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Expression of miR‑138 in cryopreserved Bovine sperm is related to their fertility potential

Journal of Animal Science and Biotechnology 2023年第6期14卷 2383-2394页

作者： Albert Salas‑Huetos Jordi Ribas‑Maynou Yentel Mateo‑Otero Carolina Tamargo Marc Llavanera Marc Yeste Unit of Cell Biology Department of BiologyFaculty of SciencesUniversity of GironaGirona 17003Spain. Biotechnology of Animal and Human Reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaGirona 17003Spain. Department of Nutrition Harvard T.H.Chan School of Public HealthHarvard UniversityBostonMA 02115USA. Consorcio CIBER M.P.Fisiopatologia de la Obesidad y Nutricion(CIBERobn)Instituto de Salud Carlos III(ISCIII)Madrid 28029Spain. Present Address:Unit of Preventive Medicine and Public Health Faculty of Medicine and Health SciencesUniversitat Rovira i VirgiliReus 43201Spain. Department of Animal Selection and Reproduction The Regional Agri-Food Research and Development Service of Asturias(SERIDA)Gijon 33394Spain. Catalan Institution for Research and Advanced Studies(ICREA) Barcelona 08010Spain.

Background MicroRNAs(miRNAs)are small,single-stranded,non-coding RNA molecules of 22–24 nucleotides that regulate gene expression.In the last decade,miRNAs have been described in sperm of several mammals,including cattle.It is known that miRNAs can act as key gene regulators of early embryogenesis in mice and humans;however,little is known about the content,expression,and function of sperm-borne miRNAs in early Bovine embryo.In this study,total sperm RNA was isolated from 29 cryopreserved sperm samples(each coming from a separate bull)using a RNeasy kit and treatment with DNase I.RNA concentration and purity were determined through an Epoch spectrophotometer and an Agilent Bioanalyzer.The expression of 10 candidate miRNAs in Bovine sperm(bta-miR-10a,bta-miR-10b,bta-miR-138,bta-miR-146b,bta-miR-19b,bta-miR-26a,bta-miR-34a,bta-miR-449a,bta-miR-495 and btamiR-7),previously identified in testis and/or epididymis,was evaluated with RT-qPCR.The cel-miR-39-3p was used as a spike-in exogenous control.Nonparametric Mann–Whitney tests were run to evaluate which miRNAs were differentially expressed between bulls with high fertility[HF;non-return rates(NRR)ranging from 39.5 to 43.5]and those with subfertility(SF;NRR ranging from 33.3 to 39.3).Several sperm functionality parameters(e.g.,viability,membrane stability or oxygen consumption,among others)were measured by multiplexing flow cytometry and oxygen sensing technologies.Results RNA concentration and purity(260/280 nm ratio)(mean±SD)from the 29 samples were 99.3±84.6 ng/μL and 1.97±0.72,respectively.Bioanalyzer results confirmed the lack of RNA from somatic cells.In terms of the presence or absence of miRNAs,and after applying the Livak method,8 out of 10 miRNAs(bta-miR-10b,-138,-146b,-19b,-26a,-449a,-495,-7)were consistently detected in Bovine sperm,whereas the other two(bta-miR-10a,and-34a)were absent.Interestingly,the relative expression of one miRNA(bta-miR-138)in sperm was significantly lower in the SF than in the HF group(P=0.038).In addition to being associated to

关键词： Bovine Fertility miRNAs Sperm

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Localization of urea transporter B in the developing Bovine rumen

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Animal Nutrition 2022年第3期10卷 216-222页

作者： Chongliang Zhong Tamsin Lyons Orla Heussaff Evelyn Doyle Eoin O'Hara Sinead M.Waters David Kenny Gavin S.Stewart School of Biology and Environmental Science University College DublinBelfieldDublin 4Ireland Animal and Bioscience Research Department Animal and Grassland Research and Innovation CentreTeagascGrangeCounty MeathIreland Department of Agriculture Foodand Nutritional SciencesUniversity of AlbertaEdmontonAlbertaCanada

Urea nitrogen secreted from blood to rumen is a crucial factor shaping the symbiotic relationship between host ruminants and their microbial populations.Passage of urea across rumen epithelia is facilitated by urea transporter B(UT-B),but the long-term regulation of these proteins remains unclear.As ruminal function develops over a period of months,the developing rumen is an excellent model with which to investigate this regulation.Using rumen epithelium samples of calves from birth to 96 d of age,this study performed immunolocalization studies to localize and semi-quantify UT-B protein development.As expected,preliminary experiments confirmed that ruminal monocarboxylate transporter 1(MCT1)short chain fatty acid transporter protein abundance increased with age(P<0.01,n=4).Further investigation revealed that ruminal UT-B was present in the first few weeks of life and initially detected in the basolateral membrane of stratum basale cells.Over the next 2 months,UT-B staining spread to other epithelial layers and semi-quantification indicated that UT-B abundance significantly increased with age(P<0.01,n=4 or 6).These changes were in line with the development of rumen function after the advent of solid feed intake and weaning,exhibiting a similar pattern to both MCT1 transporters and papillae growth.This study therefore confirmed age-dependent changes of in situ ruminal UT-B protein,adding to our understanding of the long-term regulation of ruminal urea transporters.

关键词： UT-B Immunolocalization Rumen Bovine Development

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Modification of alternative splicing in Bovine somatic cell nuclear transfer embryos using engineered CRISPR-Cas13d

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Science China(Life Sciences) 2022年第11期65卷 2257-2268页

作者： Rui Cheng Xiaoman Zheng Yingmei Wang Xing Ma Xin Liu Wenjun Xu Mengyun Wang Yuanpeng Gao Xupeng Xing Chuan Zhou Hongzheng Sun Zekun Guo Fusheng Quan Jun Liu Song Hua Yongsheng Wang Yong Zhang Xu Liu College of Veterinary Medicine Northwest A&F UniversityKey Laboratory of Animal Biotechnology of the Ministry of AgricultureYangling 712100China

Animal cloning can be achieved by somatic cell nuclear transfer(SCNT),but the resulting live birth rate is relatively low.We previously improved the efficiency of Bovine SCNT by exogenous melatonin treatment or by overexpression of lysine-specific demethylase 4D(KDM4D)and 4E(KDM4E).In this study,we revealed abundant alternative splicing(AS)transitions during fertilization and embryonic genome activation,and demonstrated abnormal AS in Bovine SCNT embryos compared with in vitro fertilized embryos.We used the CRISPR-Cas13d RNA-targeting system to target cis-elements of ABI2 and ZNF106 pre-m RNA to modify AS,thus reducing the ratio of abnormal-isoform SCNT embryos by nearly 50%and achieving a high survival rate(11%–19%).These results indicate that this system may provide an efficient method for Bovine cloning,while also paving the way for further improvements in the efficiency of SCNT.

关键词： animal cloning alternative splicing CRISPR-Cas13d Bovine

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Calcitriol induces post-thawed Bovine sperm capacitation

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BIOCELL 2023年第5期47卷 1135-1143页

作者： ANA CECILIA LIAUDAT FRANCISCA EBEL BIANCA ANA OPIZZO MARINA AURORA GONZALEZ DAMIÁN BLOIS PABLO BOSCH NANCY RODRIGUEZ Departamento de Biología Molecular Instituto de Biotecnología Ambiental y Salud(INBIAS)Río Cuarto5800Argentina

Background:Capacitation is a set of physiological changes sperms undergo to acquire fertilizing capacity.In vivo,this process is directly associated with high calcium levels in sperm cytoplasm.Calcitriol,the vitamin D hypercalcemic metabolite,is related to human sperm motility,capacitation,and acrosome reaction.This work aimed to study the effect of calcitriol on bull sperm quality parameters and capacitation.Methods:One million freezethawed spermatozoa were obtained from different bulls and treated with 20 nM of calcitriol for 30 min.Untreated cells(negative control)and treated ones with calcitriol or heparin(100μg/mL,positive capacitation control)were evaluated for motility,viability,and functional parameters.Menadione(70μM,30 min)treatment was included as a reactive oxygen species(ROS)positive sperm agent.Results:The results elucidated that sperm exposed to 20 nM calcitriol showed higher viability,vigor,and capacitation than their positive and negative controls.The percentage of sperm with intact plasma and acrosome membranes,mitochondrial membrane potential(ΔΨm),and phosphatidylserine externalization was similar in all the conditions evaluated,while ROS production was higher with heparin and menadione-treated groups than the calcitriol group or negative control.Conclusion:Our results indicate that calcitriol induces the capacitation of thawed bull spermatozoa and maintains acceptable values of progressive motility,viability,and vigor without altering key biological parameters such as redox status,ΔΨm,and cell death.

关键词： Bovine Sperm capacitation Calcitriol Cell viability Plasma membrane integrity Reactive oxygen species

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Denaturation studies on Bovine serum albumin–bile salt system:Bile salt stabilizes Bovine serum albumin through hydrophobicity

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Journal of Pharmaceutical Analysis 2018年第1期8卷 27-36页

作者： Karpagaraj Malarkani Ivy Sarkar Susithra Selvama Department of Chemistry Vel Tech UniversityAvadiChennai 600062India Department of Chemistry Indian Institute of Technology MadrasChennai 600036India Department of Chemistry PSG College of TechnologyPeelameduCoimbatore 641004India

Protein denaturation is under intensive research, since it leads to neurological disorders of severe consequences. Avoiding denaturation and stabilizing the proteins in their native state is of great importance,especially when proteins are used as drug molecules or vaccines. It is preferred to add pharmaceutical excipients in protein formulations to avoid denaturation and thereby stabilize them. The present study aimed at using bile salts(BSs), a group of well-known drug delivery systems, for stabilization of proteins.Bovine serum albumin(BSA) was taken as the model protein, whose association with two BSs, namely sodium cholate(Na C) and sodium deoxycholate(Na DC), was studied. Denaturation studies on the preformed BSA-BS systems were carried out under chemical and physical denaturation conditions. Urea was used as the chemical denaturant and BSA-BS systems were subjected to various temperature conditions to understand the thermal(physical) denaturation. With the denaturation conditions prescribed here,the data obtained is informative on the association of BSA-BS systems to be hydrophobic and this effect of hydrophobicity plays an important role in stabilizing the serum albumin in its native state under both chemical and thermal denaturation.

关键词： Bile salts Bovine serum albumin Chemical denaturation Thermal denaturation Tryptophan fluorescence Protein stabilization

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Multi-spectroscopic characterization of Bovine serum albumin upon interaction with atomoxetine

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Journal of Pharmaceutical Analysis 2017年第3期7卷 148-155页

作者： Arunkumar T. Buddanavar Sharanappa T. Nandibewoor P.G. Department of studies in Chemistry Karnatak UniversityDharwad 580003India

The quenching interaction of atomoxetine(ATX) with Bovine serum albumin(BSA) was studied in vitro under optimal physiological condition(pH=7.4) by multi-spectroscopic techniques. The mechanism of ATX-BSA system was a dynamic quenching process and was confirmed by the fluorescence spectra and lifetime measurements. The number of binding sites, binding constants and other binding characteristics were computed. Thermodynamic parameters ΔH^0 and ΔS^0 indicated that intermolecular hydrophobic forces predominantly stabilized the drug-protein system. The average binding distance between BSA and ATX was studied by F?rsters theory. UV-absorption, Fourier transform infrared spectroscopy(FT-IR), circular dichroism(CD), synchronous spectra and three-dimensional(3D) fluorescence spectral results revealed the changes in micro-environment of secondary structure of protein upon the interaction with ATX. Displacement of site probes and the effects of some common metal ions on the binding of ATX with BSA interaction were also studied.

关键词： Atomoxetine Bovine serum albumin 3D fluorescence spectra FT-IR Energy transfer Lifetime measurement

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The interplay between non-esterified fatty acids and Bovine peroxisome proliferator-activated receptors: results of an in vitro hybrid approach

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Journal of Animal Science and Biotechnology 2021年第1期12卷 292-304页

作者： Sebastiano Busato Massimo Bionaz Department of Animal and Rangeland Sciences Oregon State UniversityCorvallisORUSA

Background: In dairy cows circulating non-esterified fatty acids(NEFA) increase early post-partum while liver and other tissues undergo adaptation to greater lipid metabolism, mainly regulated by peroxisome proliferator-activated receptors(PPAR). PPAR are activated by fatty acids(FA), but it remains to be demonstrated that circulating NEFA or dietary FA activate Bovine PPAR. We hypothesized that circulating NEFA and dietary FA activate PPAR in dairy cows.Methods: The dose-response activation of PPAR by NEFA or dietary FA was assessed using HP300 e digital dispenser and luciferase reporter in several Bovine cell types. Cells were treated with blood plasma isolated from Jersey cows before and after parturition, NEFA isolated from the blood plasma, FA released from lipoproteins using milk lipoprotein lipase(LPL), and palmitic acid(C16:0). Effect on each PPAR isotype was assessed using specific synthetic inhibitors.Results: NEFA isolated from blood serum activate PPAR linearly up to ~ 4-fold at 400 μmol/L in MAC-T cells but had cytotoxic effect. Addition of albumin to the culture media decreases cytotoxic effects of NEFA but also PPAR activation by ~ 2-fold. Treating cells with serum from peripartum cows reveals that much of the PPAR activation can be explained by the amount of NEFA in the serum(R~2 = 0.91) and that the response to serum NEFA follows a quadratic tendency, with peak activation around 1.4 mmol/L. Analysis of PPAR activation by serum in MAC-T, BFH-12 and BPAEC cells revealed that most of the activation is explained by the activity of PPARδ and PPARγ, but not PPARα. Palmitic acid activated PPAR when added in culture media or blood serum but the activation was limited to PPARδ and PPARα and the response was nil in serum from post-partum cows. The addition of LPL to the serum increased > 1.5-fold PPAR activation.Conclusion: Our results support dose-dependent activation of PPAR by circulating NEFA in Bovine, specifically δand γ isotypes. Data also support the possibility of increasing PPAR activation by die

关键词： Albumin Blood serum Bovine Gene reporter Hepatocytes Lipoprotein lipase Mammary cells Non-esterified fatty acids Peroxisome proliferator-activated receptor

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Sexing Bovine Embryos Using PCR Amplification of Bovine SRY Sequence

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Science China Chemistry 1994年第2期37卷 170-176页

作者：曾溢滔张美兰陈美珏周霞娣黄英任兆瑞黄淑帧胡明信吴学清高建明张斌徐慧如 Shanghai Institute of Medical Genetics Shanghai Children's Hospital Shanghai 200040 PRC) Department of Animal Husbandry Beijing Agriculture College Beijing 102208 PRC

This study analyses the Bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for Bovine SRY. Using PCR amplification of Bovine SRY gene, the embryo sex was de... 详细信息

关键词： Bovine embryo sex determination PCR SRY.

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Methylation patterns in 5' terminal regions of pluripotency-related genes in Bovine in vitro fertilized and cloned embryos

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Journal of Genetics and Genomics 2010年第5期37卷 297-304页

作者： Jie Lan Song Hua Hailin Zhang Yongli Song Jun Liu Yong Zhang Key Laboratory of Animal Reproductive Physiology & Embryo Technology Institution of biotechnology College of Veterinary Medicine Northwest A & F University Yangling 712100 China

In order to investigate DNA methylation profiles of five pluripotency-related genes （Oct4, Sox2, Nanog, Rexl and Fgf4） during Bovine maternal to zygotic transition （MZT） in both in vitro fertilized （IVF） and nuclear transfer （NT） embryos, sodium bisulfite sequencing method was used to detect DNA methylation levels, accompanied by the statistical analysis of embryo developmental rates. The results showed that Oct4, Nanog, Rexl and Fgf4 were respectively demethylated by 25.22% （P 〈 0.01）, 3.84% （P 〉 0.05）, 31.82% （P 〈 0.01） and 10% （P 〉 0.05） while Sox2 retained unmethylafion during MZT in IVF embryos. By contrast, Oct4 and Rexl respectively underwent demethylation by 23.04% （P 〈 0.01） and 6.02% （P 〉 0.05）, and, reversely, Sox2, Nanog and Fgf4 respectively experienced remethylation by 0.84% （P 〉 0.05）, 5.39% （P 〉 0.05） and 5.46% （P 〉 0.05） during MZT in NT embryos. Interestingly, the CpG 14 site of Sox2 was specifically methylated in both 8-cell and morula NT embryos. In addition, the development of blastocysts between IVF and NT embryos showed no significant difference. DNA methylation analysis showed that only Oct4 and Sox2 underwent the correct methylation reprogramming process, which may be responsible for the development of blastocysts of NT embryos to a certain extent. In conclusion, the five genes respectively experienced demethylation to different extents and incomplete DNA methylation reprogramming during Bovine MZT in both IVF and NT embryos, suggesting that they may be used as indicators for Bovine embryo developmental competence.

关键词： Bovine DNA methylation Fgf4 Nanog Oct4 Rex1 Sox2

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Association between mitochondrial DNA haplotype compatibility and increased efficiency of Bovine intersubspecies cloning

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Journal of Genetics and Genomics 2011年第1期38卷 21-28页

作者： Hao Yan Zhonghai Yan Qingwen Ma Fei Jiao Shuzhen Huang Fanyi Zeng Yitao Zeng Shanghai Institute of Medical Genetics Shanghai Jiao Tong University 24/1400 West Beijing Road Shanghai 200040 China The Key Lab of Embryo Molecular Biology Ministry of Health China and Shanghai Lab of Embryo and Reproduction Engineering 24/1400 West Beijing Road Shanghai 200040 China Institute of Medical Science Shanghai Jiao Tong University School of Medicine 280 South Chongqing Road Shanghai 200025 China

Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer （SCNT） have poorer developmental potential than those from intrasubspecies SCNT. Based on our previous study that Holstein dairy Bovine （HD） mitochondrial DNA （mtDNA） haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development, we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer （LY） as recipient cytoplast according to mtDNA haplotypes determined by polymerase chain reaction- restriction fragment length polymorphism （PCR-RFLP） analysis. The results demonstrated that intersubspecies mtDNA homotype SCNT embryos had higher pre- and post-implantation developmental competence than intrasubspecies mtDNA heterotype embryos as well as improved blastocyst reprogramming status, including normal H3K9 dimethylation pattern and promoter hypomethylation of pluripotent genes such as Oct4 and Sox2, suggesting that intersubspecies SCNT using LY oocytes maintains HD cloning efficiency and may reprogram HD nuclei to develop into a normal cloned animal ultimately. Our results indicated that karyoplast-cytoplast interactions and mtDNA haplotype compatibility may affect Bovine intersubspecies SCNT efficiency. This study on Bovine intersubspecies SCNT is valuable for understanding the mechanisms of mtDNA haplotype compatibility between karyoplast and cytoplast impacting the Bovine SCNT efficiency, and provides an alternative and economic resource for HD cloning.

关键词： Somatic cell nuclear transfer （SCNT） Mitochondria mtDNA haplotype Epigenetic modification Bovine Intersubspecis Intrasubspecis Developmental competence

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