Publications by authors named "Yunsheng Li"

86 Publications

A small-molecule pan-class I glucose transporter inhibitor reduces cancer cell proliferation in vitro and tumor growth in vivo by targeting glucose-based metabolism.

Cancer Metab 2021 Mar 26;9(1):14. Epub 2021 Mar 26.

Department of Biological Sciences, Ohio University, Athens, OH, 45701, USA.

Background: Cancer cells drastically increase the uptake of glucose and glucose metabolism by overexpressing class I glucose transporters (GLUT1-4) to meet their energy and biomass synthesis needs and are very sensitive and vulnerable to glucose deprivation. Although targeting glucose uptake via GLUTs has been an attractive anticancer strategy, the relative anticancer efficacy of multi-GLUT targeting or single GLUT targeting is unclear. Here, we report DRB18, a synthetic small molecule, is a potent anticancer compound whose pan-class I GLUT inhibition is superior to single GLUT targeting.

Methods: Glucose uptake and MTT/resazurin assays were used to measure DRB18's inhibitory activities of glucose transport and cell viability/proliferation in human lung cancer and other cancer cell lines. Four HEK293 cell lines expressing GLUT1-4 individually were used to determine the IC values of DRB18's inhibitory activity of glucose transport. Docking studies were performed to investigate the potential direct interaction of DRB18 with GLUT1-4. Metabolomics analysis was performed to identify metabolite changes in A549 lung cancer cells treated with DRB18. DRB18 was used to treat A549 tumor-bearing nude mice. The GLUT1 gene was knocked out to determine how the KO of the gene affected tumor growth.

Results: DRB18 reduced glucose uptake mediated via each of GLUT1-4 with different ICs, which match with the docking glidescores with a correlation coefficient of 0.858. Metabolomics analysis revealed that DRB18 altered energy-related metabolism in A549 cells by changing the abundance of metabolites in glucose-related pathways in vitro and in vivo. DRB18 eventually led to G1/S phase arrest and increased oxidative stress and necrotic cell death. IP injection of DRB18 in A549 tumor-bearing nude mice at 10 mg/kg body weight thrice a week led to a significant reduction in the tumor volume compared with mock-treated tumors. In contrast, the knockout of the GLUT1 gene did not reduce tumor volume.

Conclusions: DRB18 is a potent pan-class I GLUT inhibitor in vitro and in vivo in cancer cells. Mechanistically, it is likely to bind the outward open conformation of GLUT1-4, reducing tumor growth through inhibiting GLUT1-4-mediated glucose transport and metabolisms. Pan-class I GLUT inhibition is a better strategy than single GLUT targeting for inhibiting tumor growth.
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http://dx.doi.org/10.1186/s40170-021-00248-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004435PMC
March 2021

Natural Compound α-PGG and Its Synthetic Derivative 6Cl-TGQ Alter Insulin Secretion: Evidence for Diminishing Glucose Uptake as a Mechanism.

Diabetes Metab Syndr Obes 2021 24;14:759-772. Epub 2021 Feb 24.

Department of Internal Medicine, Division of Endocrinology, Diabetes and Metabolism, University of California at Davis (UC Davis) School of Medicine, UC Davis Health Science, Sacramento, CA, 95817, USA.

Purpose: Previously we showed that natural compound α-penta-galloyl-glucose (α-PGG) and its synthetic derivative 6-chloro-6-deoxy-1,2,3,4-tetra-O-galloyl-α-D-glucopyranose (6Cl-TGQ) act to improve insulin signaling in adipocytes by increasing glucose transport. In this study, we investigated the mechanism of actions of α-PGG and 6Cl-TGQ on insulin secretion.

Methods: Mouse islets and/or INS-1832/13 beta-cells were used to test the effects of our compounds on glucose-stimulated insulin secretion (GSIS), intracellular calcium [Ca] using fura-2AM, glucose transport activity via a radioactive glucose uptake assay, intracellular ATP/ADP, and extracellular acidification (ECAR) and mitochondrial oxygen consumption rates (OCAR) using Seahorse metabolic analysis.

Results: Both compounds reduced GSIS in beta-cells without negatively affecting cell viability. The compounds primarily diminished glucose uptake into islets and beta-cells. Despite insulin-like effects in the peripheral tissues, these compounds do not act through the insulin receptor in islets. Further interrogation of the stimulus-secretion pathway showed that all the key metabolic factors involved in GSIS including ECAR, OCAR, ATP/ADP ratios, and [Ca] of INS-1832/13 cells were diminished after the compound treatment.

Conclusion: The compounds suppress glucose uptake of the beta-cells, which consequently slows down the rates of glycolysis and ATP synthesis, leading to decrease in [Ca] and GSIS. The difference between adipocytes and beta-cells in effects on glucose uptake is of great interest. Further structural and functional modifications could produce new compounds with optimized therapeutic potentials for different target cells. The higher potency of synthetic 6Cl-TGQ in enhancing insulin signaling in adipocytes but lower potency in reducing glucose uptake in beta-cells compared to α-PGG suggests the feasibility of such an approach.
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http://dx.doi.org/10.2147/DMSO.S284295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917315PMC
February 2021

miR-130a-3p regulates steroid hormone synthesis in goat ovarian granulosa cells by targeting the PMEPA1 gene.

Theriogenology 2021 Apr 19;165:92-98. Epub 2021 Feb 19.

College of Animal Science and Technology, Anhui Agricultural University, Anhui Hefei, 230036, China; Local Animal Genetic Resources Conservation and Biobreeding Laboratory of Anhui Province, Anhui Hefei, 230036, China. Electronic address:

MicroRNAs (miRNAs) are key epigenomic regulators of proliferation, differentiation, and secretion in cells involved in follicular development. We here studied the functional role of one such molecule, miR-130a-3p, in goat ovarian granulosa cells (GCs). High expression of this miRNA was evident in goat GCs by fluorescence in situ hybridization and suppressed estradiol and progesterone secretion from these cells, as determined by ELISA. miR-130a-3p was predicted to have a binding site for the 3' UTR of the prostate transmembrane protein androgen induced 1 gene (PMEPA1), and this was verified by a dual-luciferase reporter assay. PMEPA1 mRNA and protein expression were both found to be regulated by miR-130a-3p in GCs. Moreover, the overexpression or knockdown of PMEPA1 enhanced or suppressed estradiol and progesterone secretion from these cells, respectively. Furthermore, the secretion of estradiol and progesterone did not change significantly after the offsetting of PMEPA1 overexpression in GCs by miR-130a-3p. In summary, our present data indicate that miR-130a-3p inhibits the secretion of estradiol and progesterone in GCs by targeting PMEPA1. Our study thus provides seminal data and important new insights into the regulation of reproductive mechanisms in the nanny goat and other female mammals.
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http://dx.doi.org/10.1016/j.theriogenology.2021.02.012DOI Listing
April 2021

METTL3-mediated m6A methylation negatively modulates autophagy to support porcine blastocyst development.

Biol Reprod 2021 Feb 16. Epub 2021 Feb 16.

Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China.

N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Here, we show that METTL3-mediated m6A methylation sustains porcine blastocyst development via negatively modulating autophagy. We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 (a key regulator of autophagy) and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3'UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell qPCR revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Taken together, these results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.
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http://dx.doi.org/10.1093/biolre/ioab022DOI Listing
February 2021

Black and Green Tea Supplements Ameliorate Male Infertility in a Murine Model of Obesity.

J Med Food 2020 Dec 13;23(12):1303-1311. Epub 2020 Nov 13.

State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, China.

Obesity, a chronic metabolic disorder, can affect male reproductive function. As a functional beverage, tea has many biological activities and potential in the treatment of obesity. However, its effects on male reproductive damage induced by obesity are not yet clear. In this study, a murine model of obesity was established by feeding with high-fat diet (HF). A total of 24 male mice were divided into four groups: normal diet (control), HF, HF supplemented with 5% green tea powder (HF+G), and HF supplemented with 5% black tea powder (HF+B). The results showed that the HF + B significantly reduced the mouse body weight gain and testicular coefficient and lowered the serum insulin and leptin levels compared with the HF group. The sperm malformation rate of mice in the HF group had a significant increase when compared with the control group, the HF + B group had a significant decrease compared with the HF group, and no difference from the control group. The HF + G and HF + B significantly increased testosterone levels in serum compared with the HF group. The testosterone production-related gene cytochrome P450 family 11 subfamily a member () and cytochrome p450 family 17 subfamily a member 1 () expressions in testis were significantly increased in the HF + G group compared with HF group. In addition, the HF + G and HF + B abolished the effects of HF on superoxide dismutase (SOD), malondialdehyde, and glutathione levels in testis and antioxidant-related gene expressions of and . Overall, our findings have provided evidence that black and green tea has a positive effect on reducing reproductive damage in a male murine model of obesity, and that black tea is more effective than green tea.
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http://dx.doi.org/10.1089/jmf.2020.4784DOI Listing
December 2020

Proteomic analysis of healthy and atretic porcine follicular granulosa cells.

J Proteomics 2021 02 31;232:104027. Epub 2020 Oct 31.

Anhui Provincial Laboratory of Animal Genetic Resources Protection and Breeding, College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang West Road, Hefei, Anhui 230036, China; Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Bio-Breeding, 130 Changjiang West Road, Hefei, Anhui 230036, China; Department of Veterinary Medicine, College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang West Road, Hefei, Anhui 230036, China. Electronic address:

Follicular atresia is initiated with the apoptosis of granulosa cells (GCs) after birth in mammals. The molecular mechanisms underlying GC apoptosis during follicular selection are unclear at present. The objective of this study is to identify the proteins and pathways that may be involved in porcine follicular atresia. Proteins isolated from GCs collected from healthy and atretic follicles were detected by tandem mass tag (TMT) protein labeling and LC-MS/MS. A total of 4591 proteins in the healthy follicle granulosa cell (HFGC) and atretic follicle granulosa cell (AFGC) groups were identified, and 399 differentially abundant proteins were found between the HFGC and AFGC groups; of which 262 proteins were significantly up-regulated and 137 proteins were significantly down-regulated. Differential protein enrichment analysis showed that proteins involved in proteolysis, protein destabilization, phagocytosis, and engulfment were more abundant in the AFGC group. Also, these proteins were mainly involved in the lysosome, phagosome, autophagy, and apoptosis pathways. Specially, PTGFRN is potential important regulated protein in the development of the antral follicle, and down-regulation of PTGFRN in GCs may lead to follicular atresia. Our study shows that the identified proteins and their related signaling pathways may play crucial roles during health follicle develop to atretic follicle. SIGNIFICANCE: Follicular atresia during 'selection' reduces the reproductive potential of sows. In this study, we found 399 proteins differentially abundant. between the HFGC and AFGC groups. These results establish a foundation for elucidating the mechanism of follicular atresia in swine.
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http://dx.doi.org/10.1016/j.jprot.2020.104027DOI Listing
February 2021

CLAUDIN7 modulates trophectoderm barrier function to maintain blastocyst development in pigs.

Theriogenology 2020 Dec 3;158:346-357. Epub 2020 Oct 3.

Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, China. Electronic address:

Trophectoderm (TE) barrier function is an essential prerequisite for blastocyst development. CLAUDIN7 (CLDN7), a member of CLAUDINS family, is involved in regulating intercellular exchange and cell polarity in epithelium cells. However, the role of CLDN7 in porcine early embryo development is yet to be explored. Here, we found that CLDN7 was highly conserved in different species and was widely expressed in different tissues. Remarkably, CLDN7 expression maintained a low level from GV oocyte to 4-cell stage whereas its expression exhibited a higher level from 8-cell stage onwards. Microinjection of siRNA into cytoplasm effectively knocked down expression of CLDN7 mRNA and protein in porcine embryos. CLDN7 knockdown not only significantly reduced blastocyst rates of embryos derived from parthenogenetic activation and in vitro fertilization, but also reduced number of total cells and TE cells in the resulting blastocysts. Furthermore, CLDN7 knockdown led to a significant reduction in expression of multiple genes associated with tight junction assembly and fluid accumulation. A permeability assay revealed that CLDN7 knockdown disrupted tight junction assembly and paracellular sealing in the TE epithelium. Taken together, these results demonstrate that CLDN7 regulates porcine blastocyst development via modulating trophectoderm barrier function.
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http://dx.doi.org/10.1016/j.theriogenology.2020.09.038DOI Listing
December 2020

Subthalamic nucleus deep brain stimulation improves sleep in Parkinson's disease patients: a retrospective study and a meta-analysis.

Sleep Med 2020 10 10;74:301-306. Epub 2020 Aug 10.

Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. Electronic address:

Background: Most Parkinson's patients suffered from sleep problems. There is increasing evidence that Subthalamic Nucleus Deep Brain Stimulation (STN-DBS) has a positive effect on several sleep parameters, improving overall sleep quality in patients with PD. However, the results are controversial.

Methods: We performed a retrospective study and meta-analysis to assess the Parkinson's disease sleep scale (PDSS) in Parkinson's patients.

Results: We reviewed our data of patients who underwent STN-DBS, and then extracted five other trials to perform a meta-analysis. The pooled results showed an advantage on post-operative PDSS in both our medical center and pooled results (MD = 20.41, 95% CI = [13.03, 27.79], I = 61%, P < 0.001). There was a significant difference in Unified Parkinson's Disease Rating Scale (UPDRS)-Ⅲ score between pre and post-operation (MD = -12.59, 95% CI = [-14.70, -10.49], I = 90%, P < 0.001). What's more, Parkinsonian medication was significantly lower in the post-operative groups after DBS (MD = -314.71, 95% CI = [-468.13, -161.28], I = 53%, P < 0.001).

Conclusion: In the retrospective study and meta-analysis of 6 trials, we found that DBS can significantly increase sleep quality. Furthermore, motor function improved and Parkinsonian medication was significantly decreased postoperatively. The sample size was enough and no further investigations would change the conclusion.
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http://dx.doi.org/10.1016/j.sleep.2020.07.042DOI Listing
October 2020

Single-Cell Transcriptome Profiling Revealed That Vitrification of Somatic Cloned Porcine Blastocysts Causes Substantial Perturbations in Gene Expression.

Front Genet 2020 24;11:640. Epub 2020 Jul 24.

Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, China.

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http://dx.doi.org/10.3389/fgene.2020.00640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394247PMC
July 2020

RNA-Seq Reveals miRNA Role Shifts in Seven Stages of Skeletal Muscles in Goat Fetuses and Kids.

Front Genet 2020 7;11:684. Epub 2020 Jul 7.

College of Animal Science and Technology, Anhui Agricultural University, Hefei, China.

MicroRNAs (miRNAs) are indispensable for the regulation of skeletal muscle. We performed RNA sequencing (RNA-seq) to establish a comprehensive miRNA profiling of goats in seven stages, namely, 45- (F45), 65- (F65), 90- (F90), 120- (F120), and 135-day (F135) fetuses, newborn (B1), and 90-day-old (B90) kids. In total, 421 known miRNAs and 228 goat novel miRNAs were identified in the data, and the average abundance of 19 miRNAs in seven stages exceeds 10,000 reads per million. Furthermore, 420 differentially expressed miRNAs (DEmiRNAs) were identified in all comparison group at seven stages, 80 of which were uniquely differentially expressed in the B1 and B90 comparison groups. Pathway analysis indicated that this group was associated with the release of muscle hypertrophy and regulation of myoblast proliferation. Besides, 305 DEmiRNAs were clustered into three significantly enriched profiles (profiles 11, 16, and 19). Function analysis revealed that profile 16 was related to muscle hypertrophy and differentiation. Profile 11 was involved in multiple enzyme activities and metabolic processes in muscle cells. And profile 19 was involved in material transport and structural stability. Two highly expressed miRNAs and three key miRNAs (chi-miR-328-3p, chi-miR-767, and chi-miR-150) of these profiles were verified to be consistent with the data by quantitative real-time PCR. These results provided a catalog of goat muscle-associated miRNAs, allowing us to better understand the transformation of miRNA roles during mammalian muscle development.
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http://dx.doi.org/10.3389/fgene.2020.00684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7358459PMC
July 2020

miRNA-182/Deptor/mTOR axis regulates autophagy to reduce intestinal ischaemia/reperfusion injury.

J Cell Mol Med 2020 07 8;24(14):7873-7883. Epub 2020 Jun 8.

Department of Anesthesiology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.

It had been reported miR-182 was down-regulated after intestinal ischaemia/reperfusion (I/R) damage. However, its role and potential mechanisms are still unknown. This study was aimed to elucidate the function of miR-182 in intestinal I/R injury and the underlying mechanisms. The model of intestinal injury was constructed in wild-type and Deptor knockout (KO) mice. Haematoxylin-eosin staining, Chiu's score and diamine oxidase were utilized to detect intestinal damage. RT-qPCR assay was used to detected miR-182 expression. Electronic microscopy was used to detect autophagosome. Western blot was applied to detect the expression of Deptor, S6/pS6, LC3-II/LC3-I and p62. Dual-luciferase reporter assay was used to verify the relationship between miR-182 and Deptor. The results showed miR-182 was down-regulated following intestinal I/R. Up-regulation of miR-182 reduced intestinal damage, autophagy, Deptor expression and enhanced mTOR activity following intestinal I/R. Moreover, suppression of autophagy reduced intestinal damage and inhibition of mTOR by rapamycin aggravated intestinal damage following intestinal I/R. Besides, damage of intestine was reduced and mTOR activity was enhanced in Deptor KO mice. In addition, Deptor was the target gene of miR-182 and was indispensable for the protection of miR-182 on intestine under I/R condition. Together, our research implicated up-regulation of miR-182 inhibited autophagy to alleviate intestinal I/R injury via mTOR by targeting Deptor.
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http://dx.doi.org/10.1111/jcmm.15420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348187PMC
July 2020

The efficacy of transversus abdominis plane block with or without dexmedetomidine for postoperative analgesia in renal transplantation. A randomized controlled trial.

Int J Surg 2020 Jul 2;79:196-201. Epub 2020 Jun 2.

Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhongshan 2nd Road, 510080, Guangzhou, China. Electronic address:

Background: Current options for effective postoperative analgesia after renal transplantation are limited, due to altered renal clearance and the risk of renal damage. This study compared the analgesic effect of the transversus abdominis plane block, with or without dexmedetomidine, in renal transplant recipients.

Materials And Methods: This prospective randomized double-blinded clinical trial was performed from November 2014 to March 2017. Patients were randomly divided into group C (morphine intravenous patient-controlled analgesia), group R (morphine intravenous patient-controlled analgesia and transversus abdominis plane block), and group RD (morphine intravenous patient-controlled analgesia and transversus abdominis plane block with 1 μg/kg dexmedetomidine). Morphine consumption, time to first request for analgesia, pain, sedation, nausea, vomiting, respiratory depression, and bradycardia were measured at 2, 4, 6, 12 and 24 h after surgery.

Results: The visual analogue pain score in group C was the highest among the three groups at the 2nd and 4th hour. Morphine consumption was the highest in group C at all assessed time intervals (p < 0.01). By the 12th hour and 24th hour, morphine consumption (calculated by time interval) was the lowest in group RD (p < 0.05), while no statistical difference was found between groups C and R. The average time to first request of analgesia was the longest and shortest in group RD and group C, respectively (p < 0.01). The overall incidence of nausea and vomiting was the highest in group C (p < 0.05).

Conclusions: The transversus abdominis plane block reduced morphine consumption in the first 24 h following renal transplantation, and the addition of dexmedetomidine provided a more effective analgesic effect.
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http://dx.doi.org/10.1016/j.ijsu.2020.05.073DOI Listing
July 2020

Inhibition of germinal vesicle breakdown using IBMX increases microRNA-21 in the porcine oocyte.

Reprod Biol Endocrinol 2020 May 11;18(1):39. Epub 2020 May 11.

Department of Animal Science, 2356 Kildee Hall, Iowa State University, Ames, IA, 50011, USA.

Background: Germinal vesicle breakdown (GVBD) occurs during oocyte meiotic maturation, a period when transcriptional processes are virtually inactive. Thus, the maturing oocyte is reliant on processes such as post-transcriptional gene regulation (PTGR) to regulate the mRNA and protein repertoire. MicroRNA (miRNA) are a class of functional small RNA that target mRNA to affect their abundance and translational efficiency. Of particular importance is miRNA-21 (MIR21) due to its role in regulating programmed cell death 4 (PDCD4). The objective of this study was to characterize the abundance and regulation of MIR21 in relation to GVBD.

Methods: Oocytes were collected from aspirated porcine tertiary follicles. Relative abundance of mature MIR21 was quantified at 0, 8, 16, 24, 32, and 42 h of in vitro (IVM) with or without treatment with 3-isobutyl-1-methylxanthine (IBMX).

Results: IBMX increased abundance of MIR21 at 24 h approximately 30-fold compared to control oocytes (P < 0.05), and the induced increase in MIR21 abundance at 24 h was concomitant with premature depletion of PDCD4 protein abundance. To characterize the effect of artificially increasing MIR21 on oocyte competence without inhibiting GVBD, a MIR21 mimic, scrambled microRNA negative control, or nuclease free water was micro-injected into denuded oocytes at 21 h of IVM. The maturation rate of oocytes injected with synthetic MIR21 (63.0 ± 7.5%) was higher than oocytes injected with negative controls (P < 0.05).

Conclusions: Inhibition of nuclear meiotic maturation via IBMX significantly increased MIR21 and decreased its target, PDCD4. Injection of a MIR21 mimic increased oocyte maturation rate. Our results indicate MIR21 is active and important during meiotic maturation of the oocyte.
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http://dx.doi.org/10.1186/s12958-020-00603-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212575PMC
May 2020

Transcription profiles of oocytes during maturation and embryos during preimplantation development in vivo in the goat.

Reprod Fertil Dev 2020 Apr;32(7):714-725

Anhui Province Key Laboratory of Local Livestock and Poultry Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China; and Corresponding authors. Emails:

RNA sequencing performed on goat matured oocytes and preimplantation embryos generated invivo enabled us to define the transcriptome for goat preimplantation embryo development. The largest proportion of changes in gene expression in goat was found at the 16-cell stage, not as previously defined at the 8-cell stage, and is later than in other mammalian species. In all, 6482 genes were identified to be significantly differentially expressed across all consecutive developmental stage comparisons, and the important signalling pathways involved in each development transition were determined. In addition, we identified genes that appear to be transcribed only at a specific stage of development. Using weighted gene coexpression network analysis, we found nine stage-specific modules of coexpressed genes that represent the corresponding stage of development. Furthermore, we identified conserved key members (or hub genes) of the goat transcriptional networks. Their association with other embryo genes suggests that they may have important regulatory roles in embryo development. Our cross-mammalian species transcriptomic comparisons demonstrate both conserved and goat-specific features of preimplantation development.
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http://dx.doi.org/10.1071/RD19391DOI Listing
April 2020

Effect of GABA-T on Reproductive Function in Female Rats.

Animals (Basel) 2020 Mar 27;10(4). Epub 2020 Mar 27.

Anhui Provincial Laboratory of Animal Genetic Resources Protection and Breeding, College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang West Road, Hefei 230036, China.

This study explored the role of γ-aminobutyric acid transaminase (GABA-T) in the puberty and reproductive performance of female rats. Immunofluorescence technique, quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the distribution of GABA-T and the expression of genes and hormones in female rats, respectively. The results showed that GABA-T was mainly distributed in the arcuate nucleus (ARC), paraventricular nucleus (PVN) and periventricular nucleus (PeN) of the hypothalamus, and in the adenohypophysis, ovarian granulosa cells and oocytes. mRNA level at 28 d was lowest in the hypothalamus and the pituitary; at puberty, it was lowest in the ovary. mRNA level was highest in adults in the hypothalamus; at infancy and puberty, it was highest in the pituitary; and at 21 d it was highest in the ovary. After vigabatrin (GABA-T irreversible inhibitor) was added to hypothalamus cells, the levels of mRNA and mRNA were significantly reduced, but mRNA increased at the dose of 25 and 50 μg/mL; mRNA was significantly increased but mRNA was reduced at the 50 μg/mL dose. In prepubertal rats injected with vigabatrin, puberty onset was delayed. mRNA, mRNA and mRNA levels were significantly reduced, but mRNA level increased in the hypothalamus. Vigabatrin reduced the concentrations of GABA-T, luteinizing hormone (LH) and progesterone (P), and the ovarian index. Lactation performance was reduced in adult rats with vigabatrin treatment. Four hours after vigabatrin injection, the concentrations of GABA-T and LH were significantly reduced in adult and 25 d rats, but follicle-stimulating hormone (FSH) increased in 25 d rats. In conclusion, GABA-T affects the reproductive function of female rats by regulating the levels of , and in the hypothalamus as well as the concentrations of LH and P.
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http://dx.doi.org/10.3390/ani10040567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222393PMC
March 2020

Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in SCID Pigs.

Front Immunol 2020 6;11:100. Epub 2020 Feb 6.

Department of Animal Science, Iowa State University, Ames, IA, United States.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T B NK SCID pigs through site directed CRISPR/Cas9 mutagenesis of within a naturally occurring genetic background. We confirmed pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed injections of cultured human CD34 selected cord blood cells into the fetal SCID pigs. At birth, human CD45 CD3ε cells were detected in cord and peripheral blood of injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.
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http://dx.doi.org/10.3389/fimmu.2020.00100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017803PMC
March 2021

Anti-Müllerian hormone inhibits luteinizing hormone-induced androstenedione synthesis in porcine theca cells.

Theriogenology 2020 Jan 2;142:421-432. Epub 2019 Nov 2.

Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, China. Electronic address:

AMH (Anti-Müllerian Hormone) is involved in the regulation of follicle growth initiation and inhibits FSH-induced aromatase expression and estrogen production in granulosa cells. However, the function of AMH in steroidogenesis by theca cells remains unclear. The aim of this study is to investigate the role of AMH as a regulator of the basal and stimulated steroid production by pig granulosa cells (pGCs) and theca cells (pTCs). PGCs and pTCs were incubated with hormones AMH, LH (luteinizing hormone), FSH (follicle stimulating hormone), individually or in combination. The expression of CYP19A1, HSD3B1, CYP11A1, LHCGR, and CYP17A1 mRNA were evaluated by quantitative reverse transcriptase PCR. In pGCs, 10 ng/mL AMH significantly decreased the FSH-stimulated effect on FSHR and CYP19A1 expression and estradiol production. In pTCs, LH treatment significantly increased the expression of HSD3B1, CYP11A1, LHCGR, and androstenedione or progesterone production (P < 0.05). Additionally, 10 ng/mL AMH also significantly decreased the LH-stimulated effects on the expression of HSD3B1, CYP11A1, CYP17A1, LHCGR and androstenedione production. Transfection with siAMHR2-I abolished the suppressive effects of AMH on LH-induced HSD3B1 expression and androstenedione production. Taken together, these results demonstrate that AMH is involved in FSH induced estradiol production in pGCs and LH induced androstenedione production in pTCs by regulating the steroidogenesis pathway.
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http://dx.doi.org/10.1016/j.theriogenology.2019.10.037DOI Listing
January 2020

Suppression of TRPM2 reduces renal fibrosis and inflammation through blocking TGF-β1-regulated JNK activation.

Biomed Pharmacother 2019 Dec 23;120:109556. Epub 2019 Oct 23.

Department of Nephropathy, Wenling First People's Hospital of Zhejiang, Wenling, 317500, China.

Chronic kidney disease (CKD) is a major cause of death. Renal fibrosis and inflammation are common pathways contributing to the development of this disease. However, the molecular mechanisms underlying CKD are not fully understood. TRPM2 (Transient receptor potential melastatin-2) was previously identified as a potential target in various diseases due to its multiple functions. In the study, mice with unilateral urethral obstruction (UUO) were used to explore the effects of TRPM2 on renal injury. First, TRPM2 expression was up-regulated in kidney of mice after UUO. Renal histological analysis using H&E and PAS staining showed that histological changes induced by UUO were markedly alleviated in TRPM2-deficient mice. In addition, TRPM2 knockout markedly improved renal dysfunction, as evidenced by the reduced serum creatine, blood urea nitrogen (BUN), kidney injury molecule 1 (KIM-1) expression and enhanced Nephrin levels. TRPM2 ablation significantly attenuated renal interstitial fibrosis in mice with UUO via decreasing transforming growth factor (TGF)-β1 expression, accompanied with the reduction of fibrotic genes, such as α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), fibronectin (FN) and Collagen 1 alpha 1 (Col1α1). Suppressing TRPM2 expression also suppressed inflammatory cell infiltration and release of pro-inflammatory factors in UUO-triggered renal fibrosis. Further, TRPM2 deficiency inhibited IκBα/nuclear factor (NF)-κB signaling in UUO-treated mice. Moreover, c-Jun N-terminal kinase (JNK) signaling was blocked by TRPM2 knockout in UUO mice. Surprisingly, the in vitro results indicated that blocking JNK activation resulted in the suppression of TGF-β1-induced fibrosis and inflammation. Together, these findings demonstrate that the inhibition of TRPM2 might protect against renal fibrosis and inflammation through impeding JNK activation regulated by TGF-β1.
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http://dx.doi.org/10.1016/j.biopha.2019.109556DOI Listing
December 2019

Ischemic Preconditioning Preventing Downregulation of miR-182 Protects Intestine Against Ischemia/Reperfusion Injury by Inhibiting Apoptosis.

Arch Med Res 2019 07 5;50(5):241-248. Epub 2019 Oct 5.

Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. Electronic address:

Background: Intestinal ischemia/reperfusion (I/R) injury is a severe condition associated with high morbidity and mortality. Ischemic preconditioning (IPC) had been found to be the most promising strategies against I/R injury. However, the potential molecular mechanisms underlying the protective effect of IPC have not been fully disclosed. MicroRNA182 (miR-182) is closely related to apoptosis and plays an important role in I/R injury. Our recent study demonstrated that miR-182 was down-regulated in the intestinal mucosa after I/R injury. However, whether miR-182 is involved in the protective effects of IPC in the setting of intestinal I/R injury is unknown.

Aims: To investigate the role of miR-182 in the protective effect of IPC in intestine after I/R injury and potential mechanisms.

Methods: AntagomiR-182 was pretreated before IPC in mice with intestinal I/R injury. MiR-182 mimic was administered before oxygen and glucose deprivation and reperfusion (OGD/R) in mice intestinal mucosa epithelial (MIME) cells.

Results: IPC partially prevented the downregulation of miR-182 in mice, which was blocked by pretreatment with antagomiR-182. Compared with the IPC group, pretreatment with antagomiR-182 further increased Chiu's scores and diamine oxidase activities. Meanwhile, apoptotic cells and cleaved caspase-3 expression were increased. Compared with the OGD/R group, pretreatment with miR-182 mimic prevented the downregulation of miR-182, improved cell survival, reduced apoptosis and cleaved caspase-3 expression in MIME cells.

Conclusions: The downregulation of miR-182 was partially prevented by IPC, which was involved in IPC induced intestinal protection, and the mechanisms may be associated with inhibition of apoptosis.
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http://dx.doi.org/10.1016/j.arcmed.2019.08.013DOI Listing
July 2019

Resveratrol reverts Streptozotocin-induced diabetic nephropathy.

Front Biosci (Landmark Ed) 2020 01 1;25:699-709. Epub 2020 Jan 1.

Department of Nephrology, The First people's Hospital of Wenling and The Affiliated Wenling Hospital of Wenzhou Medical university. Wenling, 317500, Zhejiang, P. R. China,

Diabetes causes diabetic nephropathy (DN) which is associated with increased morbidity and mortality in diabetic patients. We tested whether Resveratrol (Res) reverses the systemic effect of Streptozotocin (STZ) induced diabetes and DN. Res treatment opposed the effect of STZ on kidney weight, 24 h urinary albumin excretion, blood urea nitrogen (BUN) and serum creatinine (Scr). Res also decreased DN induced mTOR/ULK1-mediated autophagy and apoptosis and significantly reduced STZ mediated lipid deposition in nephrons, likely by decreasing the levels of lipogenic related proteins (SREBP-1c, ACS) and increased lipidolysis related proteins (PPARα, CPT-1). Together, these findings show the potential of Res in prevention of diabetic nephropathy.
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January 2020

Extracellular and macropinocytosis internalized ATP work together to induce epithelial-mesenchymal transition and other early metastatic activities in lung cancer.

Cancer Cell Int 2019 1;19:254. Epub 2019 Oct 1.

1Department of Biological Sciences, Ohio University, Athens, OH 45701 USA.

Background: Extracellular ATP (eATP) was shown to induce epithelial-mesenchymal transition (EMT), a very important early process in metastasis, in cancer cells via purinergic receptor signaling. However, the exact induction mechanisms are far from fully known. We previously described that eATP is internalized by cancer cells in vitro and in vivo by macropinocytosis in human non-small cell lung cancer A549 and other cancer cells, drastically elevates intracellular ATP levels, enhances cell proliferation and resistance to anticancer drugs. In this study, we tested the hypothesis that eATP and macropinocytosis-internalized eATP also induces EMT and other early steps of metastasis.

Methods: Floating cells, fencing, and transwell assays were used to show that ATP induces cell detachment, new colony formation, migration and invasion in human A549 and other lung cancer cells. Western blots were used to detect ATP-induced changes in EMT-related proteins; Confocal microscopy was used to demonstrate ATP-induced metastasis-related cell morphological changes. Inhibitors and siRNA knockdowns were used to determine P2X7's involvement in the ATP-induced EMT. CRISPR-Cas9 knockout of the SNX5 gene was used to identify macropinocytosis' roles in EMT and cancer cell growth both in vitro and in vivo. Student t-test and one-way ANOVA were used to determine statistical significance, P < 0.05 was considered significant.

Results: eATP potently induces expression of matrix metallopeptidases (MMPs), and detachment, EMT, migration, and invasion of lung cancer cells. The induction was independent of TGF-β and semi-independent of P2X7 activation. eATP performs these functions not only extracellularly, but also intracellularly after being macropinocytically internalized to further enhance P2X7-mediated EMT, filopodia formation and other early steps of metastasis. The knockout of macropinocytosis-associated SNX5 gene significantly reduces macropinocytosis, slows down tumor growth, and changes tumor morphology in nude mice.

Conclusions: Collectively, these results show that eATP's functions in these processes not only from outside of cancer cells but also inside after being macropinocytotically internalized. These findings shed light on eATP's initiator and effector roles in almost every step in early metastasis, which calls for rethinking and rebalancing energy equations of intracellular biochemical reactions and the Warburg effect, and identifies eATP and macropinocytosis as novel targets for potentially slowing down EMT and preventing metastasis.
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http://dx.doi.org/10.1186/s12935-019-0973-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771108PMC
October 2019

Circular RNA profiling in the oocyte and cumulus cells reveals that circARMC4 is essential for porcine oocyte maturation.

Aging (Albany NY) 2019 09 28;11(18):8015-8034. Epub 2019 Sep 28.

Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China.

Thousands of circular RNAs (circRNAs) have been recently discovered in cumulus cells and oocytes from several species. However, the expression and function of circRNA during porcine oocyte meiotic maturation have been never examined. Here, we separately identified 7,067 and 637 circRNAs in both cumulus cells and oocytes via deep sequencing and bioinformatic analysis. Further analysis revealed that a faction of circRNAs is differentially expressed (DE) in a developmental stage-specific manner. The host genes of DE circRNAs are markedly enriched to multiple signaling pathways associated with cumulus cell function and oocyte maturation. Additionally, most DE circRNAs harbor several miRNA targets, suggesting that these DE circRNAs potentially act as miRNA sponge. Importantly, we found that maternal knockdown by siRNA microinjection caused a severely impaired chromosome alignment, and significantly inhibited first polar body extrusion and early embryo development. Taken together, these results demonstrate for the first time that circRNAs are abundantly and dynamically expressed in a developmental stage-specific manner in cumulus cells and oocytes, and maternally expressed is essential for porcine oocyte meiotic maturation and early embryo development.
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http://dx.doi.org/10.18632/aging.102315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781969PMC
September 2019

Knockdown of TUG1 by shRNA inhibited renal cell carcinoma formation by miR-299-3p/VEGF axis in vitro and in vivo.

Eur J Pharmacol 2019 Oct 13;860:172536. Epub 2019 Jul 13.

Department of Nephrology, The First People's Hospital of Wenling &The Affiliated Wenling Hospital of Wenzhou Medical University. Wenling, 317500, Zhejiang, PR China. Electronic address:

Renal cell carcinoma (RCC) is one of the top ten deadly malignancies in the world. The long non-coding RNA taurine up-regulated gene 1 (TUG1) is a transcript that is up-regulated by taurine. There is ample evidence that TUG1 plays a crucial role in the progression of various cancers. This study aimed to investigate the role of TUG1 in RCC and its underlying molecular mechanisms. In the current study, knockdown of TUG1 by shRNA (sh-TUG1) significantly inhibited proliferation, invasion, migration and EMT processes of ACHN cells and OS-RC-2 cells, and induced apoptosis. Besides, bioinformatics analysis revealed that miR-299-3p is a target of TUG1. TUG1 overexpression (LV-TUG1) significantly inhibited the expression of miR-299-3p, whereas sh-TUG1 showed the opposite effect. Dual luciferase reporter assay further confirmed the targeting relationship between TUG1 and miR-299-3p. In addition, vascular endothelial growth factor (VEGFA) is a target of miR-299-3p. Knockdown of VEGFA (si-VEGFA) significantly inhibited the proliferation and motility of ACHN cells, and induced apoptosis. RT-qPCR results showed that sh-TUG1 similarly inhibited VEGFA expression. Further functional analysis indicated that sh-TUG1 inhibited tumorigenesis by down-regulating VEGFA levels. However, LV-TUG1 showed the opposite effects. Furthermore, animal experiments have shown that sh-TUG1 inhibited tumor growth and metastasis and induces apoptosis in vivo. These results indicate that sh-TUG1 inhibited renal cell carcinoma formation by miR-299-3p/VEGF axis in vitro and in vivo. Taken together, all of these results reveal a novel mechanism of TUG1 in RCC tumorigenesis, suggesting that targeted drugs for TUG1 provides a new direction for the treatment of RCC.
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http://dx.doi.org/10.1016/j.ejphar.2019.172536DOI Listing
October 2019

Inhibition of Autophagy Attenuated Intestinal Injury After Intestinal I/R via mTOR Signaling.

J Surg Res 2019 11 2;243:363-370. Epub 2019 Jul 2.

Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. Electronic address:

Background: Intestinal ischemia/reperfusion (I/R) is a grave condition related to high morbidity and mortality. Autophagy, which can induce a new cell death named type II programmed cell death, has been reported in some intestinal diseases, but little is known in I/R-induced intestinal injury. In this study, we aimed to explore the role of autophagy in intestinal injury induced by I/R and its potential mechanisms.

Materials And Methods: The rats pretreated with rapamycin or 3-methyladenine had intestinal I/R injury. After reperfusion, intestinal injury was measured by Chiu's score, intestinal mucosal wet-to-dry ratio, and lactic acid level. Intestinal mucosal oxidative stress level was measured by malondialdehyde and superoxide dismutase. Autophagosome, LC3, and p62 were detected to evaluate autophagy level. Mammalian target of rapamycin (mTOR) was detected to explore potential mechanism.

Results: Chiu's score, intestinal mucosal wet-to-dry ratio, lactic acid level, malondialdehyde level, autophagosomes, and LC3-II/LC3-I were significantly increased, and superoxide dismutase level and expression of p62 were significantly decreased in intestinal mucosa after intestinal ischemia/reperfusion. Pretreatment with rapamycin significantly aggravated intestinal injury evidenced by increased Chiu's score, intestinal mucosal wet-to-dry ratio and lactic acid level, increased autophagy level evidenced by increased autophagosomes and LC3-II/LC3-I and decreased expression of p62, and downregulated expression of p-mTOR/mTOR. On the contrary, pretreatment with 3-methyladenine significantly attenuated intestinal injury and autophagy level and upregulated expression of p-mTOR/mTOR.

Conclusions: In summary, autophagy was significantly enhanced in intestinal mucosa after intestinal ischemia/reperfusion, and inhibition of autophagy attenuated intestinal injury induced by I/R through activating mTOR signaling.
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http://dx.doi.org/10.1016/j.jss.2019.05.038DOI Listing
November 2019

Semantic Fisher Scores for Task Transfer: Using Objects to Classify Scenes.

IEEE Trans Pattern Anal Mach Intell 2020 12 3;42(12):3102-3118. Epub 2020 Nov 3.

The transfer of a neural network (CNN) trained to recognize objects to the task of scene classification is considered. A Bag-of-Semantics (BoS) representation is first induced, by feeding scene image patches to the object CNN, and representing the scene image by the ensuing bag of posterior class probability vectors (semantic posteriors). The encoding of the BoS with a Fisher vector (FV) is then studied. A link is established between the FV of any probabilistic model and the Q-function of the expectation-maximization (EM) algorithm used to estimate its parameters by maximum likelihood. This enables 1) immediate derivation of FVs for any model for which an EM algorithm exists, and 2) leveraging efficient implementations from the EM literature for the computation of FVs. It is then shown that standard FVs, such as those derived from Gaussian or even Dirichlet mixtures, are unsuccessful for the transfer of semantic posteriors, due to the highly non-linear nature of the probability simplex. The analysis of these FVs shows that significant benefits can ensue by 1) designing FVs in the natural parameter space of the multinomial distribution, and 2) adopting sophisticated probabilistic models of semantic feature covariance. The combination of these two insights leads to the encoding of the BoS in the natural parameter space of the multinomial, using a vector of Fisher scores derived from a mixture of factor analyzers (MFA). A network implementation of the MFA Fisher Score (MFA-FS), denoted as the MFAFSNet, is finally proposed to enable end-to-end training. Experiments with various object CNNs and datasets show that the approach has state-of-the-art transfer performance. Somewhat surprisingly, the scene classification results are superior to those of a CNN explicitly trained for scene classification, using a large scene dataset (Places). This suggests that holistic analysis is insufficient for scene classification. The modeling of local object semantics appears to be at least equally important. The two approaches are also shown to be strongly complementary, leading to very large scene classification gains when combined, and outperforming all previous scene classification approaches by a sizable margin.
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http://dx.doi.org/10.1109/TPAMI.2019.2921960DOI Listing
December 2020

Melatonin improves developmental competence of oocyte-granulosa cell complexes from porcine preantral follicles.

Theriogenology 2019 Jul 2;133:149-158. Epub 2019 May 2.

Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, China. Electronic address:

Melatonin has been reported to improve the survival rate of mouse and goat preantral follicles cultured in vitro. However, the role of melatonin in the development of oocyte-granulosa cell complexes (OGCs) isolated from preantral follicles remains unclear. Cumulus-oocyte complexes were isolated from OGCs cultured in vitro for 18.5 days and were then maturated in vitro for 42 h. The matured oocytes were parthenogenetically activated and were further cultured up to the blastocyst stage. We found that the developmental capacity of oocytes from in vitro cultured OGCs was significantly inferior to that from in vivo grown counterparts. Additionally, a 10 M dose of melatonin added to the medium during in vitro culture of OGCs did not improve oocyte meiotic maturation but enhanced blastocyst rate of parthenogenetically activated embryos. Besides, these beneficial effects could be reversed by luzindole treatment, a melatonin membrane receptor antagonist. mRNA sequencing analysis further revealed that melatonin caused differential expression of 76 genes of which 75 were upregulated and 1 was downregulated in OGCs. Twelve of the 76 genes were identified as potential regulators of metabolic pathways by functional analysis. Taken together, these results indicate that melatonin improves developmental competence of porcine oocyte-granulosa cell complexes.
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http://dx.doi.org/10.1016/j.theriogenology.2019.05.003DOI Listing
July 2019

HASPIN kinase mediates histone deacetylation to regulate oocyte meiotic maturation in pigs.

Reproduction 2019 06;157(6):501-510

Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, China.

HASPIN kinase-catalyzed phosphorylation of histone H3 on threonine 3 (H3T3p) directs the activity and localization of chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC) to regulate chromosome condensation and segregation in both mitosis and meiosis. However, the function of HASPIN kinase in the meiotic maturation of porcine oocytes is not yet known. Here, we found that HASPIN mRNA is constantly expressed in porcine oocyte maturation and subsequent early embryo development. H3T3p is highly enriched on chromosomes at germinal vesicle breakdown (GVBD) stage and thereafter maintains a low level in progression through metaphase I (MI) to metaphase II (MII). Correspondingly, H3T3p was completely abolished in oocytes treated with an inhibitor of HASPIN kinase. Functionally, inhibition of HASPIN activity led to a significant reduction in the rate of oocyte meiotic maturation and the limited cumulus expansion. Additionally, HASPIN inhibition caused both spindle disorganization and chromosome misalignment in oocytes at MI and MII stage. Importantly, HASPIN inhibition severely prevented deacetylation of several highly conserved lysine (K) residues of histone H3 and H4 including H3K9, H3K14, H4K5, H4K8, H4K12 and H4K16 on the metaphase chromosomes during oocyte meiotic maturation. Taken together, these results demonstrate that HASPIN kinase regulates porcine oocyte meiotic maturation via modulating histone deacetylation.
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http://dx.doi.org/10.1530/REP-18-0447DOI Listing
June 2019

Stimulatory effects of NESFATIN-1 on meiotic and developmental competence of porcine oocytes.

J Cell Physiol 2019 08 25;234(10):17767-17774. Epub 2019 Feb 25.

Department of Animal Science, Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, China.

NESFATIN-1 acts as a neuroendocrine hormone to suppress gonadotropin secretion in the female goldfish and to prevent germinal vesicle breakdown of oocytes in the zebrafish. However, the expression and function of NESFATIN-1 in meiotic maturation and development of porcine oocytes remains elusive. Genomic structure of porcine NESFATIN-1 precursor nucleobindin 2 (NUCB2) is first characterized in detail and an evolutionally closer relationship of NESFATIN-1 between pig and rat is shown by phylogenetic analysis of multiple species. Additionally, immunofluorescence analysis revealed that NESFATIN-1 is predominantly expressed and localizes on the membrane of both theca cells and granulosa cells, but not expressed in oocytes. Real-time quantitative polymerase chain reaction showed that the abundance of NESFATIN-1 transcripts in granulosa cells progressively decreases during the developmental transition from small follicles to large follicles. Correspondingly, NESFATIN-1 could significantly enhance both the cleavage and blastocyst rate of parthenogenetically activated oocytes from small follicles (p < 0.05), whereas it did not affect meiotic maturation and development of oocytes from large follicles. Interestingly, we found that NESFATIN-1 significantly improves meiotic maturation of oocytes cultured in chemically defined medium in the absence of pyruvate compared with the control group (p < 0.05), suggesting that the NESFATIN-1 as a substitute for pyruvate exerts beneficial effects on porcine oocyte maturation. In conclusion, these results demonstrate that NESFATIN-1 facilitates both meiotic maturation and development of porcine oocytes.
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http://dx.doi.org/10.1002/jcp.28402DOI Listing
August 2019

Production of porcine aminopeptidase N (pAPN) site-specific edited pigs.

Anim Sci J 2019 Mar 8;90(3):366-371. Epub 2019 Jan 8.

Anhui Provincial Laboratory of Local Livestock and Poultry Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui, China.

Porcine viral diarrhea is an acute and highly contagious enteric disease in pigs which causes huge economic losses in pig industry worldwide. Transmissible gastroenteritis virus (TGEV) is main pathogens responsible for piglets viral diarrhea. Knockout the host cellular surface receptor for TGEV may be an effective way to accelerate the breeding of resistant pigs. In this study, we applied site-specific editing pAPN which is effective in swine testis (ST) cells. Site-specific editing of pAPN reduced TGEV proliferation in ST cells by 96%-99% at different time periods post-infection. Next, the site-specific editing of pAPN porcine fetal fibroblasts were produced, and then the cell colonies were used as donor cells to generate the site-specific editing of pAPN pigs. Our research findings will not only offer a more thorough understanding of the pathogenesis of piglet diarrhea and lay the foundation for breeding TGEV-resistant piglets, but also understanding the molecular mechanisms involved in coronaviral infections.
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http://dx.doi.org/10.1111/asj.13163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7159506PMC
March 2019

Human exhaled air can efficiently support maturation of porcine oocytes and subsequent early embryonic development.

Anim Reprod 2018 Aug 16;15(1):29-38. Epub 2018 Aug 16.

Anhui Provincial Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and Breeding, College of Animal Science and Technology, , , .

Air phase is an indispensable environmental factor affecting oocyte maturation and early embryo development. Human exhaled air was previously proved to be a reliable and inexpensive atmosphere that sustains normal early development of mouse and bovine embryos. However, whether human exhaled air can support maturation (IVM) of porcine oocytes is not yet known. To evaluate the feasibility of maturing oocytes in human exhaled air, we examined oocyte morphology, expression, nuclear and cytoplasmic maturation. We found that cumulus expansion status, expression levels of important for cumulus expansion and the rate of first polar body emission were similar among human exhaled air, 5% O or 20% O in air after IVM of 44 h. Furthermore, the percentage of metaphase II (MII) oocytes showing normal cortical and sub-membranous localization of cortical granules and diffused mitochondrial distribution patterns is comparable among groups. The cleavage, blastocyst rate and total cell number were not apparently different for parthenogenetic activated and somatic cloned embryos derived from MII oocytes matured in three air phases, suggesting oocytes matured in human exhaled air obtain normal developmental competence. Taken together, human exhaled air can efficiently support maturation of porcine oocytes and subsequent early embryonic development.
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http://dx.doi.org/10.21451/1984-3143-AR2017-0027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746221PMC
August 2018