis, skeletal muscle meat production and organ size regulation, muscle hypertrophy and atrophy, muscle fiber formation and its transformation between different types, and cardiomyocytes. The roles in proliferation and regeneration are methodically summarized and analyzed comprehensively. The summary and prospect of the Hippo signaling pathway within this article will provide ideas for further improving meat production and muscle deposition and developing new molecular breeding technologies for livestock and poultry, which will be helpful for the development of animal molecular breeding.

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Prescreening of large-effect markers with multiple strategies improves the accuracy of genomic prediction

Keanning Li, Bingxing An, Mang Liang, Tianpeng Chang, Tianyu Deng, Lili Du, Sheng Cao, Yueying Du, Hongyan Li, Lingyang Xu, Lupei Zhang, Xue Gao, Junya LI, Huijiang Gao

2024, 23 (5): 1634-1643. DOI: 10.1016/j.jia.2023.11.048

Abstract （110） PDF in ScienceDirect

Presently, integrating multi-omics information into a prediction model has become a ameliorate strategy for genomic selection to improve genomic prediction accuracy. Here, we set the genomic and transcriptomic data as the training population data, using BSLMM, TWAS, and eQTL mapping to prescreen features according to |^β_b|>0, top 1% of phenotypic variation explained (PVE), expression-associated single nucleotide polymorphisms (eSNPs), and egenes (false discovery rate (FDR)<0.01), where these loci were set as extra fixed effects (named GBLUP-Fix) and random effects (GFBLUP) to improve the prediction accuracy in the validation population, respectively. The results suggested that both GBLUP-Fix and GFBLUP models could improve the accuracy of longissimus dorsi muscle (LDM), water holding capacity (WHC), shear force (SF), and pH in Huaxi cattle on average from 2.14 to 8.69%, especially the improvement of GFBLUP-TWAS over GBLUP was 13.66% for SF. These methods also captured more genetic variance than GBLUP. Our study confirmed that multi-omics-assisted large-effects loci prescreening could improve the accuracy of genomic prediction.

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Evaluating the performance of genomic selection on purebred population by incorporating crossbred data in pigs

Jun Zhou, Qing Lin, Xueyan Feng, Duanyang Ren, Jinyan Teng, Xibo Wu, Dan Wu, Xiaoke Zhang, Xiaolong Yuan, Zanmou Chen, Jiaqi Li, Zhe Zhang, Hao Zhang

2024, 23 (2): 639-648. DOI: 10.1016/j.jia.2023.09.004

Abstract （188） PDF in ScienceDirect

Genomic selection (GS) has been widely used in livestock, which greatly accelerated the genetic progress of complex traits. The population size was one of the significant factors affecting the prediction accuracy, while it was limited by the purebred population. Compared to directly combining two uncorrelated purebred populations to extend the reference population size, it might be more meaningful to incorporate the correlated crossbreds into reference population for genomic prediction. In this study, we simulated purebred offspring (P_AS and P_BS) and crossbred offspring (C_AB) base on real genotype data of two base purebred populations (P_A and P_B), to evaluate the performance of genomic selection on purebred while incorporating crossbred information. The results showed that selecting key crossbred individuals via maximizing the expected genetic relationship (REL) was better than the other methods (individuals closet or farthest to the purebred population, CP/FP) in term of the prediction accuracy. Furthermore, the prediction accuracy of reference populations combining P_A and C_AB was significantly better only based on P_A, which was similar to combine P_A and P_AS. Moreover, the rank correlation between the multiple of the increased relationship (MIR) and reliability improvement was 0.60–0.70. But for individuals with low correlation (Cor(P_i, P_{A or B}), the reliability improvement was significantly lower than other individuals. Our findings suggested that incorporating crossbred into purebred population could improve the performance of genetic prediction compared with using the purebred population only. The genetic relationship between purebred and crossbred population is a key factor determining the increased reliability while incorporating crossbred population in the genomic prediction on pure bred individuals.

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