Publications by authors named "Deshun Shi"

84 Publications

lncSAMM50 Enhances Adipogenic Differentiation of Buffalo Adipocytes With No Effect on Its Host Gene.

Front Genet 2021 26;12:626158. Epub 2021 Mar 26.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.

Fat deposition is one of the most important traits that are mediated by a set of complex regulatory factors in meat animals. Several researches have revealed the significant role of long non-coding RNAs (lncRNAs) in fat deposition while the precise regulatory mechanism is still largely elusive. In this study, we investigated the lncRNA profiles of adipose and muscle tissues in buffalo by using the Illumina HiSeq 3000 platform. In total, 43,809 lncRNAs were finally identified based on the computer algorithm. A comparison analysis revealed 241 lncRNAs that are differentially expressed (DE) in adipose and muscle tissues. We focused on lncSAMM50, a DE lncRNA that has a high expression in adipose tissue. Sequence alignment showed that lncSAMM50 is transcribed from the antisense strand of the upstream region of sorting and assembly machinery component 50 homolog (), a gene involved in the function of mitochondrion and is subsequently demonstrated to inhibit the adipogenic differentiation of 3T3-L1 adipocyte cells in this study. lncSAMM50 is highly expressed in adipose tissue and upregulated in the mature adipocytes and mainly exists in the nucleus. Gain-of-function experiments demonstrated that lncSAMM50 promotes the adipogenic differentiation by upregulating adipogenic markers but with no effect on its host gene in buffalo adipocytes. These results indicate that lncSAMM50 enhances fat deposition in buffalo and provide a new factor for the regulatory network of adipogenesis.
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http://dx.doi.org/10.3389/fgene.2021.626158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033173PMC
March 2021

HDAC6 Is Involved in the Histone Deacetylation of In Vitro Maturation Oocytes and the Reprogramming of Nuclear Transplantation in Pig.

Reprod Sci 2021 Mar 16. Epub 2021 Mar 16.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, 530005, Guangxi, China.

It remained unknown whether HDAC6 affected the histone deacetylation of in vitro maturation oocytes and the reprogramming of nuclear transplantation in pig. Our results indicated that HDAC6 specific inhibition did not affect overall HDAC activity and meiosis process, which increased histone H3K9/K14 and H4K8 acetylation of porcine in vitro maturation oocytes and pseudo-pronucleus embryos. HDAC6 inhibition also significantly enhanced the cleavage and blastocyst of nuclear transfer embryos (0.81 ± 0.12 vs. 0.68 ± 0.12 and 0.46 ± 0.19; 0.73 ± 0.13 vs. 0.63 ± 0.18 and 0.40 ± 0.16, P<0.05). The inhibition of HDAC6 significantly enhanced histone H3K9/K14 and H4K8 acetylation, and upregulated the OCT4 and CDX2 expressions (1.83 ± 0.16 vs. 1.00 ± 0.00 %; 2.07 ± 0.09 vs. 1.00 ± 0.00; P<0.05) in porcine SCNT blastocysts. Interestingly, HDAC6 inhibition significantly increased the pseudo-pronucleus volume during somatic cell reprogramming. Thus, HDAC6 was required for porcine histone deacetylation during the in vitro maturation and pseudo-pronucleus stages. HDAC6 inhibition improved the in vitro development of nuclear transfer embryos. HDAC6 may restrict the reprogramming of somatic nuclear transfer by regulating pseudo-pronucleus expansion. We need further research to confirm this in the future.
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http://dx.doi.org/10.1007/s43032-021-00533-2DOI Listing
March 2021

LC-MS/MS Based Metabolomics Reveal Candidate Biomarkers and Metabolic Changes in Different Buffalo Species.

Animals (Basel) 2021 Feb 20;11(2). Epub 2021 Feb 20.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530005, China.

Consumers have shown more and more interest in high-quality and healthy dairy products and buffalo milk is commercially more viable than other milks in producing superior dairy products due to its higher contents of fat, crude protein, and total solids. Metabolomics is one of the most powerful strategies in molecular mechanism research however, little study has been focused on the milk metabolites in different buffalo species. Therefore, the aim of this study was to explore the underlying molecular mechanism of the fatty synthesis and candidate biomarkers by analyzing the metabolomic profiles. Milk of three groups of buffaloes, including 10 Mediterranean, 12 Murrah, and 10 crossbred buffaloes (Murrah × local swamp buffalo), were collected and UPLC-Q-Orbitrap HRMS was used to obtain the metabolomic profiles. Results showed that milk fatty acid in Mediterranean buffalo was significantly higher than Murrah buffalo and crossbred buffalo. A total of 1837/726 metabolites was identified in both positive and negative electrospray ionization (ESI±) mode, including 19 significantly different metabolites between Mediterranean and Murrah buffalo, and 18 different metabolites between Mediterranean and crossbred buffalo. We found 11 of the different metabolites were both significantly different between Mediterranean vs. Murrah group and Mediterranean vs crossbred group, indicating that they can be used as candidate biomarkers of Mediterranean buffalo milk. Further analysis found that the different metabolites were mainly enriched in fat synthesis related pathways such as fatty acid biosynthesis, unsaturated fatty acid biosynthesis, and linoleic acid metabolism, indicating that the priority of different pathways affected the milk fat content in different buffalo species. These specific metabolites may be used as biomarkers in the identification of milk quality and molecular breeding of high milk fat buffalo.
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http://dx.doi.org/10.3390/ani11020560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924386PMC
February 2021

AQP8 participates in oestrogen-mediated buffalo follicular development by regulating apoptosis of granulosa cells.

Reprod Domest Anim 2021 Feb 27. Epub 2021 Feb 27.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China.

Aquaporins (AQPs), a family of small membrane-spanning proteins, are involved in fluid transport, cell signalling and reproduction. Regulating AQP8 expression influences apoptosis of granulosa cells (GCs), ovarian folliculogenesis, oogenesis and early embryonic development in mice, but its role has never been investigated in other species. The aim of the present study was to characterize the AQP8 function in buffalo follicular development. The expression pattern of AQP8 in buffalo follicle was analysed by immunohistochemistry method. 17β-Estradiol (E2) or oestrogen receptor antagonist ICI182780 was used to treat GCs cultured in vitro, and the expression of AQP8 was detected using qRT-PCR. Its roles in apoptosis of buffalo GCs were investigated by shRNA technology. AQP8 was found to be expressed higher in secondary follicles (p < .05), and its mRNA level in GCs was upregulated by E2 via receptor-mediated mechanism in a dose-dependent manner. A 732-bp buffalo AQP8 coding region was obtained, which was highly conserved at the amino acid level among different species. AQP8-shRNA2 had more effective inhibition on target gene than AQP8-shRNA1 (66.49% vs. 58.31%) (p < .05). Knockdown of AQP8 induced GCs arrested at G2/M stage and occurred apoptosis. Compared with the control group, higher Caspase9 expression were observed in AQP8-shRNA2 lentivirus infected GCs (p < .05), while Bcl-2 and Bax expression levels had no obvious change (p > .05). Altogether, the above results indicate that AQP8 is involved in oestrogen-mediated regulation of buffalo follicular development by regulating cell cycle progression and apoptosis of GCs.
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http://dx.doi.org/10.1111/rda.13921DOI Listing
February 2021

The role of FATP1 in lipid accumulation: a review.

Mol Cell Biochem 2021 Apr 24;476(4):1897-1903. Epub 2021 Jan 24.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530004, Guangxi, China.

Lipid accumulation in mammals has been widely studied for decades due to its significant association with obesity in humans and meat quality in livestock animals. Fatty acid transport 1 (FATP1) is an evolutionarily conserved protein that localizes to the plasma membrane to enhance the transportation of fatty acids (FAs). In line with this function, FATP1 is involved in the metabolism of FAs, including their esterification and oxidation. In addition, the expression of FATP1 can be regulated by several energy-related factors, such as insulin and PPAR activators and transcription factors. These events connect FATP1 with cellular lipid accumulation. Recently, several studies have suggested that FATP1 acts as a facilitator in cellular lipid accumulation, whereas others hold a contrary view. Here, we will review these data and probe the possibility that FATP1 acts as a regulator in lipid accumulation, which will provide effective information for studies on the relationship between FATP1 and obesity in humans and meat quality in livestock animals.
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http://dx.doi.org/10.1007/s11010-021-04057-wDOI Listing
April 2021

Histone Demethylase Could Improve the Developmental Competence of Buffalo () Somatic Cell Nuclear Transfer (SCNT) Embryos.

Microsc Microanal 2021 Jan 22:1-11. Epub 2021 Jan 22.

Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning530005, P.R. China.

Somatic cell nuclear transfer (SCNT) holds vast potential in agriculture. However, its applications are still limited by its low efficiency. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as an epigenetic barrier for this. Histone demethylase KDM4D could regulate the level of H3K9me3. However, its effects on buffalo SCNT embryos are still unclear. Thus, we performed this study to explore the effects and underlying mechanism of KDM4D on buffalo SCNT embryos. The results revealed that compared with the IVF embryos, the expression level of KDM4D in SCNT embryos was significantly lower at 8- and 16-cell stage, while the level of H3K9me3 in SCNT embryos was significantly higher at 2-cell, 8-cell, and blastocyst stage. Microinjection of KDM4D mRNA could promote the developmental ability of buffalo SCNT embryos. Furthermore, the expression level of ZGA-related genes such as ZSCAN5B, SNAI1, eIF-3a, and TRC at the 8-cell stage was significantly increased. Meanwhile, the pluripotency-related genes like POU5F1, SOX2, and NANOG were also significantly promoted at the blastocyst stage. The results were reversed after KDM4D was inhibited. Altogether, these results revealed that KDM4D could correct the H3K9me3 level, increase the expression level of ZGA and pluripotency-related genes, and finally, promote the developmental competence of buffalo SCNT embryos.
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http://dx.doi.org/10.1017/S1431927620024964DOI Listing
January 2021

Expression patterns of ZO-1/2 and their effects on porcine oocyte in vitro maturation and early embryonic development.

Theriogenology 2021 Feb 13;161:262-270. Epub 2020 Dec 13.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, 530005, PR China. Electronic address:

Zonula occludens (ZO)-1 and ZO-2 are involved in epithelial polarity maintenance, gene transcription, cell proliferation and tumor cell metastasis. Regulating ZO-1/2 expression influences the early embryonic development of mice, but whether they are involved in oocyte maturation is still poorly understood. In the present study, the expression patterns of ZO-1 and ZO-2 in porcine cumulus cells and oocytes matured in vitro and early embryos from parthenogenetic activation were detected by qRT-PCR or Western blot, and then their roles in porcine oocyte maturation and early embryo development were investigated by shRNA technology. ZO-1 and ZO-2 were found to be expressed in cumulus cells, oocytes and early embryos, while ZO-1α was expressed only in cumulus cells, morula and blastocysts. During in vitro maturation (IVM), the abundance of ZO-1 and ZO-2 in oocytes was significantly higher than that in cumulus cells at 0 h (P < 0.01), and their mRNA and protein levels displayed relatively higher expression at 0 and 18 h, respectively. Compared with the control groups, cumulus cell expansion, oocyte nucleus maturation, and subsequent cleavage were not influenced by treatment of the cumulus-oocyte complexes (COCs) with ZO-1-shRNA1, ZO-2-shRNA2 or combined ZO-1-shRNA1 and ZO-2-shRNA2 lentivirus (P > 0.05). However, the blastocyst rate was reduced by treatment of COCs with ZO-1-shRNA1 but not ZO-2-shRNA2. The total cell number of blastocysts was decreased by downregulation of ZO-1 and ZO-2 (P < 0.05). Downregulation of ZO-1 and ZO-2 also resulted in a significant decrease (P < 0.05) in the expression of Cx43, Cx45, PTX3 and PTGS2 in cumulus cells, Cx45, BMP15, ZP3 and C-KIT in MII oocytes, and Nanog in blastocysts, with the exception of HAS2 expression in cumulus cells and Oct4 expression in blastocysts (P > 0.05). Altogether, the above results indicate that ZO-1 and ZO-2 display similar expression patterns during porcine oocyte IVM and are critical to porcine oocyte maturation and early embryonic development.
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http://dx.doi.org/10.1016/j.theriogenology.2020.12.009DOI Listing
February 2021

Circular RNA Profiling Reveals an Abundant circEch1 That Promotes Myogenesis and Differentiation of Bovine Skeletal Muscle.

J Agric Food Chem 2021 Jan 21;69(1):592-601. Epub 2020 Dec 21.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China.

Beef is considered to be a good quality meat product because it contains linoleic acid and specific proteins, which can bring significant benefits to health. Circular RNAs (circRNAs) have been reported to regulate skeletal myogenesis. RNA-seq was used to investigate the circRNA molecular regulatory mechanisms with respect to differences in muscle quality between buffalo and cattle. A total of 10,449 circRNA candidates were detected, and 1128 of these were found to be differentially expressed between cattle and buffalo muscle tissue libraries. Differentially expressed 23 circRNAs were verified by qPCR. CircEch1, one of the most up-regulated circRNAs during muscle development, was subsequently characterized. CCK-8 (65.05 ± 2.33%, < 0.0001), EdU (72.99 ± 0.04%, < 0.001), and Western blotting assays showed that overexpression of circEch1 inhibited the proliferation of bovine myoblasts but promoted differentiation. studies suggested that circEch1 stimulates skeletal muscle regeneration. These results demonstrate that the novel regulator circEch1 induces myoblast differentiation and skeletal muscle regeneration. They also provide new insights into the mechanisms of circRNA regulation of beef quality.
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http://dx.doi.org/10.1021/acs.jafc.0c06400DOI Listing
January 2021

Cell synchronization by Rapamycin improves the developmental competence of buffalos (Bubalus bubalis) somatic cell nuclear transfer embryos.

Reprod Domest Anim 2021 Feb 3;56(2):313-323. Epub 2020 Dec 3.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China.

This study mainly explored the effects of Rapamycin on the growth of the Buffalo ear fibroblast (BEF) and embryonic developmental competence of somatic cell nuclear transfer (SCNT). The results show that the appropriate concentration (1 μM) of Rapamycin could significantly improve the proportion of the G0/G1 phase in BEF cells treated at a certain time (72 hr). Simultaneously, the percentage of the G0/G1 phase also was significantly higher than the serum starvation and control group. This may be related to Rapamycin inhibiting the phosphorylation of mTOR and affecting the expression of cell cycle-related genes (CDK2, CDK4, P27, CycleD1, and CycleD3). Besides, compared with the control group and serum-starved group, Rapamycin significantly decreased BEF cell apoptosis by reducing ROS generation. Moreover, these results also indicated that the proportion of BEF cells with normal chromosome multiples treated by Rapamycin is significantly higher than that of the serum-starved group (p < .05). Finally, this study explored the effects of Rapamycin and serum starvation on the embryonic developmental competence of SCNT. The results show that Rapamycin significantly increased the rate of 8-cell and blastocyst, compared with the control group and serum starvation group (p < .05). To summarize, these results indicate that Rapamycin improved the embryonic development competence of SCNT, which may be related to Rapamycin increasing the percentage of G0/G1 phase and maintaining BEF cell quality.
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http://dx.doi.org/10.1111/rda.13868DOI Listing
February 2021

Aberrant regulation of RNA methylation during spermatogenesis.

Reprod Domest Anim 2021 Jan 1;56(1):3-11. Epub 2020 Dec 1.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Animal Science and Technology, Guangxi University, Nanning, China.

Natural modifications of cellular RNA include various chemical modifications, such as N6-methyladenosine (m A), which enable the orderly metabolism and function of RNA structural diversity, thereby affecting gene expression. Spermatogenesis is a complex differentiating developmental process, which includes the proliferation of spermatogonial stem cells, spermatocyte meiosis and sperm maturation. Emerging evidence has shown that RNA methylation can influence RNA splicing, exportation and translation, which are controlled in the male germline in order to ensure coordinated gene expression. In this review, we summarize the typical characteristics of different types of RNA methylation during the process of spermatogenesis. In particular, we emphasize the functions of the RNA methylation effectors during the male germ cell development.
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http://dx.doi.org/10.1111/rda.13856DOI Listing
January 2021

Comparative transcriptome analysis reveals that PCK1 is a potential gene affecting IMF deposition in buffalo.

BMC Genomics 2020 Oct 12;21(1):710. Epub 2020 Oct 12.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530005, Guangxi, China.

Background: In China, although buffaloes are abundant, beef is mainly obtained from cattle, and this preference is mainly attributed to the low intramuscular fat (IMF) content of buffalo. Genetic factors are an important driver that affects IMF deposition.

Results: To reveal the intrinsic factors responsible for the low IMF content of buffalo, mRNA expression patterns in muscle and adipose tissue between buffalo and cattle were characterized by RNA sequencing analysis. The IMF content in Nanyang cattle was higher than that in Xinyang buffalo. A total of 1566 mRNAs expressed in adipose tissue showed differential expression between the longissimus dorsi muscles of buffalo and cattle. Functional annotation suggested a difference in the glycolysis/gluconeogenesis pathway between the two species. The results of RT-qPCR analysis and gain-of-function experiments confirmed the positive association between the IMF content and phosphoenolpyruvate carboxykinase 1 (PCK1) expression in buffalo. In both mouse C2C12 cells and cultured bovine myocytes, the activity of the PCK1 promoter in buffalo is lower than that in cattle. However, in mouse 3T3-L1 adipocytes and cultured bovine adipocytes, the activity of PCK1 in buffalo promoter is higher than that in cattle.

Conclusions: These results indicate the important role of PCK1 in buffalo IMF deposition and illustrate the differences between buffalo and cattle promoter activity that drive PCK1 expression. This research helps to establish a foundation for further studies investigating IMF deposition in buffalo.
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http://dx.doi.org/10.1186/s12864-020-07120-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7552535PMC
October 2020

Granulosa cell-conditioned medium enhances steroidogenic competence of buffalo (Bubalus bubalis) theca cells.

In Vitro Cell Dev Biol Anim 2020 Oct 30;56(9):799-807. Epub 2020 Sep 30.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China.

Granulosa cells (GCs) and theca cells (TCs) are the main components of follicles, and the interactions between GCs and TCs play a significant role in steroidogenesis, follicular growth, and atresia. However, the effects of GCs in the form of conditioned medium on steroidogenesis in buffalo TCs remain unclear. In the present study, the impacts of GC-conditioned medium (GCCM) on androgen synthesis in buffalo TCs were examined. The results showed that GCCM collected at 48 h promoted both the expression levels of androgen synthesis-related genes (CYP11A1, CYP17A1, 3β-HSD, and Star) and the secretion levels of testosterone in TCs. The treatment time of 48 h in GCCM improved both the expression levels of androgen synthesis-related genes (CYP11A1, CYP17A1, 3β-HSD, and Star) and the secretion levels of testosterone in TCs. Furthermore, GCCM that was collected at 48 h and applied to TCs for 48 h (48 h and 48 h) promoted the sensitivity of buffalo TCs to LH. This study indicated that GCCM (48 h and 48 h) enhanced the steroidogenic competence of TCs mainly through facilitating the responsiveness of TCs to LH in buffalo. This study provides a basis for further exploration of interactions between GCs and TCs for steroidogenesis in the ovary.
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http://dx.doi.org/10.1007/s11626-020-00509-7DOI Listing
October 2020

PI3K inhibitor reduces in vitro maturation and developmental competence of porcine oocytes.

Theriogenology 2020 Nov 14;157:432-439. Epub 2020 Aug 14.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi, 530004, China; School of Animal Science and Technology, Guangxi University, Nanning, Guangxi, 530004, China. Electronic address:

The phosphatidylinositol -3- kinase (PI3K) signaling pathway is critical for the cell proliferation, apoptosis, metabolism, DNA repair and protein synthesis. Significant effort has focused on elucidating the relationship between PI3K signaling pathway and other nuclear signal transducers; However, little is known about the connection between PI3K signaling pathway and porcine oocyte meiotic maturation. In this study, we investigated the function of PI3K signaling pathway in porcine oocytes. PI3K signaling pathway was important during oocyte maturation. Furthermore, the PI3K signaling pathway inhibitor LY-294002 blocked porcine oocyte maturation, reducing the percentage of oocytes that first polar body (PBI) extrusion. LY-294002 also decreased the expression of oocyte proliferation-related gene PCNA and reduced the mRNA and protein levels of PI3K. What's more, LY-294002 also decreased other maturation-related genes that are predominantly expressed duringporcine oocyte maturation, including bone morphogenetic protein 15 (BPM15), growth differentiation factor 9 (GDF9), cell division cycle protein 2 (CDC2), phosphatase and tensin homolog (PTEN), CyclinB1, MOS and Akt. LY-294002 treatment decreased the developmental potential of blastocysts following parthenogenetic activation, increased the level of cell apoptosis and reduced the level of cell-cycle. This study revealed that inhibiting the PI3K signaling pathway could reduce in vitro maturation and developmental competence of porcine oocytes, probably by reducing cell cycle arrest and proliferation, promoting the oocyte apoptosis, and altering the expression of other maternal genes.
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http://dx.doi.org/10.1016/j.theriogenology.2020.08.019DOI Listing
November 2020

Improved early development potence of in vitro fertilization embryos by treatment with tubacin increasing acetylated tubulin of matured porcine oocytes.

Mech Dev 2020 12 21;164:103631. Epub 2020 Aug 21.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, Guangxi 530005, China. Electronic address:

To improve the developmental potential of in vitro embryos is a long-term concern field for human assisted reproduction and animal in vitro embryo production practice. In the current study, we examined the effects and mechanism of an HDAC6 inhibitor, tubacin, on the maturation of porcine oocytes and in vitro development of porcine IVF embryos. It has been demonstrated the effect of tubacin on the acetylation level of α-tubulin in porcine oocytes. As a result, the maturation rate of porcine oocytes was significantly improved (P < 0.05), and the following development potent of blastocysts forming rate was also significantly increased (P < 0.05). We found that the increased acetylation of α-tubulin significantly reduced the abnormal rate of microtubule, furthermore, the proportion of mitochondria in the vicinity of in vitro fertilization (IVF) nucleus was significantly enhanced in Metaphase I (MI) and Metaphase II (MII) stages. The expression levels of microtubule assembly genes (TUBA1A, αTAT1 and MAP2) significantly up-regulated in MI and MII stages. Together, these results suggest that treatment of porcine oocytes during maturation with tubacin could promote their IVF embryos developmental competence by altering spindle formation, mitochondrial concentration and genes expression patterns of matured porcine oocytes.
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http://dx.doi.org/10.1016/j.mod.2020.103631DOI Listing
December 2020

Theca cell-conditioned medium added to in vitro maturation enhances embryo developmental competence of buffalo (Bubalus bubalis) oocytes after parthenogenic activation.

Reprod Domest Anim 2020 Nov 28;55(11):1501-1510. Epub 2020 Oct 28.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China.

Theca cells (TCs) play a key role in follicular growth and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to estrogens needed for oocyte maturation. However, the effects of TCs in the form of conditioned medium on in vitro maturation (IVM) and developmental competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of TC-conditioned medium (TCCM) on maturation efficiency and embryo development of buffalo oocytes after parthenogenic activation (PA). Our results showed that TCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2 days & 20%) exerted no significant effect on IVM rate (43.06% vs. 44.71%), but significantly (p  < .05) enhanced embryo development (oocyte cleavage, 80.93% vs. 69.66%; blastocyst formation, 39.85% vs. 32.84%) of buffalo oocytes after PA compared with the control group. However, monolayer TC significantly (p < .05) promoted both maturation efficiency (48.84% vs. 44.53%) and embryo development (oocyte cleavage, 80.39% vs. 69.32%; blastocyst formation, 35.38% vs. 29.25%) of buffalo oocytes after PA compared to that in the control group. Furthermore, TCs secreted some testosterone into the conditioned medium, which significantly (p < .05) promoted the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 17β-HSD) in buffalo cumulus-oocyte complexes (COCs). Our study indicated that TCCM (2 days & 20%) did not significantly affect IVM efficiency, but enhanced embryo developmental competence of oocytes after PA principally by stimulating the secretion of testosterone and facilitating estradiol synthesis of buffalo COCs.
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http://dx.doi.org/10.1111/rda.13799DOI Listing
November 2020

Theca cell-conditioned medium enhances steroidogenesis competence of buffalo (Bubalus bubalis) granulosa cells.

Reprod Domest Anim 2021 Feb 10;56(2):254-262. Epub 2020 Dec 10.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China.

Theca cells (TCs) play a crucial role in follicular development and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to oestrogens needed for follicular growth. However, the effects of TCs in the form of conditioned medium on steroidogenesis in buffalo GCs remain unclear. In the present study, the impacts of TC-conditioned medium (TCCM) on oestrogen synthesis in buffalo GCs were examined. The results showed that TCs secreted principally testosterone, but almost no androstenedione or oestradiol into TCCM. TCs at passage 3 had a stronger secretion capacity of testosterone in TCCM. Furthermore, TCCM collected at 72 hr improved both the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1, 3β-HSD and 17β-HSD) and the secretion levels of estradiol in GCs. The treatment of 72 hr in TCCM promoted both the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 3β-HSD) and the secretion levels of estradiol in GCs. Besides, TCCM that was collected at 72 hr and applied to GCs for 72 hr (72 & 72 hr) improved the sensitivity of buffalo GCs to FSH. This study indicates that TCCM (72 & 72 hr) enhances the steroidogenesis competence of GCs mainly through facilitating the responsiveness of GCs to FSH in buffalo.
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http://dx.doi.org/10.1111/rda.13792DOI Listing
February 2021

Activation of Wnt/β-Catenin Signaling Pathway Enhances the Derivation of Buffalo () Embryonic Stem Cell-Like Cells.

Cell Reprogram 2020 08 14;22(4):217-225. Epub 2020 Jul 14.

Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Guangxi, China.

Wnt/β-Catenin signaling pathway plays an important role in maintaining self-renewal and pluripotency of human and mouse embryonic stem cells (ESCs). Activation of Wnt/β-Catenin signaling pathway by glycogen synthase kinase-3 (GSK3) inhibitor, the Wnt signaling agonist, could maintain the pluripotency of human and mouse ESCs in the presence of serum. However, the role of signaling pathway in the derivation of buffalo ESCs remains unclear. In this study, we used GSK3 inhibitors (6-bromoindirubin-3'-oxime [BIO] and CHIR99021) and investigated the effect of Wnt/β-Catenin activation on colony formation, proliferation, self-renewal, and pluripotency of Chinese swamp buffalo (buffalo) embryonic stem cell-like cells (ES-like cells), which were isolated from blastocysts. The results showed that buffalo ES-like cells displayed typical morphological characteristics of pluripotent stem cells: positive for alkaline phosphatase staining, expression of pluripotent markers, including , , SSEA-1, SSEA-4, , , , and the proliferative markers, and . Furthermore, activation of Wnt/β-Catenin signaling pathway by GSK3 inhibitors could promote colony formation and proliferation of buffalo ES-like cells and maintain their undifferentiated state, and upregulate the expression levels of pluripotent-related genes and proliferation-related genes. These results indicated that Wnt/β-Catenin signaling pathway plays an important role in the derivation and pluripotency of buffalo ES-like cells.
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http://dx.doi.org/10.1089/cell.2020.0027DOI Listing
August 2020

Hypoxia Enhances Mesenchymal Characteristics Maintenance of Buffalo Bone Marrow-Derived Mesenchymal Stem Cells.

Cell Reprogram 2020 06 21;22(3):167-177. Epub 2020 May 21.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Animal Reproduction Institute, Guangxi University, Nanning, People's Republic of China.

Bone marrow-derived mesenchymal stem cells (BMSCs) from livestock are valuable resources for veterinary therapeutics and animal reproduction. Previous studies have shown that hypoxic conditions were beneficial in maintaining the mesenchymal feature of BMSCs. However, the effects of hypoxia on buffalo BMSCs (bBMSCs) remain unclear. In this study, the effects of hypoxic conditions on cell morphology, migration, polarity, and karyotype of bBMSCs were examined. The results showed that hypoxia (5% oxygen) enhanced colony formation and stress fiber synthesis of bBMSCs. Under the hypoxic culture conditions, the migration capacity and normal karyotype rate of bBMSCs were significantly improved ( < 0.05), which resulted in weakened cell polarity and enhanced karyotype stability in bBMSCs. In addition, it was significantly ( < 0.05) upregulated in the expression levels of signaling pathway axis-related genes (, , , , , , , ). The axis of bBMSCs was also activated in hypoxia. Finally, it was more effective and easier to maintain the mesenchymal feature of bBMSCs in hypoxic conditions. These findings not only provide theoretical guidance to elucidate the detailed regulation mechanism of hypoxia on mesenchymal nature maintenance of bBMSCs, but also provide positive support to further establish the stable culture system of bBMSCs.
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http://dx.doi.org/10.1089/cell.2019.0097DOI Listing
June 2020

Comparison of Long Non-Coding RNA Expression Profiles of Cattle and Buffalo Differing in Muscle Characteristics.

Front Genet 2020 26;11:98. Epub 2020 Feb 26.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.

Buffalo meat consist good qualitative characteristics as it contains "thined tender" which is favorable for cardavascular system. However, the regulatory mechanisms of long non-coding RNA (lncRNA), differences in meat quality are not well known. The chemical-physical parameters revealed the muscle quality of buffalo that can be equivalent of cattle, but there are significant differences in shearing force and muscle fiber structure. Then, we examined lncRNA expression profiles of buffalo and cattle skeletal muscle that provide first insights into their potential roles in buffalo myogenesis. Here, we profiled the expression of lncRNA in cattle and buffalo skeletal muscle tissues, and 16,236 lncRNA candidates were detected with 865 up-regulated lncRNAs and 1,296 down-regulated lncRNAs when comparing buffalo to cattle muscle tissue. We constructed coexpression and ceRNA networks, and found lncRNA MSTRG.48330.7, MSTRG.30030.4, and MSTRG.203788.46 could be as competitive endogenous RNA (ceRNA) containing potential binding sites for miR-1/206 and miR-133a. Tissue expression analysis showed that MSTRG.48330.7, MSTRG.30030.4, and MSTRG.203788.46 were highly and specifically expressed in muscle tissue. Present study may be used as a reference tool for starting point investigations into the roles played by several of those lncRNAs during buffalo myogenesis.
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http://dx.doi.org/10.3389/fgene.2020.00098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054449PMC
February 2020

Histone hyperacetylation may improve the preimplantation development and epigenetic status of cloned embryos.

Reprod Biol 2020 Jun 20;20(2):237-246. Epub 2020 Feb 20.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, Guangxi, 530004, China. Electronic address:

The current study investigated the mechanism of mini pig fetal fibroblasts in improving the epigenetic modification and preimplantation development of cloned embryos. The results showed that the increased AcH3K14 level was dose- and time-dependent. Histone hyperacetylation had no significant effect on cell morphology, cell viability, cell cycle, and relative gene (HDAC1, HAT1, DNMT3A, and BAX) expression. The treated cloned embryos had significantly higher development rates and the total nuclei number than the control (27.62 ± 6.94 % vs. 16.14 ± 10.55 %; 43.90 ± 18.39 vs. 33.06 ± 15.87; P < 0.05). The AcH3K14 level in the treated cloned blastocysts was close to that of IVF blastocysts (5.17 ± 0.93 vs. 5.45 ± 1.91, P > 0.05). The gene transcription (CDX2 and OCT4) of the treated cloned blastocysts was significantly up-regulated than the control (3.32 ± 0.51 vs. 2.05 ± 0.30; 1.21 ± 0.18 vs. 0.81 ± 0.09; P < 0.05). The improvement in the cloned embryo development and the partial correction of abnormal acetylation modification were not necessarily related to the cellular characteristics. This could be caused by histone hyperacetylation of mini pig fetal fibroblasts.
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http://dx.doi.org/10.1016/j.repbio.2020.02.005DOI Listing
June 2020

Male fertility in Mus musculus requires the activity of TRYX5 in sperm migration into the oviduct.

J Cell Physiol 2020 09 4;235(9):6058-6072. Epub 2020 Feb 4.

National Institute of Biological Sciences (NIBS), Beijing, China.

Nowadays, abnormal loss of serine proteases appears very frequently in male patients with unexplained sterility. In fact, many testis-specific serine proteases, the largest family among the four protease families implicated in murine spermatogenesis, are indispensable for reproduction. In the present study, we demonstrate that the previously uncharacterized testis-specific serine protease TRYX5 (1700074P13Rik) is required for male fertility in mice. Tryx5 male mice are sterile, yet they have normal spermatogenesis and normal sperm parameters. In vivo fertilization experiments showed that the fertilization rate of Tryx5 sperm was almost zero. Sperm counting and analysis of paraffin sections of oviducts revealed that Tryx5 sperm were unable to migrate into the oviduct, which is likely the cause of the observed infertility of the Tryx5 male mice. Importantly, we also found that there was almost no mature ADAM3 present in Tryx5 sperm and almost no ADAM3 precursor in Tryx5 elongated spermatids of S13-16 stage, even though testes of Tryx5 and wild type mice had the same amount of the total precursor ADAM3. Collectively, our results demonstrate that Tryx5 is essential for male fertility in mice and suggest that TRYX5 functions in the stability or localization of ADAM3 precursor in elongated spermatids S13-16 stage, thereby regulating the ability of sperm to migrate from the uterus into the ampulla of the oviduct, the site of fertilization.
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http://dx.doi.org/10.1002/jcp.29534DOI Listing
September 2020

Promotes Differentiation and Apoptosis of Bovine Skeletal Muscle-Derived Mesenchymal Stem Cells.

Cell Reprogram 2020 02;22(1):22-29

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Nanning, Guangxi, China.

In this study, Squalene epoxidase () overexpression vector was transfected into bovine skeletal muscle-derived mesenchymal stem/stromal cells (MSCs) to study the molecular mechanism of regulating meat quality through myogenesis. We initially profiled the expression of in cattle embryos and adults, in the muscle tissue of four different cattle varieties, and in 11 different tissues/organs of Guangxi cattle variety. Subsequently, we isolated and cultured bovine skeletal muscle-derived MSCs and detected the expression of during cell proliferation and differentiation. Then, we constructed a bovine overexpression vector and transfected it into bovine skeletal muscle-derived MSCs by liposome transfection. Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU), flow cytometry, immunofluorescence, and quantitative polymerase chain reaction assays were used to characterize cell proliferation and differentiation in detail. The results showed that the relative expression level of bovine gene in brain tissue was the highest, and in adult muscle tissue was significantly higher than that in embryonic stage. Especially, the expression of was significantly upregulated in cell differentiation stage. Furthermore, the proliferation, cell cycle, apoptosis, and myoblast differentiation assays indicated that significantly promoted the differentiation and apoptosis of bovine skeletal muscle-derived MSCs, but inhibited their proliferation. In conclusion, our study reveals the role of in myoblast differentiation. These results will provide new clues for the regulation network of bovine muscle development.
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http://dx.doi.org/10.1089/cell.2019.0077DOI Listing
February 2020

Anti-silencing factor 1A is associated with genome stability maintenance of mouse preimplantation embryos†.

Biol Reprod 2020 04;102(4):817-827

Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China and.

Genome stability is critical for the normal development of preimplantation embryos, as DNA damages may result in mutation and even embryo lethality. Anti-silencing factor 1A (ASF1A) is a histone chaperone and enriched in the MII oocytes as a maternal factor, which may be associated with the maintenance of genome stability. Thus, this study was undertaken to explore the role of ASF1A in maintaining the genome stability of early mouse embryos. The ASF1A expressed in the preimplantation embryos and displayed a dynamic pattern throughout the early embryonic development. Inhibition of ASF1A expression decreased embryonic development and increased DNA damages. Overexpression of ASF1A improved the developmental potential and decreased DNA damages. When 293T cells that had been integrated with RGS-NHEJ were co-transfected with plasmids of pcDNA3.1-ASF1A, gRNA-NHEJ, and hCas9, less cells expressed eGFP, indicating that non-homologous end joining was reduced by ASF1A. When 293T cells were co-transfected with plasmids of HR-donor, gRNA-HR, hCas9, and pcDNA3.1-ASF1A, more cells expressed eGFP, indicating that homologous recombination (HR) was enhanced by ASF1A. These results indicate that ASF1A may be associated with the genome stability maintenance of early mouse embryos and this action may be mediated by promoting DNA damage repair through HR pathway.
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http://dx.doi.org/10.1093/biolre/ioaa001DOI Listing
April 2020

The key long non-coding RNA screening and validation between germinal vesicle and metaphase II of porcine oocyte in vitro maturation.

Reprod Domest Anim 2020 Mar 23;55(3):351-363. Epub 2020 Jan 23.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.

Oocyte maturation plays a vitally important role in the reproduction of pigs. However, the roles of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) in the developmental process of porcine oocyte maturation are still largely unclear. In this study, a transcriptome analysis of germinal vesicle (GV) and metaphase II (MII) of oocytes from Chinese Duroc pigs was performed. A total of 1,753,030 and 2,486 differentially expressed (DE) mRNAs, 22,811 and 9,868 DE lncRNAs were identified between GV and MII stages, respectively. Furthermore, functional enrichment analysis showed that the common DE mRNAs and DE lncRNAs during the process of maturation were mainly involved in biological process and cellular components. Our study provides new insights of the expression changes of mRNAs and lncRNAs during GV and MII stages, which might contribute to the maturation of oocytes. These results greatly improve our understanding of the molecular mechanisms regulating the maturation of oocytes in pigs.
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http://dx.doi.org/10.1111/rda.13620DOI Listing
March 2020

A new three-dimensional glass scaffold increases the in vitro maturation efficiency of buffalo (Bubalus bubalis) oocyte via remodelling the extracellular matrix and cell connection of cumulus cells.

Reprod Domest Anim 2020 Feb 3;55(2):170-180. Epub 2020 Jan 3.

Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China.

At present, many three-dimensional (3D) culture systems have been reported, improving the oocyte quality of in vitro maturation (IVM), yet the mechanism still needs to be further explored. Here we examined the effects of a new self-made 3D glass scaffold on buffalo oocyte maturation; meanwhile, the underlying mechanism on buffalo oocyte maturation was also detected. Compared to the two-dimensional (2D) glass dish culture, results revealed that the 3D culture can improve the first polar body rate of oocytes, subsequent cleavage and blastocysts rate of parthenogenetic activation embryos (p < .05). The extracellular matrix-related proteins COL1A1, COL2A1, COL3A1, FN and cell connection-related proteins N-cadherin, E-cadherin, GJA1 were found higher in cumulus cells of 3D culture. Moreover, in cumulus cells, proteins of the PI3K/AKT pathway reported being regulated by FN and E-cadherin including PI3K P85 and p-AKT were also higher in 3D culture. Furthermore, proapoptosis proteins P53, BAX, caspase-3 were lower in both cumulus cells and oocytes in 3D culture, while proteins PCNA and BCL2 showed the opposite result. Results also showed that the apoptosis was inhibited, and the proliferation was enhanced in cumulus cells of 3D culture. Finally, the cumulus expansion-related genes HAS2, CD44, HMMR, PTX3, PTGS2 were found higher in cumulus cells of 3D culture. Taken together, the 3D culture could promote oocyte maturation by regulating proteins correlated with the ECM, cell connection and PI3K/AKT pathway, inhibiting the apoptosis of cumulus cells and oocytes, enhancing the proliferation of cumulus cells and the cumulus expansion.
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http://dx.doi.org/10.1111/rda.13602DOI Listing
February 2020

A Novel Pale-Yellow Coat Color of Rabbits Generated Mutation With CRISPR/Cas9 System.

Front Genet 2019 18;10:875. Epub 2019 Sep 18.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China.

Coat color is of great importance in animal breed characteristics; it is not only a significant productive trait but also an indispensable economic trait, especially in the rabbit industry. In the present study, the relationship between melanocortin 1 receptor () genotypes and coat color phenotypes was observed in five rabbit breeds with popular coat colors that are present in China. These breeds comprised the Lianshan black rabbit (BR), Fujian yellow rabbit (YR), New Zealand white rabbit (WR), Gray Giant rabbit (GR), and Checkered Giant rabbit (CR), which were firstly determined, and the results showed that GR had an E allele; WR, CR, and BR had a 6-bp in-frame deletion (c.281_286del6, E allele); and YR had a 30-bp deletion (c.304_333del30 E allele). To explore the feasibility of obtaining a novel rabbit coat color through the mutation of with the CRISPR/Cas9 system, two single-guide RNAs (sgRNAs) were designed for the gene, and the editing efficiency was confirmed by injection of rabbits' zygotes. Unlike the donor rabbits whose coat color was originally black, two novel pale-yellow-coated rabbits were generated in the founders. A total of six novel gene deletions were identified in the two founder rabbits, in which the longest deletion was more than 700 bp. The histological hematoxylin-and-eosin (H&E) staining results indicated that eumelanin amounts were absent in hair follicles of -knockout (KO) rabbits, when compared with that of donor BR. In addition, the messenger RNA (mRNA) levels of some key downstream genes in the MC1R pathway were all downregulated in -KO rabbits compared with BR and YR. These results further indicate that loss-of-function MC1R contributed to blocking the synthesis of eumelanin and created a novel pale-yellow coat color in the -KO rabbits, and gene editing technology may be a useful tool to generate novel phenotypes in rabbit breeding.
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http://dx.doi.org/10.3389/fgene.2019.00875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759607PMC
September 2019

Partially Reprogrammed Induced Pluripotent Stem Cells Using MicroRNA Cluster miR-302s in Guangxi Bama Minipig Fibroblasts.

Cell Reprogram 2019 10 3;21(5):229-237. Epub 2019 Sep 3.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Animal Reproduction Institute, Guangxi University, Nanning, China.

Pig-induced pluripotent stem cells (piPSCs) have great potential application in regenerative medicine. The miR-302s cluster alone has been shown to reprogram mouse and human somatic cells into induced pluripotent stem cells (iPSCs) without exogenous transcription factors. However, miR-302s alone have not been reported to reprogram cells in large livestock. In this study, we induced pig somatic cells into partially reprogrammed piPSCs using overexpression of the miR-302s cluster (miR-302s-piPSC) and investigated the early reprogramming events during the miRNA induction process. The results showed that miR-302s-piPSCs exhibited some characteristics of pluripotent stem cells including expression of pluripotency markers-particularly, efficient activation of endogenous -and differentiation to the three germ layers . During the early reprogramming process, somatic cells first underwent epithelial-mesenchymal transition and then mesenchymal-epithelial transition to eventually form miR-302s-piPSCs. These data show, for the first time, that single factor miR-302s successfully induced pig somatic cells into miR-302s-piPSCs. This study provides a new tool and research direction for the induction of pluripotent stem cells in a large livestock.
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http://dx.doi.org/10.1089/cell.2019.0035DOI Listing
October 2019

Self-assembling Rotavirus VP6 Nanoparticle Vaccines Expressed in Escherichia coli Elicit Systemic and Mucosal Responses in Mice.

Protein Pept Lett 2019 ;26(12):904-909

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi, 530005, China.

Background: Rotavirus is the most common cause of infectious diarrhea in infants and young children around the world. The inner capsid protein VP6 has been discussed as alternative vaccine as it can induce cross-protective immune responses against different RV strai. The use of ferritin nanoparticle may enhance the immunogenicity of the subunit vaccine.

Objective: In this article, our motivation is to design and obtain a self-assemble rotavirus nanoparticle vaccine which can induce efficiency immune response.

Methods: The VP6 protein was fused with ferritin and expressed in the Escherichia coli expression system. The recombinant VP6-ferritin (rVP6-ferritin) protein was purified by His-tag affinity chromatography and fast protein liquid chromatography. Transmission electron micrographic analysis was used to detect the nanostructure of the self-assembled protein. Mice were gavage with the protein and ELISA was used to detect the titer of the VP6 specific antibody.

Results: The recombined VP6-ferritin was expressed in the Escherichia coli as an inclusion body form and the purified protein has similar antigenicity to rotavirus VP6. Transmission electron micrographic analysis of rVP6-ferritin exhibited spherical architecture with a uniform size distribution, which is similar to the ferritin nanocage. Immune response analysis showed that mice immunized by rVP6-ferritin protein induced 8000 (8000±1093) anti-VP6 IgG titers or 1152 (1152±248.8) anti-VP6 IgA titers.

Conclusion: According to the above research, the rotavirus VP6-ferritin protein can be easily express and self-assemble to the nano-vaccine and induce efficiency humoral and mucosal immunity. Our research makes a foundation for the development of oral rotavirus vaccine.
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http://dx.doi.org/10.2174/0929866526666190820161328DOI Listing
January 2020