Publications by authors named "Pengxiang Qu"

19 Publications

  • Page 1 of 1

Sperm-borne proteins improve rabbit cloning efficiency via regulating embryonic cleavage and epigenetics.

Proteomics 2022 Jul 2:e2200020. Epub 2022 Jul 2.

Laboratory Animal Center, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, China.

Somatic cell nuclear transfer (SCNT) shows great application value in the generation of transgenic animals, protection of endangered species, and therapeutic cloning. However, the cloning efficiency is still very low, which greatly restricts its application. Compared to fertilized embryos, cloned embryos lack the sperm proteins, which are considered to play an important role in embryonic development. Here, we compared the sperm proteome, with that of donor fibroblasts and oocytes, and identified 342 proteins unique to sperm, with 42 being highly expressed. The 384 proteins were mainly enriched in the categories of post-translational modification and cytoskeletal arrangement. Extracts of soluble sperm or fibroblast proteins were injected into cloned embryos, and the result showed that injection of sperm protein significantly inhibited abnormal embryonic cleavage, significantly decreased the level of trimethylated histone H3 Lys9 (H3K9me3) and the apoptotic index, and increased the inner cell mass (ICM)-to-trophectoderm (TE) ratio. More importantly, the sperm proteins also significantly enhanced the birthrate. The results of in vitro and in vivo experiments demonstrate that sperm-derived proteins improve embryo cloning efficiency. Our findings not only provide new insights into ways to overcome low cloning efficiency, but also add to the understanding of sperm protein function.
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http://dx.doi.org/10.1002/pmic.202200020DOI Listing
July 2022

Loss of Renewal of Extracellular Vesicles: Harmful Effects on Embryo Development in vitro.

Int J Nanomedicine 2022 19;17:2301-2318. Epub 2022 May 19.

Laboratory Animal Center, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, People's Republic of China.

Background: Extracellular vesicles (EVs), as a promising platform for drug delivery, have attracted much attention. Degradation and regeneration of EVs maintain their homeostasis in vivo, but this regeneration is missing in the in vitro culture (IVC) system, which is likely to lead to negative effects. It is particularly concerning that most studies involving the addition of EVs in IVC seem to overlook this point.

Methods: We used rabbit embryos and oviduct fluid EVs as a model of embryo development to examine the effect of loss or gain of EV functionality in an IVC system. Embryonic development ratios were determined in each group. Malondialdehyde and ammonium ions in the culture medium were measured. RNA-seq, reactive oxygen species (ROS) staining, immunofluorescence of LC3 and H3K36me3, and qPCR of oxidative stress-related genes and autophagy-related genes of blastocysts in the in vivo group, non-EVs group, con-EVs group, and R-EVsM group was implemented.

Results: Incubation of embryos with 9.1×10 EV particles/mL had a positive effect at 48 h and 72 h, which disappeared by 96 h, however. EVs at a concentration of 9.1×10 particles/mL even showed a negative effect at 96 h. As culture time in the IVC system was increased, the amount of malondialdehyde and ammonium ions in the culture medium was increased, and there was a decrease in embryonic development activity of EVs. Lack of EV renewal in the IVC system impaired embryonic development competence, while replacement of EVs and medium during IVC could sustain embryonic development. Loss or gain of renewal in the IVC system affected EVs' influence on embryo transcriptome, embryonic ROS, autophagy, epigenetic state and apoptosis.

Conclusion: Loss of renewal in the IVC system affected EVs' role in embryonic development by causing an imbalance in ROS, autophagy, abnormal H3K36me3 levels and apoptosis, while gain of renewal in the IVC system reduced these adverse effects and ensured the beneficial function of EVs.
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http://dx.doi.org/10.2147/IJN.S354003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9126234PMC
May 2022

Current Progress and Prospects in Rabbit Cloning.

Cell Reprogram 2022 04 15;24(2):63-70. Epub 2022 Feb 15.

Laboratory Animal Center, Xi'an Jiaotong University Health Science Center, Xi'an, China.

Somatic cell nuclear transfer (SCNT) shows great value in the generation of transgenic animals, protection of endangered animals, and stem cell therapy. The combination of SCNT and gene editing has produced a variety of genetically modified animals for life science and medical research. Rabbits have unique advantages as transgenic bioreactors and human disease models; however, the low SCNT efficiency severely impedes the application of this technology. The difficulty in SCNT may be attributable to the abnormal reprogramming of somatic cells in rabbits. This review focuses on the abnormal reprogramming of cloned mammalian embryos and evaluates the progress and prospects of rabbit somatic cell cloning.
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http://dx.doi.org/10.1089/cell.2021.0090DOI Listing
April 2022

Effects of changing culture medium on preimplantation embryo development in rabbit.

Zygote 2022 Jun 29;30(3):338-343. Epub 2021 Sep 29.

Laboratory Animal Center, Xi'an Jiaotong University Health Science Center, Xi'an, 710061, Shaanxi Province, China.

Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.
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http://dx.doi.org/10.1017/S0967199421000721DOI Listing
June 2022

Sperm-borne small RNAs improve the developmental competence of pre-implantation cloned embryos in rabbit.

Zygote 2021 Oct 9;29(5):331-336. Epub 2021 Mar 9.

Laboratory Animal Center, Xi'an Jiaotong University Health Science Center, No. 76, Yanta West Road, Xi'an, 710061, Shaanxi, China.

The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.
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http://dx.doi.org/10.1017/S0967199420000805DOI Listing
October 2021

Sperm-borne miR-202 targets and regulates first cleavage of bovine embryos via cytoskeletal remodeling.

Development 2021 03 5;148(5). Epub 2021 Mar 5.

College of Veterinary Medicine, Northwest A&F University, Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Yangling 712100, Shaanxi Province, China

In mammals, sperm-borne regulators can be transferred to oocytes during fertilization and have different effects on the formation of pronuclei, the first cleavage of zygotes, the development of preimplantation embryos and even the metabolism of individuals after birth. The regulatory role of sperm microRNAs (miRNAs) in the development of bovine preimplantation embryos has not been reported in detail. By constructing and screening miRNA expression libraries, we found that miR-202 was highly enriched in bovine sperm. As a target gene of miR-202, co-injection of siRNA can partially reverse the accelerated first cleavage of bovine embryos caused by miR-202 inhibitor. In addition, both a miR-202 mimic and siRNA delayed the first cleavage of somatic cell nuclear transfer (SCNT) embryos, suggesting that miR-202- mediates the delay of first cleavage of bovine embryos. By further exploring the relationship between miR-202/SEPT7, HDAC6 and acetylated α-tubulin during embryonic development, we investigated how sperm-borne miR-202 regulates the first cleavage process of bovine embryos by and demonstrate the potential of sperm-borne miRNAs to improve the efficiency of SCNT.
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http://dx.doi.org/10.1242/dev.189670DOI Listing
March 2021

Insights into the roles of sperm in animal cloning.

Stem Cell Res Ther 2020 02 18;11(1):65. Epub 2020 Feb 18.

Laboratory Animal Center, Xi'an Jiaotong University Health Science Center, No.76, Yanta West Road, Xi'an, 710061, Shaanxi, China.

Somatic cell nuclear transfer (SCNT) has shown a wide application in the generation of transgenic animals, protection of endangered animals, and therapeutic cloning. However, the efficiency of SCNT remains very low due to some poorly characterized key factors. Compared with fertilized embryos, somatic donor cells lack some important components of sperm, such as sperm small noncoding RNA (sncRNA) and proteins. Loss of these factors is considered an important reason for the abnormal development of SCNT embryo. This study focused on recent advances of SCNT and the roles of sperm in development. Sperm-derived factors play an important role in nucleus reprogramming and cytoskeleton remodeling during SCNT embryo development. Hence, considering the role of sperm may provide a new strategy for improving cloning efficiency.
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http://dx.doi.org/10.1186/s13287-020-01599-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027237PMC
February 2020

Melatonin Protects Rabbit Somatic Cell Nuclear Transfer (SCNT) Embryos from Electrofusion Damage.

Sci Rep 2020 02 10;10(1):2186. Epub 2020 Feb 10.

Laboratory Animal Centre, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, 710061, China.

The study's objectives were to examine the effects of electrofusion on rabbit somatic cell nuclear transfer (SCNT) embryos, and to test melatonin as a protective agent against electrofusion damage to SCNT embryos. The levels of reactive oxygen species (ROS), the epigenetic state (H3K9me3), and the content of endoplasmic reticulum (ER) stress-associated transcripts (IRE-1 and CHOP) were measured. Melatonin was added during the preimplantation development period. The total blastocyst cell numbers were counted, and the fragmentation rate and apoptotic index were determined and used to assess embryonic development. Electrofusion increased (1) ROS levels at the 1-, 2-, 4-, and 8-cell stages; (2) H3K9me3 levels at the 2-, 4-, and 8-cell stage; and (3) the expression of IRE-1 and CHOP at the 8-cell, 16-cell, morula, and blastocyst stages. The treatment of SCNT embryos with melatonin significantly reduced the level of ROS and H3K9me3, and the expression levels of IRE-1 and CHOP. This treatment also significantly reduced the fragmentation rate and apoptotic index of blastocysts and increased their total cell number. In conclusion, the electrofusion of rabbit SCNT embryos induced oxidative stress, disturbed the epigenetic state, and caused ER stress, while melatonin reduced this damage. Our findings are of signal importance for improving the efficiency of SCNT and for optimizing the application of electrical stimulation in other biomedical areas.
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http://dx.doi.org/10.1038/s41598-020-59161-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010831PMC
February 2020

Extracellular vesicles and melatonin benefit embryonic develop by regulating reactive oxygen species and 5-methylcytosine.

J Pineal Res 2020 Apr 16;68(3):e12635. Epub 2020 Feb 16.

Laboratory Animal Centre, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi, China.

Embryo culture conditions are crucial as they can affect embryo quality and even offspring. Oviductal extracellular vesicles (EVs) long been considered a major factor influencing interactions between the oviduct and embryos, and thus its absence is associated with inferior embryonic development in in vitro culture. Herein, we demonstrated that melatonin is present in oviduct fluids and oviduct fluid-derived EVs. Addition of either EVs (1.87 × 10 particles/mL) or melatonin (340 ng/mL) led to a significant downregulation of reactive oxygen species (ROS) and 5-methylcytosine (5-mC), as well as an increase in the blastocyst rate of embryos, which was inhibited by the addition of luzindole-a melatonin receptor agonist. A combination of EVs (1.87 × 10 particles/mL) and melatonin (at 34.3 pg/mL) led to the same results as well as a significant decrease in the apoptosis index and increase in the inner cell mass (ICM)/trophectoderm (TE) index. These results suggest that an EV-melatonin treatment benefits embryonic development. Our findings provide insights into the role of EVs and melatonin during cell communication and provide new evidence of the communication between embryos and maternal oviduct.
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http://dx.doi.org/10.1111/jpi.12635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154726PMC
April 2020

The effects of glycine-glutamine dipeptide replaced l-glutamine on bovine parthenogenetic and IVF embryo development.

Theriogenology 2020 Jan 6;141:82-90. Epub 2019 Sep 6.

College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR China; Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, 712100, PR China. Electronic address:

Relative to alanine and serine amino acid levels, glutamine is highly abundant in follicular fluid, and is an important source of energy required for oocyte maturation and embryo development. Thus, glutamine is an essential component of in vitro embryo culture media. However, glutamine has poor stability and degrades spontaneously in solution to form ammonia and pyrrolidonecarboxylic acid. In the present study, we aimed to explore the effect of substituting l-glutamine with glycine-glutamine, a more stable glutamine, on development of early parthenogenetic embryos and in vitro fertilization (IVF) embryos in bovine. Results revealed that glycine-glutamine can significantly increase cleavage rate (parthenogenetic embryos:87.24% vs. 72.61%, IVF embryos:89.33% vs. 83.79%, P < 0.01), blastocyst number (parthenogenetic embryos:24.98% vs. 18.07%, IVF embryos:33.53% vs. 27.29%, P < 0.01), and blastocyst number (parthenogenetic embryos:96 vs. 76, IVF embryos:114 vs. 109, P < 0.01), reduce blastocyst apoptosis (parthenogenetic embryos:3.72% vs. 6.65%, IVF embryos:2.53% vs.6.23%, P < 0.01), alleviate embryo ammonia toxicity, and reduce the content of reactive oxygen species (ROS) compared with the l-glutamine. In addition, glycine-glutamine can alter epigenetic reprogramming by increasing the expression of HDAC1 (Histone Deacetylase 1) and decreasing the relative expression levels of H3K9 acetylation in early parthenogenetic embryos and IVF embryos. From our present study, we concluded that glycine-glutamine is an effective substitute of glutamine in modified synthetic oviduct fluid with amino acids (mSOFaa).
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http://dx.doi.org/10.1016/j.theriogenology.2019.09.005DOI Listing
January 2020

Use of oocytes selected by brilliant cresyl blue staining enhances rabbit cloned embryo development in vitro.

Zygote 2019 Jun 7;27(3):166-172. Epub 2019 Jun 7.

1Laboratory Animal Centre,Xi'an Jiaotong University Health Science Centre,Xi'an,Shaanxi 710061,China.

SummaryRabbits play an important role in people's lives due to their high nutritional value and high-quality hair that can be used as raw material for textiles. Furthermore, rabbits are an important animal model for human disease, as genome-edited animals are particularly valuable for studying gene functions and pathogenesis. Somatic cell nuclear transfer (SCNT) is an important technique for producing genome-edited animals and it has great value in saving endangered species and in clone stem cell therapy. However, the low efficiency of SCNT limits its application, with the selection of suitable rabbit oocytes being crucial to its success. In the present study, we collected oocytes from ovarian follicles and stained them with 26 μM brilliant cresyl blue (BCB). We then matured the oocytes in vitro and used them for SCNT. Comparison of the BCB-positive oocytes with BCB-negative oocytes and the control group showed that the BCB-positive group had a significantly higher maturation rate (81.4% vs. 48.9% and 65.3% for the negative and control groups, respectively), cleavage rate (86.6% vs. 67.9% and 77.9%), blastocyst rate (30.5% vs. 12.8% and 19.6%), total number of blastocysts (90±7.5 vs. 65.3±6.3 and 67.5±5.7), and inner cell mass (ICM)/ trophectoderm (TE) index (42.3±4.2 vs. 30.2±2.1 and 33.9±5.1) (P<0.05). The BCB-positive group had a significantly lower apoptosis index (2.1±0.6 vs. 8.2±0.9 and 6.7±1.1 for the negative and control groups, respectively) (P<0.05). These findings demonstrate that BCB-positive oocytes have a higher maturation ability and developmental competence in vitro, indicating that BCB staining is a reliable method for selecting oocytes to enhance the efficiency of SCNT.
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http://dx.doi.org/10.1017/S0967199419000200DOI Listing
June 2019

Sperm-borne small RNAs regulate α-tubulin acetylation and epigenetic modification of early bovine somatic cell nuclear transfer embryos.

Mol Hum Reprod 2019 08;25(8):471-482

Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.

Accumulated evidence indicates that sperm-borne small RNA plays a crucial role in embryonic development, especially the absence of the sperm-borne small RNA might be a major cause of the abnormal development of cloned embryos. In this study, we found that sperm-borne small RNA can affect abnormal pronuclear-like structures, postpone the timing of first embryo cleavage and enhance developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos. In addition, the supplementation of sperm-borne small RNA can significantly increase live birth rates and decrease the birth weights of cloned offspring. To investigate the underlying mechanisms, the levels of α-tubulin K40 acetylation (Ac α-tubulin K40) and histone H3 lysine 9 trimethylation (H3K9me3) during early embryo development were investigated in SCNT embryos with sperm-borne small RNA supplementation (termed as T-NT), compared to those normal SCNT embryos and embryos obtained from standard IVF. The results showed that sperm-borne small RNA can significantly decrease the H3K9me3 levels at the pronuclear and two-cell stages, while significantly increase Ac α-tubulin K40 levels at anaphase and telophase of bovine SCNT embryos during the first cleavage. Collectively, our study for the first time demonstrates that sperm-borne small RNA plays a crucial role in the developmental competence of SCNT embryos by regulating H3K9me3 and Ac α-tubulin K40. Further studies will be required to determine how sperm small RNA regulate the H3K9me3 and Acα-tubulin K40. Our study suggests that the supplementation of sperm-borne small RNA is a potential application to improve the cloning efficiency.
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http://dx.doi.org/10.1093/molehr/gaz023DOI Listing
August 2019

Extracellular vesicles derived from donor oviduct fluid improved birth rates after embryo transfer in mice.

Reprod Fertil Dev 2019 Jan;31(2):324-332

Laboratory Animal Centre, Xi'an Jiaotong University Health Science Centre, Xi'an, Shaanxi 710061, China.

Embryo transfer (ET) is an important procedure for assisted reproduction. However, the relatively lower success rate of ET hampers its application potential. In this study we aimed to elucidate the effects of extracellular vesicles derived from donor oviduct fluid (EDOF) on embryo development after ET. Extracellular vesicles from the oviduct were isolated and purified using ultracentrifugation and identified using transmission electron microscopy, NanoSight, bicinchoninic acid (BCA) protein assay and western blotting. The results revealed that extracellular vesicles were present in donor oviduct fluid in higher concentrations (P<0.05) and contained more proteins (P<0.05) than extracellular vesicles derived from recipient oviduct fluid (EROF). EDOF or EROF were supplemented in an ET medium (ETM) and the results showed that EDOF significantly improved birth rate via resisting apoptosis and promoting differentiation. In conclusion, our study indicated that there are differences in EDOF and EROF and that supplementing EDOF to ETM can improve the efficiency of ET; improved ET efficiency promotes the use of gene editing and benefits assisted reproductive technology and animal welfare.
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http://dx.doi.org/10.1071/RD18203DOI Listing
January 2019

Tauroursodeoxycholic acid (TUDCA) alleviates endoplasmic reticulum stress of nuclear donor cells under serum starvation.

PLoS One 2018 2;13(5):e0196785. Epub 2018 May 2.

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, United States of America.

Serum starvation is a routine protocol for synchronizing nuclear donor cells to G0/G1 phase during somatic cell nuclear transfer (SCNT). However, abrupt serum deprivation can cause serious stress to the cells cultured in vitro, which might result in endoplasmic reticulum (ER) stress, chromosome damage, and finally reduce the success rate of SCNT. In the present study, the effects of tauroursodeoxycholic acid (TUDCA), an effective ER stress-relieving drug, on the nuclear donor cells under serum deprivation condition as well as following SCNT procedures were first assessed in the bovine. The results showed that TUDCA significantly reduced ER stress and cell apoptosis in those nuclear donor cells. Moreover, it significantly decreased the expression of Hdac1 and Dnmt1, and increased the level of H3K9 acetylation in nuclear donor cells compared with control group. SCNT reconstructed embryos cloned from TUDCA-treated donor cells showed significantly higher fusion, cleavage, blastocyst formation rate, total cell number in day 7 blastocysts, and lower apoptotic index than that from control group. In addition, the expression of Hdac1, Dnmt1 and Bax was significantly lower in blastocysts derived from TUDCA-treated donor cells than that from control group. In conclusion, TUDCA significantly reduced the ER stress of nuclear donor cells under serum starvation condition, and significantly improved the developmental competence of following SCNT reconstructed embryos when these TUDCA-treated cells were used as the nuclear donors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196785PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931650PMC
July 2018

Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine.

Sci Rep 2017 10 17;7(1):13403. Epub 2017 Oct 17.

College of Veterinary Medicine, Northwest A&F University, Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Yangling, Shaanxi Province, 712100, PR China.

Accumulating evidence indicates the absence of paternally derived miRNAs, piwiRNAs, and proteins may be one important factor contributing to developmental failure in somatic cell cloned embryos. In the present study, we found microRNA-449b (miR-449b) was highly expressed in sperm. Target gene predictions and experimental verification indicate that several embryonic development-related genes, including CDK6, c-MYC, HDAC1 and BCL-2, are targets of miR-449b. We therefore investigated the role of miR-449b using somatic cell nuclear transfer (SCNT) embryo model. Bovine fetal fibroblasts, expressing miR-449b through a doxycycline (dox) induced expression system were used as nuclear donor cells for SCNT. The results showed that miR-449b expression in SCNT embryos significantly enhanced the cleavage rate at 48 h after activation and the levels of H3K9 acetylation at the 2-cell to 8-cell stages, meanwhile, significantly decreased the apoptosis index of blastocysts. In addition, we verified miR-449b could regulate the expression levels of CDK6, c-MYC, HDAC1 and BCL-2. In conclusion, the present study shows that miR-449b expression improves the first cleavage division, epigenetic reprogramming and apoptotic status of bovine preimplantation cloned embryos.
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http://dx.doi.org/10.1038/s41598-017-13899-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645405PMC
October 2017

MicroRNA-125b is a key epigenetic regulatory factor that promotes nuclear transfer reprogramming.

J Biol Chem 2017 09 9;292(38):15916-15926. Epub 2017 Aug 9.

From the Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China

Somatic cell nuclear transfer (SCNT)-mediated reprogramming is a rapid, efficient, and sophisticated process that reprograms differentiated somatic cells to a pluripotent state. However, many factors in this elaborate reprogramming process remain largely unknown. Here, we report that the microRNA (miR) miR-125b is an important component of SCNT-mediated reprogramming. Luciferase reporter assay, quantitative PCR, and Western blotting demonstrated that miR-125b directly binds the 3'-untranslated region of SUV39H1, encoding the histone-lysine -methyltransferase SUV39H1, to down-regulate histone H3 lysine-9 tri-methylation (H3K9me3) in SCNT embryos. Furthermore, the miR-125b/SUV39H1 interaction induced loss of SUV39H1-mediated H3K9me3, caused heterochromatin relaxation, and promoted the development of SCNT embryos. Transcriptome analyses of SCNT blastomeres indicated that HNF1 homeobox B (HNF1B), a gene encoding a transcription factor downstream of and controlled by the miR-125b/SUV39H1 axis, is important for conferring developmental competence on preimplantation embryos. We conclude that miR-125b promotes SCNT-mediated nuclear reprogramming by targeting SUV39H1 to decrease the deposition of repressive H3K9me3 modifications.
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http://dx.doi.org/10.1074/jbc.M117.796771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612121PMC
September 2017

Effects of embryo-derived exosomes on the development of bovine cloned embryos.

PLoS One 2017 28;12(3):e0174535. Epub 2017 Mar 28.

College of Veterinary Medicine, Northwest A&F University, Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Yangling, Shaanxi, PR China.

The developmental competence of in vitro cultured (IVC) embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT) embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE), as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development), but also following growth to term (in vivo development). Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174535PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5370134PMC
August 2017

Isolation of Bovine Skin-Derived Precursor Cells and Their Developmental Potential After Nuclear Transfer.

Cell Reprogram 2016 11;18(6):411-418

1 College of Veterinary Medicine, Northwest A&F University , Yangling, Shaanxi, 712100 China .

Nuclei from less differentiated stem cells yield high cloning efficiency. However, pluripotent stem cells are rather difficult to obtain from bovines. Skin-derived precursor (SKPs) cells exhibit a certain degree of pluripotency, which has been shown to enhance the efficiency of nuclear transfer (NT) in pigs. In this study, bovine SKPs were isolated and characterized. Results showed that bovine SKPs expressed nestin, fibronectin, vimentin, pluripotency-related genes, and characteristic neural crest markers, such as NGFR, PAX3, SOX9, SNAI2, and OCT4. Bovine SKPs and fibroblasts were used as NT donor cells to examine and compare the preimplantation developmental potential of reconstructed embryos after somatic cell nuclear transfer (SCNT). Bovine SKP-cloned embryos displayed higher developmental competence in terms of blastocyst formation rate and total cell number in blastocysts compared with the bovine embryonic fibroblast-cloned embryos. This study revealed that bovine SKPs may be considered excellent candidate nuclear donors for SCNT and may provide a promising platform for transgenic cattle generation.
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http://dx.doi.org/10.1089/cell.2016.0021DOI Listing
November 2016

Effects of Insulin-like Growth Factor-1 on Development of Somatic Cell Cloned Bovine Embryos.

Cell Reprogram 2016 06 2;18(3):162-70. Epub 2016 May 2.

1 College of Veterinary Medicine, Northwest A&F University , Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Yangling, Shaanxi Province, P.R. China .

The aim of this study was to assess the effect of insulin-like growth factor-1 (IGF-1) on the developmental competence of somatic cell nuclear transfer (SCNT) bovine embryos. First, the expression levels of IGF-1 receptor (IGF-1R) and IGF-1 in the oocytes and embryos of different developmental stages were examined. Then the effects of exogenous IGF-1 on the development of SCNT embryos were evaluated both in vitro and in vivo. The results showed that IGF-1 was not expressed in both IVF and SCNT embryos, whereas IGF-1R could be detected throughout the preimplantation stages in both protein and mRNA levels. Also, exogenous IGF-1 had no obvious impact on the developmental competence of IVF embryos. However, it could improve the developmental competence of SCNT embryos in terms of blastocyst developmental rate (31.3% vs. 43.2%, p < 0.05), total cell number (93.0 ± 9.9 vs. 101.0 ± 9.8, p < 0.05), ratio of inner cell mass (ICM) to trophectoderm (TE) (0.29 ± 0.006 vs. 0.39 ± 0.005, p < 0.05), and apoptosis index in day 7 blastocysts (2.5 ± 0.22 vs. 8.7 ± 0.41, p < 0.05) compared to the control group. Although no statistical difference in pregnancy rate and birth rate was observed after embryo transfer, there was an upward tendency in both examined terms in the IGF-1-supplemented group when compared with the control group. In conclusion, the present study showed that supplementing exogenous IGF-1 to the culture medium has an obvious positive effect on the development competence of SCNT embryos.
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http://dx.doi.org/10.1089/cell.2015.0079DOI Listing
June 2016
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