Background In poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying ...Background In poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying follicular ovulation in avians is a complex biological process that remains unclear.Results Critical biochemical events involved in ovulation in domestic chickens(Gallus gallus) were evaluated by transcriptomics, proteomics, and in vitro assays. Comparative transcriptome analyses of the largest preovulatory follicle(F1) and postovulatory follicle(POF1) in continuous laying(CL) and intermittent laying(IL) chickens indicated the greatest difference between CL_F1 and IL_F1, with 950 differentially expressed genes(DEGs), and the smallest difference between CL_POF1 and IL_POF1, with 14 DEGs. Additionally, data-independent acquisition proteomics revealed 252 differentially abundant proteins between CL_F1 and IL_F1. Perivitelline membrane synthesis, steroid biosynthesis, lysosomes, and oxidative phosphorylation were identified as pivotal pathways contributing to ovulation regulation. In particular, the regulation of zona pellucida sperm-binding protein 3, plasminogen activator, cathepsin A, and lactate dehydrogenase A(LDHA) was shown to be essential for ovulation. Furthermore, the inhibition of LDHA decreased cell viability and promoted apoptosis of ovarian follicles in vitro.Conclusions This study reveals several important biochemical events involved in the process of ovulation, as well as crucial role of LDHA. These findings improve our understanding of ovulation and its regulatory mechanisms in avian species.展开更多
Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other...Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.展开更多
Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell a...Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons（breeding and non-breeding season） on superovulation and the imported exogenous gene on growth.Results： In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep.Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons（P 〉 0.05）. The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group（P 〈 0.05）. The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.Conclusions： Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.展开更多
Background: Interferon-γ(IFN-γ) is critical for innate and adaptive immunity against viral and bacterial infections. IFN-γ reportedly affects the phagocytic ability of monocytes and macrophages as well as regulates...Background: Interferon-γ(IFN-γ) is critical for innate and adaptive immunity against viral and bacterial infections. IFN-γ reportedly affects the phagocytic ability of monocytes and macrophages as well as regulates pituitary function in humans and mice. The present study analyzed the impact of IFN-γ on monocyte and macrophage phagocytosis, production performance, and pituitary function in vivo and in vitro(in dwarf chickens). IFN-γ was injected into dwarf chickens through a vein, and then, the laying rate, average egg weight, and levels of follicle-stimulating hormone(FSH) and IFN-γ were measured in treatment and control groups. For the in vitro experiment, the pituitary tissues were supplemented with IFN-γ, and the m RNA expression levels of follicle-stimulating hormone beta subunit(FSH-β), interferon gamma receptor 1(IFNGR1),and interferon gamma receptor 2(IFNGR2) in the pituitary were assessed.Results: Monocyte and macrophage phagocytosis product(PP) was decreased by IFN-γ treatment in a dose-dependent manner in vitro. In the in vivo experiment, the level of IFN-γ in the treatment group was higher than that in the control group at 7 d(P < 0.05), 14 d(P < 0.01), and 21 d(P < 0.01) post-injection.Compared with the control group, monocyte and macrophage PP was lower in the treatment group after injection(P < 0.01). The laying rate was higher in the treatment group than in the control group at 2 and3 wk post-injection(P < 0.05). There was a significant difference between the treatment and control groups in the levels of FSH at 1, 3, 7, and 14 d post-injection(P < 0.01). In the in vitro experiment, increased m RNA expression levels of FSH-β, IFNGR1, and IFNGR2 were observed in the treatment group after stimulation with100 U/m L IFN-γ for 24 h compared to those in the control group(P < 0.05).Conclusions: IFN-γ inhibited the phagocytosis of monocytes and macrophages; up-regulated the m RNA expression levels of the FSH-β, IFNGR1, and IFNGR2; enhanced the secretion of FSH; and improved the laying rate. IFN-γ might be an important regulator in the trade-off between the immune effect and production performance in dwarf chickens.展开更多
Background:This study investigated the effect of melatonin(MT)on cell cycle(G1/S/G2/M)of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II(MII)oocytes and elucidated the potential mechanism of...Background:This study investigated the effect of melatonin(MT)on cell cycle(G1/S/G2/M)of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II(MII)oocytes and elucidated the potential mechanism of MT action in the first cleavage of embryos.Results:After vitrification and warming,oocytes were parthenogenetically activated(PA)and in vitro cultured(IVC).Then the spindle morphology and chromosome segregation in oocytes,the maternal mRNA levels of genes including Miss,Doc1r,Setd2 and Ythdf2 in activated oocytes,pronuclear formation,the S phase duration in zygotes,mitochondrial function at G1 phase,reactive oxygen species(ROS)level at S phase,DNA damage at G2 phase,early apoptosis in 2-cell embryos,cleavage and blastocyst formation rates were evaluated.The results indicated that the vitrification/warming procedures led to following perturbations 1)spindle abnormalities and chromosome misalignment,alteration of maternal mRNAs and delay in pronucleus formation,2)decreased mitochondrial membrane potential(MMP)and lower adenosine triphosphate(ATP)levels,increased ROS production and DNA damage,G1/S and S/G2 phase transition delay,and delayed first cleavage,and 3)increased early apoptosis and lower levels of cleavage and blastocyst formation.Our results further revealed that such negative impacts of oocyte cryopreservation could be alleviated by supplementation of warming,recovery,PA and IVC media with 10^(−9) mol/L MT before the embryos moved into the 2-cell stage of development.Conclusions:MT might promote cell cycle progression via regulation of MMP,ATP,ROS and maternal mRNA levels,potentially increasing the first cleavage of parthenogenetic zygotes developed from vitrified-warmed mouse oocytes and their subsequent development.展开更多
Background Abdominal fat deposition depends on both the proliferation of preadipocytes and their maturation into adipocytes,which is a well-orchestrated multistep process involving many regulatory molecules.Circular R...Background Abdominal fat deposition depends on both the proliferation of preadipocytes and their maturation into adipocytes,which is a well-orchestrated multistep process involving many regulatory molecules.Circular RNAs(circRNAs)have emergingly been implicated in mammalian adipogenesis.However,circRNA-mediated regulation in chicken adipogenesis remains unclear.Our previous circRNA sequencing data identified a differentially expressed novel circRNA,8:27,886,180|27,889,657,during the adipogenic differentiation of chicken abdominal preadipocytes.This study aimed to investigate the regulatory role of circDOCK7 in the proliferation and adipogenic differentiation of chicken abdominal preadipocytes,and explore its molecular mechanisms of competing endogenous RNA underlying chicken adipogenesis.Results Our results showed that 8:27,886,180|27,889,657 is an exonic circRNA derived from the head-to-tail splicing of exons 19–22 of the dedicator of cytokinesis 7(DOCK7)gene,abbreviated as circDOCK7.CircDOCK7 is mainly distributed in the cytoplasm of chicken abdominal preadipocytes and is stable because of its RNase R resistance and longer half-life.CircDOCK7 is significantly upregulated in the abdominal fat tissues of fat chickens compared to lean chickens,and its expression gradually increases during the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.Functionally,the gain-and loss-of-function experiments showed that circDOCK7 promoted proliferation,G0/G1-to S-phase progression,and glucose uptake capacity of chicken abdominal preadipocytes,in parallel with adipogenic differentiation characterized by remarkably increased intracellular lipid droplet accumulation and triglyceride and acetyl coenzyme A content in differentiated chicken abdominal preadipocytes.Mechanistically,a pull-down assay and a dual-luciferase reporter assay confirmed that circDOCK7 interacted with gga-miR-301b-3p,which was identified as an inhibitor of chicken abdominal adipogenesis.Moreover,the ACSL1 gene was demonstrated to be a direct target of gga-miR-301b-3p.Chicken ACSL1 protein is localized in the endoplasmic reticulum and mitochondria of chicken abdominal preadipocytes and acts as an adipogenesis accelerator.Rescue experiments showed that circDOCK7 could counteract the inhibitory effects of gga-miR-301b-3p on ACSL1 mRNA abundance as well as the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.Conclusions CircDOCK7 serves as a miRNA sponge that directly sequesters gga-miR-301b-3p away from the ACSL1 gene,thus augmenting adipogenesis in chickens.These findings may elucidate a new regulatory mechanism underlying abdominal fat deposition in chickens.展开更多
Background: Tibetan pigs, which inhabit the Tibetan Plateau, exhibit distinct phenotypic and physiological characteristics from those of lowland pigs and have adapted well to the extreme conditions at high altitude.Ho...Background: Tibetan pigs, which inhabit the Tibetan Plateau, exhibit distinct phenotypic and physiological characteristics from those of lowland pigs and have adapted well to the extreme conditions at high altitude.However, the genetic and epigenetic mechanisms of hypoxic adaptation in animals remain unclear.Methods: Whole-genome DNA methylation data were generated for heart tissues of Tibetan pigs grown in the highland(TH, n = 4) and lowland(TL, n = 4), as well as Yorkshire pigs grown in the highland(YH, n = 4) and lowland(YL, n = 4), using methylated DNA immunoprecipitation sequencing.Results: We obtained 480 million reads and detected 280679, 287224, 259066, and 332078 methylation enrichment peaks in TH, YH, TL, and YL, respectively. Pairwise TH vs. YH, TL vs. YL, TH vs. TL, and YH vs. YL comparisons revealed6829, 11997, 2828, and 1286 differentially methylated regions(DMRs), respectively. These DMRs contained 384, 619,192, and 92 differentially methylated genes(DMGs), respectively. DMGs that were enriched in the hypoxia-inducible factor 1 signaling pathway and pathways involved in cancer and hypoxia-related processes were considered to be important candidate genes for high-altitude adaptation in Tibetan pigs.Conclusions: This study elucidates the molecular and epigenetic mechanisms involved in hypoxic adaptation in pigs and may help further understand human hypoxia-related diseases.展开更多
Background:Excessive abdominal fat deposition in commercial broilers presents an obstacle to profitable meat quality,feed utilization,and reproduction.Abdominal fat deposition depends on the proliferation of preadipoc...Background:Excessive abdominal fat deposition in commercial broilers presents an obstacle to profitable meat quality,feed utilization,and reproduction.Abdominal fat deposition depends on the proliferation of preadipocytes and their maturation into adipocytes,which involves a cascade of regulatory molecules.Accumulating evidence has shown that microRNAs(miRNAs)serve as post-transcriptional regulators of adipogenic differentiation in mammals.However,the miRNA-mediated molecular mechanisms underlying abdominal fat deposition in chickens are still poorly understood.This study aimed to investigate the biological functions and regulatory mechanism of miRNAs in chicken abdominal adipogenesis.Results:We established a chicken model of abdominal adipocyte differentiation and analyzed miRNA and mRNA expression in abdominal adipocytes at different stages of differentiation(0,12,48,72,and 120 h).A total of 217 differentially expressed miRNAs(DE-miRNAs)and 3520 differentially expressed genes were identified.Target prediction of DE-miRNAs and functional enrichment analysis revealed that the differentially expressed targets were significantly enriched in lipid metabolism-related signaling pathways,including the PPAR signaling and MAPK signaling pathways.A candidate miRNA,gga-miR-106-5p,exhibited decreased expression during the proliferation and differentiation of abdominal preadipocytes and was downregulated in the abdominal adipose tissues of fat chickens compared to that of lean chickens.gga-miR-106-5p was found to inhibit the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.A dual-luciferase reporter assay suggested that the KLF15 gene,which encodes a transcriptional factor,is a direct target of gga-miR-106-5p.gga-miR-106-5p suppressed the posttranscriptional activity of KLF15,which is an activator of abdominal preadipocyte proliferation and differentiation,as determined with gain-and loss-of-function experiments.Conclusions:gga-miR-106-5p functions as an inhibitor of abdominal adipogenesis by targeting the KLF15 gene in chickens.These findings not only improve our understanding of the specific functions of miRNAs in avian adipogenesis but also provide potential targets for the genetic improvement of excessive abdominal fat deposition in poultry.展开更多
Background： Resveratrol, an important phyto-antioxidant commonly found in grapes, mulberry, and other plants,has a variety of functions including anti-aging, anti-cancer and anti-inflammatory activities. In the curre...Background： Resveratrol, an important phyto-antioxidant commonly found in grapes, mulberry, and other plants,has a variety of functions including anti-aging, anti-cancer and anti-inflammatory activities. In the current study, we investigated the beneficial effects of resveratrol on in vitro porcine oocyte maturation under heat stress（HS）. The effect of resveratrol, melatonin and their combination on alleviating HS was compared according to the maturation rate of oocytes and the development competence of embryos after parthenogenetic activation（PA）.Results： Supplementation with resveratrol（2.0 μmol/L） not only improved the nuclear maturation but also raised the blastocyst rate of porcine embryos＇ PA from oocytes that underwent HS by increasing their glutathione（GSH）level, reducing reactive oxygen species（ROS） and up-regulating the expression of Sirtuin 1（SIRT1）. It was also found that melatonin（10^-7mol/L） and the combination of resveratrol（2.0 μmol/L） plus melatonin（10^-7mol/L） exhibited more potent effects than resveratrol alone regarding their protective activities on oocyte maturation under HS.Conclusions： This study compared the efficiencies of resveratrol, melatonin and their combination for protecting porcine oocytes from heat stress. The mechanisms are attributed to the fact that each treatment may have different ability to regulate the synthesis of steroid hormones and the expression of mature related genes.展开更多
Background:Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health.Toll-like receptor 4(TLR4)plays a crucial role in host defence against Gram-negative bacteria...Background:Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health.Toll-like receptor 4(TLR4)plays a crucial role in host defence against Gram-negative bacteria.Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections.In a previous study,we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance.The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood.Results:In the present study,we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model.The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection.This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis.Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4,Src signalling,or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria,weakened the ability of the monocytes to eliminate the bacteria,and increased the pH of the endosomes.Conclusion:Together,our results reveal the effects of TLR4 on the control of E.coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model,providing theoretical support for breeding disease resistance in the future.Furthermore,Src and caveolin 1(CAV1)could be potentially valuable targets for the control of infectious diseases.展开更多
Sperm cryopreservation is useful in assisted reproductive technology and male fertility preservation.However,freezing and thawing significantly reduces the total and progressive motility of human spermatozoa.In the pr...Sperm cryopreservation is useful in assisted reproductive technology and male fertility preservation.However,freezing and thawing significantly reduces the total and progressive motility of human spermatozoa.In the present study,we explored the effects of peroxiredoxin 6(PRDX6)on total and progressive motility of human spermatozoa after cryopreservation.Semen samples of 20 males with normal parameters were collected and frozen in media supplemented with different concentrations of PRDX6(0 mM,10−5 mM,10−7 mM,and 10−9 mM,respectively).Postthaw total and progressive motility of sperms were measured.The results showed that in comparison with 0 mM,the concentrations of 10−5 mM,10−7 mM,and 10−9 mM of PRDX6 all significantly improved total motility and progressive motility of sperms(p<0.05).The 10−7 mM of PRDX6 showed the best performance.In conclusion,the supplementation of PRDX6 helps to maintain the total and progressive motility of human spermatozoa.展开更多
Background:Tan sheep,an important local sheep breed in China,is famous for their fur quality.One-month-old Tan sheep have white,curly hair with beautiful flower spikes,commonly known as“nine bends”,which has high ec...Background:Tan sheep,an important local sheep breed in China,is famous for their fur quality.One-month-old Tan sheep have white,curly hair with beautiful flower spikes,commonly known as“nine bends”,which has high economic value.However,the“nine bends”characteristic gradually disappears with age;consequently,the economic value of the Tan sheep decreases.Age-related changes in DNA methylation have been reported and may be responsible for age-induced changes in gene expression.Until now,no genome-wide surveys have been conducted to identify potential DNA methylation sites involved in different sheep growth stages.In this study we investigated the dynamic changes of genome-wide DNA methylation profiles in Tan sheep using DNA from skin and deep whole-genome bisulfite sequencing,and compared the DNA methylation levels at three different growth stages:1,24,and 48 months old(mon1,mon24,and mon48,respectively).Results:In this study,11 skin samples from three growth stages(four for mon1,four for mon24,and three for mon48)were used for DNA methylation analysis and gene expression profiling.There were 52,288 and 236 differentially methylated genes(DMGs)identified between mon1 and mon24,mon1 and mon48,and mon24 and mon48,respectively.Of the differentially methylated regions,1.11%,7.61%,and 7.65% were in the promoter in mon1 vs.mon24,mon24 vs.mon48,and mon1 vs.mon48,respectively.DMGs were enriched in the MAPK and WNT signaling pathways,which are related to age growth and hair follicle morphogenesis processes.There were 51 DMGs associated with age growth and curly fleece formation.Four DMGs between mon1 and mon48(KRT71,CD44,ROR2 and ZDHHC13)were further validated by bisulfite sequencing.Conclusions:This study revealed dynamic changes in the genomic methylation profiles of mon1,mon24,and mon48 sheep,and the percentages of methylated cytosines were 3.38%,2.85% and 4.17%,respectively.Of the DMGs,KRT71 and CD44 were highly methylated in mon1,and ROR2 and ZDHHC13 were highly methylated in mon48.These findings provide foundational information that may be used to develop strategies for potentially retaining the lamb fur and thus improving the economic value of Tan sheep.展开更多
The CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPR-associated)adaptive immune system,which was discovered in bacteria and archaea,can specifically degrade invasive viral and plasmid DNA by...The CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPR-associated)adaptive immune system,which was discovered in bacteria and archaea,can specifically degrade invasive viral and plasmid DNA by base pairing between crRNAs(CRISPR RNAs)and the target DNA[1,2].Recently,the Streptococcus pyogenes type II CRISPR system was shown to be able to perform efficient targeted gene disruption by employing three fundamental components:(1)Cas9 endonuclease.展开更多
基金supported by the National Key Research and Development Program of China (2022YFD1600902)Key Research and Development Program of Shandong (2022LZGC013)China Agriculture Research System (CARS-40)。
文摘Background In poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying follicular ovulation in avians is a complex biological process that remains unclear.Results Critical biochemical events involved in ovulation in domestic chickens(Gallus gallus) were evaluated by transcriptomics, proteomics, and in vitro assays. Comparative transcriptome analyses of the largest preovulatory follicle(F1) and postovulatory follicle(POF1) in continuous laying(CL) and intermittent laying(IL) chickens indicated the greatest difference between CL_F1 and IL_F1, with 950 differentially expressed genes(DEGs), and the smallest difference between CL_POF1 and IL_POF1, with 14 DEGs. Additionally, data-independent acquisition proteomics revealed 252 differentially abundant proteins between CL_F1 and IL_F1. Perivitelline membrane synthesis, steroid biosynthesis, lysosomes, and oxidative phosphorylation were identified as pivotal pathways contributing to ovulation regulation. In particular, the regulation of zona pellucida sperm-binding protein 3, plasminogen activator, cathepsin A, and lactate dehydrogenase A(LDHA) was shown to be essential for ovulation. Furthermore, the inhibition of LDHA decreased cell viability and promoted apoptosis of ovarian follicles in vitro.Conclusions This study reveals several important biochemical events involved in the process of ovulation, as well as crucial role of LDHA. These findings improve our understanding of ovulation and its regulatory mechanisms in avian species.
基金Key Research and Development Project of Hainan Province(ZDYF2021XDNY174)Science and Technology Major Project of Inner Mongolia(2021ZD0023–1)National Transgenic Key Project of the Ministry of Agriculture of China(2018ZX0800801B)。
文摘Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.
基金supported by grants from National Transgenic Creature Breeding Grand Project (2014ZX08008-005)
文摘Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons（breeding and non-breeding season） on superovulation and the imported exogenous gene on growth.Results： In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep.Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons（P 〉 0.05）. The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group（P 〈 0.05）. The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.Conclusions： Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.
基金supported by grants from National Natural Science Foundation of China(3157130570)Young Scientist Supporting Project,Program for Changjiang Scholars and Innovative Research Team in University(IRT_15R62)+1 种基金Farm Animals Germplasm Resource Platform,National Transgenic Creature Breeding Grand Project(2016ZX08008-003)Innovation Base Cultivation and Development Project-research on precise genetic modify in sheep(Z171100002217072)
文摘Background: Interferon-γ(IFN-γ) is critical for innate and adaptive immunity against viral and bacterial infections. IFN-γ reportedly affects the phagocytic ability of monocytes and macrophages as well as regulates pituitary function in humans and mice. The present study analyzed the impact of IFN-γ on monocyte and macrophage phagocytosis, production performance, and pituitary function in vivo and in vitro(in dwarf chickens). IFN-γ was injected into dwarf chickens through a vein, and then, the laying rate, average egg weight, and levels of follicle-stimulating hormone(FSH) and IFN-γ were measured in treatment and control groups. For the in vitro experiment, the pituitary tissues were supplemented with IFN-γ, and the m RNA expression levels of follicle-stimulating hormone beta subunit(FSH-β), interferon gamma receptor 1(IFNGR1),and interferon gamma receptor 2(IFNGR2) in the pituitary were assessed.Results: Monocyte and macrophage phagocytosis product(PP) was decreased by IFN-γ treatment in a dose-dependent manner in vitro. In the in vivo experiment, the level of IFN-γ in the treatment group was higher than that in the control group at 7 d(P < 0.05), 14 d(P < 0.01), and 21 d(P < 0.01) post-injection.Compared with the control group, monocyte and macrophage PP was lower in the treatment group after injection(P < 0.01). The laying rate was higher in the treatment group than in the control group at 2 and3 wk post-injection(P < 0.05). There was a significant difference between the treatment and control groups in the levels of FSH at 1, 3, 7, and 14 d post-injection(P < 0.01). In the in vitro experiment, increased m RNA expression levels of FSH-β, IFNGR1, and IFNGR2 were observed in the treatment group after stimulation with100 U/m L IFN-γ for 24 h compared to those in the control group(P < 0.05).Conclusions: IFN-γ inhibited the phagocytosis of monocytes and macrophages; up-regulated the m RNA expression levels of the FSH-β, IFNGR1, and IFNGR2; enhanced the secretion of FSH; and improved the laying rate. IFN-γ might be an important regulator in the trade-off between the immune effect and production performance in dwarf chickens.
基金supported by the National Natural Science Foundation of China(grant no.32072735,31572398)the Natural Science Fund of Qinghai Province(2020-ZJ-902)by the China Agriculture Research System(grant no.CARS-36).
文摘Background:This study investigated the effect of melatonin(MT)on cell cycle(G1/S/G2/M)of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II(MII)oocytes and elucidated the potential mechanism of MT action in the first cleavage of embryos.Results:After vitrification and warming,oocytes were parthenogenetically activated(PA)and in vitro cultured(IVC).Then the spindle morphology and chromosome segregation in oocytes,the maternal mRNA levels of genes including Miss,Doc1r,Setd2 and Ythdf2 in activated oocytes,pronuclear formation,the S phase duration in zygotes,mitochondrial function at G1 phase,reactive oxygen species(ROS)level at S phase,DNA damage at G2 phase,early apoptosis in 2-cell embryos,cleavage and blastocyst formation rates were evaluated.The results indicated that the vitrification/warming procedures led to following perturbations 1)spindle abnormalities and chromosome misalignment,alteration of maternal mRNAs and delay in pronucleus formation,2)decreased mitochondrial membrane potential(MMP)and lower adenosine triphosphate(ATP)levels,increased ROS production and DNA damage,G1/S and S/G2 phase transition delay,and delayed first cleavage,and 3)increased early apoptosis and lower levels of cleavage and blastocyst formation.Our results further revealed that such negative impacts of oocyte cryopreservation could be alleviated by supplementation of warming,recovery,PA and IVC media with 10^(−9) mol/L MT before the embryos moved into the 2-cell stage of development.Conclusions:MT might promote cell cycle progression via regulation of MMP,ATP,ROS and maternal mRNA levels,potentially increasing the first cleavage of parthenogenetic zygotes developed from vitrified-warmed mouse oocytes and their subsequent development.
基金the National Key Research and Development Program of China(2021YFD1200803 and 2022YFD1600902)China Agriculture Research System(CARS-40).
文摘Background Abdominal fat deposition depends on both the proliferation of preadipocytes and their maturation into adipocytes,which is a well-orchestrated multistep process involving many regulatory molecules.Circular RNAs(circRNAs)have emergingly been implicated in mammalian adipogenesis.However,circRNA-mediated regulation in chicken adipogenesis remains unclear.Our previous circRNA sequencing data identified a differentially expressed novel circRNA,8:27,886,180|27,889,657,during the adipogenic differentiation of chicken abdominal preadipocytes.This study aimed to investigate the regulatory role of circDOCK7 in the proliferation and adipogenic differentiation of chicken abdominal preadipocytes,and explore its molecular mechanisms of competing endogenous RNA underlying chicken adipogenesis.Results Our results showed that 8:27,886,180|27,889,657 is an exonic circRNA derived from the head-to-tail splicing of exons 19–22 of the dedicator of cytokinesis 7(DOCK7)gene,abbreviated as circDOCK7.CircDOCK7 is mainly distributed in the cytoplasm of chicken abdominal preadipocytes and is stable because of its RNase R resistance and longer half-life.CircDOCK7 is significantly upregulated in the abdominal fat tissues of fat chickens compared to lean chickens,and its expression gradually increases during the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.Functionally,the gain-and loss-of-function experiments showed that circDOCK7 promoted proliferation,G0/G1-to S-phase progression,and glucose uptake capacity of chicken abdominal preadipocytes,in parallel with adipogenic differentiation characterized by remarkably increased intracellular lipid droplet accumulation and triglyceride and acetyl coenzyme A content in differentiated chicken abdominal preadipocytes.Mechanistically,a pull-down assay and a dual-luciferase reporter assay confirmed that circDOCK7 interacted with gga-miR-301b-3p,which was identified as an inhibitor of chicken abdominal adipogenesis.Moreover,the ACSL1 gene was demonstrated to be a direct target of gga-miR-301b-3p.Chicken ACSL1 protein is localized in the endoplasmic reticulum and mitochondria of chicken abdominal preadipocytes and acts as an adipogenesis accelerator.Rescue experiments showed that circDOCK7 could counteract the inhibitory effects of gga-miR-301b-3p on ACSL1 mRNA abundance as well as the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.Conclusions CircDOCK7 serves as a miRNA sponge that directly sequesters gga-miR-301b-3p away from the ACSL1 gene,thus augmenting adipogenesis in chickens.These findings may elucidate a new regulatory mechanism underlying abdominal fat deposition in chickens.
基金supported by the National Natural Science Foundation of China(No.31560615)the National Key Technology Research and Development Program(No.2015BAD03B02)+1 种基金the Program for Changjiang Scholars and Innovation Research Team in University(No.IRT_15R62)the Innovation Base Cultivation and Development Project(No.Z171100002217072)
文摘Background: Tibetan pigs, which inhabit the Tibetan Plateau, exhibit distinct phenotypic and physiological characteristics from those of lowland pigs and have adapted well to the extreme conditions at high altitude.However, the genetic and epigenetic mechanisms of hypoxic adaptation in animals remain unclear.Methods: Whole-genome DNA methylation data were generated for heart tissues of Tibetan pigs grown in the highland(TH, n = 4) and lowland(TL, n = 4), as well as Yorkshire pigs grown in the highland(YH, n = 4) and lowland(YL, n = 4), using methylated DNA immunoprecipitation sequencing.Results: We obtained 480 million reads and detected 280679, 287224, 259066, and 332078 methylation enrichment peaks in TH, YH, TL, and YL, respectively. Pairwise TH vs. YH, TL vs. YL, TH vs. TL, and YH vs. YL comparisons revealed6829, 11997, 2828, and 1286 differentially methylated regions(DMRs), respectively. These DMRs contained 384, 619,192, and 92 differentially methylated genes(DMGs), respectively. DMGs that were enriched in the hypoxia-inducible factor 1 signaling pathway and pathways involved in cancer and hypoxia-related processes were considered to be important candidate genes for high-altitude adaptation in Tibetan pigs.Conclusions: This study elucidates the molecular and epigenetic mechanisms involved in hypoxic adaptation in pigs and may help further understand human hypoxia-related diseases.
基金supported by the National Key Research and Development Program of China(2021YFD1200803)Sanya Yazhou Bay Science and Technology City Administration(SYND-2022-28)China Agriculture Research System of MOF and MARA(CARS-40).
文摘Background:Excessive abdominal fat deposition in commercial broilers presents an obstacle to profitable meat quality,feed utilization,and reproduction.Abdominal fat deposition depends on the proliferation of preadipocytes and their maturation into adipocytes,which involves a cascade of regulatory molecules.Accumulating evidence has shown that microRNAs(miRNAs)serve as post-transcriptional regulators of adipogenic differentiation in mammals.However,the miRNA-mediated molecular mechanisms underlying abdominal fat deposition in chickens are still poorly understood.This study aimed to investigate the biological functions and regulatory mechanism of miRNAs in chicken abdominal adipogenesis.Results:We established a chicken model of abdominal adipocyte differentiation and analyzed miRNA and mRNA expression in abdominal adipocytes at different stages of differentiation(0,12,48,72,and 120 h).A total of 217 differentially expressed miRNAs(DE-miRNAs)and 3520 differentially expressed genes were identified.Target prediction of DE-miRNAs and functional enrichment analysis revealed that the differentially expressed targets were significantly enriched in lipid metabolism-related signaling pathways,including the PPAR signaling and MAPK signaling pathways.A candidate miRNA,gga-miR-106-5p,exhibited decreased expression during the proliferation and differentiation of abdominal preadipocytes and was downregulated in the abdominal adipose tissues of fat chickens compared to that of lean chickens.gga-miR-106-5p was found to inhibit the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.A dual-luciferase reporter assay suggested that the KLF15 gene,which encodes a transcriptional factor,is a direct target of gga-miR-106-5p.gga-miR-106-5p suppressed the posttranscriptional activity of KLF15,which is an activator of abdominal preadipocyte proliferation and differentiation,as determined with gain-and loss-of-function experiments.Conclusions:gga-miR-106-5p functions as an inhibitor of abdominal adipogenesis by targeting the KLF15 gene in chickens.These findings not only improve our understanding of the specific functions of miRNAs in avian adipogenesis but also provide potential targets for the genetic improvement of excessive abdominal fat deposition in poultry.
基金supported by programs for the 973 National Basic Research Program of China (2014CB138505)the Open Foundation of State Key Laboratory of Animal Nutrition (2004DA115184F1415)
文摘Background： Resveratrol, an important phyto-antioxidant commonly found in grapes, mulberry, and other plants,has a variety of functions including anti-aging, anti-cancer and anti-inflammatory activities. In the current study, we investigated the beneficial effects of resveratrol on in vitro porcine oocyte maturation under heat stress（HS）. The effect of resveratrol, melatonin and their combination on alleviating HS was compared according to the maturation rate of oocytes and the development competence of embryos after parthenogenetic activation（PA）.Results： Supplementation with resveratrol（2.0 μmol/L） not only improved the nuclear maturation but also raised the blastocyst rate of porcine embryos＇ PA from oocytes that underwent HS by increasing their glutathione（GSH）level, reducing reactive oxygen species（ROS） and up-regulating the expression of Sirtuin 1（SIRT1）. It was also found that melatonin（10^-7mol/L） and the combination of resveratrol（2.0 μmol/L） plus melatonin（10^-7mol/L） exhibited more potent effects than resveratrol alone regarding their protective activities on oocyte maturation under HS.Conclusions： This study compared the efficiencies of resveratrol, melatonin and their combination for protecting porcine oocytes from heat stress. The mechanisms are attributed to the fact that each treatment may have different ability to regulate the synthesis of steroid hormones and the expression of mature related genes.
基金supported by National Science and Technology Major Project of China(2016ZX08008-003).
文摘Background:Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health.Toll-like receptor 4(TLR4)plays a crucial role in host defence against Gram-negative bacteria.Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections.In a previous study,we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance.The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood.Results:In the present study,we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model.The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection.This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis.Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4,Src signalling,or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria,weakened the ability of the monocytes to eliminate the bacteria,and increased the pH of the endosomes.Conclusion:Together,our results reveal the effects of TLR4 on the control of E.coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model,providing theoretical support for breeding disease resistance in the future.Furthermore,Src and caveolin 1(CAV1)could be potentially valuable targets for the control of infectious diseases.
基金Peking Post-doctoral Research Fund(EE 2019-50)Peking University International Hospital Research Funds(No.YN2019QN13)NHC Key Laboratory of Family Planning and Healthy/key laboratory of reproductive medicine of Hebei provincial(SZ-202006).
文摘Sperm cryopreservation is useful in assisted reproductive technology and male fertility preservation.However,freezing and thawing significantly reduces the total and progressive motility of human spermatozoa.In the present study,we explored the effects of peroxiredoxin 6(PRDX6)on total and progressive motility of human spermatozoa after cryopreservation.Semen samples of 20 males with normal parameters were collected and frozen in media supplemented with different concentrations of PRDX6(0 mM,10−5 mM,10−7 mM,and 10−9 mM,respectively).Postthaw total and progressive motility of sperms were measured.The results showed that in comparison with 0 mM,the concentrations of 10−5 mM,10−7 mM,and 10−9 mM of PRDX6 all significantly improved total motility and progressive motility of sperms(p<0.05).The 10−7 mM of PRDX6 showed the best performance.In conclusion,the supplementation of PRDX6 helps to maintain the total and progressive motility of human spermatozoa.
基金supported by the talent cultivation and developmental support program of China Agricultural University,an award to study the cultivation of high-quality mutton sheep varieties(or lines)from Ningxia province(NXNYYZ20150101)the Natural Science Foundation of Hebei Province of China for Youths(C2019402261).
文摘Background:Tan sheep,an important local sheep breed in China,is famous for their fur quality.One-month-old Tan sheep have white,curly hair with beautiful flower spikes,commonly known as“nine bends”,which has high economic value.However,the“nine bends”characteristic gradually disappears with age;consequently,the economic value of the Tan sheep decreases.Age-related changes in DNA methylation have been reported and may be responsible for age-induced changes in gene expression.Until now,no genome-wide surveys have been conducted to identify potential DNA methylation sites involved in different sheep growth stages.In this study we investigated the dynamic changes of genome-wide DNA methylation profiles in Tan sheep using DNA from skin and deep whole-genome bisulfite sequencing,and compared the DNA methylation levels at three different growth stages:1,24,and 48 months old(mon1,mon24,and mon48,respectively).Results:In this study,11 skin samples from three growth stages(four for mon1,four for mon24,and three for mon48)were used for DNA methylation analysis and gene expression profiling.There were 52,288 and 236 differentially methylated genes(DMGs)identified between mon1 and mon24,mon1 and mon48,and mon24 and mon48,respectively.Of the differentially methylated regions,1.11%,7.61%,and 7.65% were in the promoter in mon1 vs.mon24,mon24 vs.mon48,and mon1 vs.mon48,respectively.DMGs were enriched in the MAPK and WNT signaling pathways,which are related to age growth and hair follicle morphogenesis processes.There were 51 DMGs associated with age growth and curly fleece formation.Four DMGs between mon1 and mon48(KRT71,CD44,ROR2 and ZDHHC13)were further validated by bisulfite sequencing.Conclusions:This study revealed dynamic changes in the genomic methylation profiles of mon1,mon24,and mon48 sheep,and the percentages of methylated cytosines were 3.38%,2.85% and 4.17%,respectively.Of the DMGs,KRT71 and CD44 were highly methylated in mon1,and ROR2 and ZDHHC13 were highly methylated in mon48.These findings provide foundational information that may be used to develop strategies for potentially retaining the lamb fur and thus improving the economic value of Tan sheep.
基金supported by the Genetically Modified Organisms Breeding Major Projects of China(2013ZX08008-005).
文摘The CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPR-associated)adaptive immune system,which was discovered in bacteria and archaea,can specifically degrade invasive viral and plasmid DNA by base pairing between crRNAs(CRISPR RNAs)and the target DNA[1,2].Recently,the Streptococcus pyogenes type II CRISPR system was shown to be able to perform efficient targeted gene disruption by employing three fundamental components:(1)Cas9 endonuclease.
