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Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos

  • Author Footnotes
    * These authors contributed equally to this work.
    H.Y. Chang
    Footnotes
    * These authors contributed equally to this work.
    Affiliations
    Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

    College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China
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  • Author Footnotes
    * These authors contributed equally to this work.
    R.X. Xie
    Footnotes
    * These authors contributed equally to this work.
    Affiliations
    Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

    College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China
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  • Author Footnotes
    * These authors contributed equally to this work.
    L. Zhang
    Footnotes
    * These authors contributed equally to this work.
    Affiliations
    Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

    College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China
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  • L.Z. Fu
    Affiliations
    Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

    College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China
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  • C.T. Zhang
    Affiliations
    Animal Husbandry and Veterinary Station of Xining, Xining 810003, Qinghai, China
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  • H.H. Chen
    Affiliations
    Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

    College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China
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  • Z.Q. Wang
    Affiliations
    Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

    College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China
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  • Y. Zhang
    Correspondence
    Corresponding authors
    Affiliations
    Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

    College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China
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  • F.S. Quan
    Correspondence
    Corresponding authors
    Affiliations
    Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China

    College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China
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  • Author Footnotes
    * These authors contributed equally to this work.
Open ArchivePublished:March 14, 2019DOI:https://doi.org/10.3168/jds.2018-15072

      ABSTRACT

      Accumulating studies have suggested that microRNA play a part in regulating multiple cellular processes, such as cell proliferation, apoptosis, the cell cycle, and embryo development. This study explored the effects of miR-101-2 on donor cell physiological status and the development of Holstein cow somatic cell nuclear transfer (SCNT) embryos in vitro. Holstein cow bovine fetal fibroblasts (BFF) overexpressing miR-101-2 were used as donor cells to perform SCNT; then, cleavage rate, blastocyst rate, inner cell mass-to-trophectoderm ratio, and the expression of some development- and apoptosis-related genes in different groups were analyzed. The miR-101-2 suppressed the expression of inhibitor of growth protein 3 (ING3) at mRNA and protein levels, expedited cell proliferation, and decreased apoptosis in BFF, suggesting that ING3, a target gene of miR-101-2, is a potential player in this process. Moreover, by utilizing donor cells overexpressing miR-101-2, the development of bovine SCNT embryos in vitro was significantly enhanced; the apoptotic rate in SCNT blastocysts was reduced, and the inner cell mass-to-trophectoderm ratio and SOX2, POU5F1, and BCL2L1 expression significantly increased, whereas BAX and ING3 expression decreased. Collectively, these findings suggest that miR-101-2 promotes BFF proliferation and vitality, reduces their apoptosis, and improves the early development of SCNT embryos.

      Key words

      INTRODUCTION

      Since the time the first successful clone, Dolly the sheep, was created, many mammalian species have been successfully cloned by somatic cell nuclear transfer (SCNT;
      • Wakayama T.
      • Perry A.C.
      • Zuccotti M.
      • Johnson K.R.
      • Yanagimachi R.
      Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.
      ;
      • Baguisi A.
      • Behboodi E.
      • Melican D.T.
      • Pollock J.S.
      • Destrempes M.M.
      • Cammuso C.
      • Williams J.L.
      • Nims S.D.
      • Porter C.A.
      • Midura P.
      Production of goats by somatic cell nuclear transfer.
      ;
      • Woods G.L.
      • White K.L.
      • Vanderwall D.K.
      • Li G.-P.
      • Aston K.I.
      • Bunch T.D.
      • Meerdo L.N.
      • Pate B.J.
      A mule cloned from fetal cells by nuclear transfer.
      ). Somatic cell nuclear transfer technology brings good prospects for propagation of elite genetic merit livestock. Somatic cell nuclear transfer is also a promising technology because of its potential applications in biomedicine (
      • Le R.
      • Kou Z.
      • Jiang Y.
      • Li M.
      • Huang B.
      • Liu W.
      • Li H.
      • Kou X.
      • He W.
      • Rudolph K.L.
      Enhanced telomere rejuvenation in pluripotent cells reprogrammed via nuclear transfer relative to induced pluripotent stem cells.
      ). Low efficiency of SCNT clone embryos, however, is a significant barrier to the use of this technology. Many factors have been reported to influence the efficiency of SCNT, of which nuclear donor cells are one of the crucial factors. Tissue specificity, cell type, age, status, and the progression of the cell cycle of donor cells were shown to affect the development of cloned embryos (
      • Inoue K.
      • Noda S.
      • Ogonuki N.
      • Miki H.
      • Inoue S.
      • Katayama K.
      • Mekada K.
      • Miyoshi H.
      • Ogura A.
      Differential developmental ability of embryos cloned from tissue-specific stem cells.
      ).
      MicroRNA (miRNA) are small, highly conserved, noncoding RNA that usually suppress gene expression through partially complementary binding to the 3′-untranslated region (UTR) of target gene mRNA (
      • Luo C.
      • Merz P.R.
      • Chen Y.
      • Dickes E.
      • Pscherer A.
      • Schadendorf D.
      • Eichmüller S.B.
      MiR-101 inhibits melanoma cell invasion and proliferation by targeting MITF and EZH2.
      ;
      • Agarwal V.
      • Bell G.W.
      • Nam J.-W.
      • Bartel D.P.
      Predicting effective microRNA target sites in mammalian mRNAs.
      ). Researchers found that decreasing miRNA-145 expression could improve the development of SCNT embryos in vitro (
      • Li W.
      • Xiong Y.
      • Wang F.
      • Liu X.
      • Gao Y.
      • Wang Y.
      • Zhang Y.
      • Jin Y.
      MicroRNA-145 inhibitor significantly improves the development of bovine somatic cell nuclear transfer embryos in vitro.
      ). Recent reports have also shown that the quality of SCNT embryos was enhanced via using donor cells expressing sperm-borne miR-449b or overexpressing miR-148a (
      • Wang M.
      • Gao Y.
      • Qu P.
      • Qing S.
      • Qiao F.
      • Zhang Y.
      • Mager J.
      • Wang Y.
      Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine.
      ,
      • Wang P.
      • Li X.
      • Cao L.
      • Huang S.
      • Li H.
      • Zhang Y.
      • Yang T.
      • Jiang J.
      • Shi D.
      MicroRNA-148a overexpression improves the early development of porcine somatic cell nuclear transfer embryos.
      ). Furthermore, our previous research demonstrated that miR-125b promotes SCNT-mediated nuclear reprogramming by targeting SUV39H1 (
      • Zhang J.
      • Qu P.
      • Zhou C.
      • Liu X.
      • Ma X.
      • Wang M.
      • Wang Y.
      • Su J.
      • Liu J.
      • Zhang Y.
      microRNA-125b is a key epigenetic regulatory factor that promotes nuclear transfer reprogramming.
      ). These indicated miRNA play a part in the development of SCNT embryos.
      We previously analyzed high-throughput sequencing data for differences between cloned and normal in vitro fertilized (IVF) preimplantation embryos (F. S. Quan, unpublished data) and found that miR-101-2 was aberrantly downregulated in cloned embryos compared with the IVF embryos. From a bioinformatics analysis, it is predicted that ING3 is a high-ranking target gene of miR-101-2, which was abnormally highly expressed in cloned embryos compared with the IVF embryos. Ubiquitously expressed in mammalian tissues, ING3 is particularly highly expressed in mouse, rhesus monkey, and human oocytes and is one of the candidate oocyte reprogramming factors (
      • Awe J.P.
      • Byrne J.A.
      Identifying candidate oocyte reprogramming factors using cross-species global transcriptional analysis.
      ). In addition, numerous studies have demonstrated that miR-101 can regulate cell proliferation and apoptosis; it plays different roles in disparate kinds of cells by targeting several genes (
      • Liang X.
      • Liu Y.
      • Zeng L.
      • Yu C.
      • Hu Z.
      • Zhou Q.
      • Yang Z.
      miR-101 inhibits the G1-to-S phase transition of cervical cancer cells by targeting Fos.
      ;
      • Lin X.
      • Guan H.
      • Li H.
      • Liu L.
      • Liu J.
      • Wei G.
      • Huang Z.
      • Liao Z.
      • Li Y.
      miR-101 inhibits cell proliferation by targeting Rac1 in papillary thyroid carcinoma.
      ;
      • Zhou X.
      • Xia Y.
      • Li L.
      • Zhang G.
      MiR-101 inhibits cell growth and tumorigenesis of Helicobacter pylori related gastric cancer by repression of SOCS2.
      ). Therefore, it is hypothesized that overexpression of miR-101-2 would enhance the developmental potential of SCNT embryos in vitro.
      To test this hypothesis, a stable cell line overexpressing miR-101-2 was established and effects on ING3 expression, progression of the cell cycle, and apoptosis of bovine fetal fibroblasts (BFF) were examined. This stable cell line was subsequently used as a source of nuclear donor cells to produce SCNT embryos, and development rate, total cell numbers, apoptosis-related genes expression, apoptotic rate, and expression of development-related genes were examined.

      MATERIALS AND METHODS

      Ethics Statement

      The experimental procedure followed was approved by the Animal Care and Use Committee of Northwest A&F University, China, and performed in accordance with the animal welfare and ethics guidelines. Bovine ovaries were collected from slaughtered mature cattle at a local abattoir in Xi'An, China. Frozen-thawed spermatozoa were bought from Bright Farming (Shanghai, China).

      Reagents

      All chemicals and reagents were purchased from Sigma Aldrich (St. Louis, MO) unless specifically indicated otherwise. Disposable, sterile plastic ware was purchased from Corning (Corning, NY).

      Experiment Design

      The experiments undertaken will be briefly outlined in this section, and the details of each of the procedures undertaken will be described in the sections that follow. Experiment 1 was performed to determine whether miR-101-2 targets ING3. Experiment 2 was performed to determine whether miR-101-2 overexpression affects donor cell status and expression of ING3, TP53, CDKN1A, BAX, and BCL2L1 mRNA. Experiment 3 was performed to measure the miR-101-2 expression in 2-cell, 8-cell, and Day-7 blastocysts from IVF, 513-B1-nuclear transfer (513-B1-NT; SCNT embryos produced using BFF transfected with empty vectors as donor cells), and miR-101-NT (SCNT embryos produced by using BFF with miR-101-2 overexpression as donor cells) treatments. Experiment 4 was performed to examine the effects of miR-101-2 overexpression on the quality of SCNT embryos and the expression of key developmental genes.

      Cell Isolation and Culture

      The BFF were established from the bovine fetal back skin tissue of approximately 90-d-old Holstein cow fetuses (
      • Pan S.
      • Chen W.
      • Liu X.
      • Xiao J.
      • Wang Y.
      • Liu J.
      • Du Y.
      • Wang Y.
      • Zhang Y.
      Application of a novel population of multipotent stem cells derived from skin fibroblasts as donor cells in bovine SCNT.
      ). Briefly, fetal skin tissue samples were rinsed several times with PBS containing penicillin and streptomycin and then minced into 1-mm3 pieces. Tissues were cultivated for 1 to 2 wk in 60-mm Petri dishes (Corning) containing Dulbecco's modified Eagle's medium/F12 high-glucose (Invitrogen, Carlsbad, CA) medium supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY), 100 mg/mL of streptomycin, and 100 IU/mL of penicillin, in saturation humidity with 5% CO2 at 37°C. When the BFF were at 90% confluence, they were trypsinized, rinsed, and recultivated in several new 60-mm Petri dishes with fresh culture medium for further passaging.
      Human HEK293T cells were used for the luciferase reporter assay, and stored at the Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, and cultured in Dulbecco's modified Eagle's high-glucose medium containing 10% FBS, in saturation humidity with 5% CO2 at 37°C.

      Vector Construction

      The DNA fragments corresponding to the miR-101-2 precursor sequence were amplified by PCR from genomic DNA of BFF and cloned into the BamHI and EcoRI restriction sites of pCDH-Promoter-MCS-EF1 Lentivector (CD513B-1, SBI, Mountain View, CA), resulting in the generation of the vector pCDH-miR101. The plasmids Psi-ING3-3′-UTR-wild type (WT) and Psi-ING3-3′-UTR-mutant (Mut) were generated by inserting ING3-3′-UTR-WT or ING3-3′-UTR-Mut into the Psi-check-2 XhoI and NotI restriction sites, respectively. All constructed plasmids were verified by DNA sequencing. Primer sequences are listed in Supplemental Table S1 (https://doi.org/10.3168/jds.2018-15072), and the sequences of the insert fragments are listed in Supplemental Table S2 (https://doi.org/10.3168/jds.2018-15072).

      Cell Transfection and Clone Selection

      Upon reaching 80% confluence on 60-mm Petri dishes, cells were resuspended in transfer buffer, placed in a 4-mm cuvette gap with 6 µg of pCDH-miR101 or pCDH-513-B9 (empty vector), and then electroporated at 510 V with 3 pulses of 2-ms duration using a BTX Electro Cell Manipulator ECM2001 (BTX, Holliston, MA). The cells were placed in 90-mm Petri dishes to which fresh cell culture medium was added in advance at an average of 5 × 105 cells. After 24 h, the medium containing 2 µg/mL of Puro (Sigma Aldrich) was replaced by a medium containing 1 µg/mL of Puro when floating cells constituted approximately 50% of the total. Approximately 7 d later, Puro-resistant clone cells began to emerge and exhibited green fluorescence under UV light. When the clone cells became large enough for transfer, they were moved into new 60-mm Petri dishes and cultured in 500 ng/mL of Puro. Cells were passaged in several new 60-mm Petri dishes or frozen in 10% dimethylsulfoxide and 90% FBS upon reaching 90% confluence.

      Luciferase Reporter Assay

      For the luciferase reporter assay, 5 × 104 293T cells were plated in each well of a 24-well plate. The cells were co-transfected with 400 ng of pCDH-miR-101-2 or pCDH-513B and 40 ng of Psi-ING3-3′-UTR-WT or Psi-ING3-3′-UTR-Mut. Using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's instructions, cells were collected 48 h after transfection and analyzed using the Dual-Luciferase Reporter Assay System (TransGen Biotech, Beijing, China). Relative luciferase activity was normalized to Renilla luciferase activity. Transfections were performed in duplicate and repeated at least thrice in independent experiments.

      Western Blot Analysis

      Western blot was carried out as previously described with minor modifications (
      • Wang X.
      • Zou P.
      • He Y.
      • Meng K.
      • Quan F.
      • Zhang Y.
      Effect of luteinizing hormone on goat theca cell apoptosis and steroidogenesis through activation of the PI3K/AKT pathway.
      ). The cells were collected and washed with PBS, and then lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime Inst. Biotech, Beijing, China) with 1% PMSF and protease inhibitor on ice for 30 min. A bicinchoninic acid protein assay kit (TransGen Biotech) was used for protein quantification. Proteins were separated by 10% SDS PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were then blocked in 10% nonfat milk (wt/vol in TBST) for 4 h and incubated with primary antibodies anti-ING3 (1:1,000, ab84645; Abcam, Cambridge, UK) and internal reference anti-β-actin (1:1,000, ab8227; Abcam) overnight at 4°C. After washing 3 times in TBST, membranes were incubated with the secondary antibodies goat anti-rabbit (Beyotime Inst. Biotech, Beijing, China) for 2 h, which followed by washes for 3 times in TBST. After using the chemiluminescence reagent (Beyotime Inst. Biotech), immunoreactive proteins were visualized by autography using SuperSignal West Pico Substrate (Thermo Fisher Scientific, Waltham, MA). All steps were performed at room temperature unless stated otherwise. The gray level of the image was analyzed using Image J (https://imagej.nih.gov/ij/) software.

      RNA Extraction, Reverse-Transcription PCR, and Quantitative Real-Time PCR

      For measurement of the expression of mRNA in BFF, total RNA was extracted using Trizol reagent (TransGen Biotech) according to the manufacturer's instructions. First-strand cDNA were prepared from 1 µg of RNA using a reverse transcription kit (TransGen Biotech). The miRNA samples were isolated using the mir-Vana miRNA Isolation Kit and reverse-transcribed using the miScript II RT Kit (Qiagen, Hilden, Germany).
      For measurement of the expression of mRNA in embryos, a Cells-to-Signal kit (Ambion Co., Austin, TX) was used to isolate total RNA from IVF and SCNT embryos (n = 18 embryos per pool) according to the manufacturer's instructions. The M-MLV RT reagent included in the kit was used for reverse transcription. Samples of miRNA (n = 55 embryos per pool) were isolated using the miScript Single Cell qPCR Kit (Qiagen) and reverse transcribed using a miScript II RT Kit (Qiagen).
      The expression levels of mRNA were measured using SYBR Premix Ex Taq (TaKaRa, Dalian, China) according to the manufacturer's instructions. The GAPDH was selected by GeNorm and NormFinder software as housekeeping gene to normalize mRNA expression levels (Supplemental Tables S3 and S4; https://doi.org/10.3168/jds.2018-15072;
      • Perez S.
      • Royo L.J.
      • Astudillo A.
      Identifying the most suitable endogenous control for determining gene expression in hearts from organ donors.
      ). Mature miRNA expression was quantified using a miScript SYBR Green PCR Kit containing 10 × miScript Universal Primer (Qiagen), according to the manufacturer's instructions. U6 was used as an internal control to normalize miRNA expression levels. The results were expressed as fold change = 2−ΔΔCt. ABI StepOnePlus PCR system (Applied Biosystems, Foster City, CA) was used to perform quantitative real-time PCR (qRT-PCR). The PCR cycle conditions were as follows: 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Primer sequences were designed to span an intron splice site, and are summarized in Supplemental Table S5 (https://doi.org/10.3168/jds.2018-15072).

      Cell Cycle and Apoptosis Analyses

      The progression of the cell cycle and apoptosis were assessed by flow cytometry (Becton Dickinson, Oxford, UK). For analyzing the cell cycle, miR-101-2 and vector clone cells in a 60-mm dish were collected and rinsed with cold PBS and then fixed in 75% ethanol overnight at 4°C. Fixed cells were then washed and resuspended in 200 to 500 µL of cold PBS supplemented with 20 µL of RNase A and then incubated at 37°C for 30 min. This was followed by supplementation of 400 mL of propidium iodide (100 mg/mL) staining solution into the resultant cell suspension and then incubated for 0.5 to 1 h at 4°C. Results were analyzed by flow cytometry. For analyzing cell apoptosis, miR-101-2 or vector clone cells were collected and washed twice with cold PBS. Then, they were supplemented with 400 mL of 1_Annexin V (BB-4101, BestBio, Shanghai, China) and 5 mL of Annexin V-EGFP staining medium (BB-4101, BestBio). This cell suspension was mixed slightly and incubated in the dark for 15 min at 2 to 8°C. This mixture was then supplemented with 10 mL of propidium iodide (10 mg/mL) for 5 min before flow cytometry.

      Cell Proliferation Analysis

      Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8; Beyotime Inst. Biotech) according to the manufacturer's instructions. Briefly, the miR-101-2 clone cells, 513-B1 clone cells, and ordinary fibroblasts were seeded in 96-well plates, with each type of cell having the same density of 2,000 cells per well, being grown at 37°C for 24 h. The medium in each well was replaced with a new medium, which was then incubated with 10 µL of CCK-8 for 2 h at 37°C. For each group, there were 3 replicates, along with the use of the medium at the same amount as in CCK-8 as a control. Finally, the absorbance was determined at 450 nm using a microplate reader.

      Oocyte Collection, In Vitro Maturation, NT, and In Vitro Reconstituted Embryo Culture

      Oocyte collection, in vitro maturation, and NT were performed as previously described with minor modifications (
      • Wang Y.S.
      • Tang S.
      • An Z.X.
      • Li W.Z.
      • Liu J.
      • Quan F.S.
      • Hua S.
      • Zhang Y.
      Effect of mSOF and G1.1/G2.2 media on the developmental competence of SCNT-derived bovine embryos.
      ). Briefly, slaughterhouse-derived ovaries were stored in sterile saline at 20 to 25°C, and transported to the laboratory within 6 h. Cumulus-oocyte complexes (COC) were aspirated from antral follicles (diameter, 2–8 mm) with a 10-mL syringe, and only those with multilayered compact cumulus, uniformly dark, and evenly granulated cytoplasm were selected. These oocytes were rinsed thrice in PBS containing 3% FBS, and cultured for 20 h in bicarbonate-buffered tissue culture medium 199 (TCM-199, Gibco, BRL, Grand Island, NY) supplemented with 10% FBS, 1 mg/mL of 17β-estradiol, and 0.075 IU/mL of human menopausal gonadotropin in saturation humidity with 5% CO2 at 38.5°C.
      The COC were then transferred into PBS with 0.1% bovine testicular hyaluronidase to disperse the cumulus cells from oocytes. Oocytes with uniform, granulated ooplasm and an extruded first polar body were selected for SCNT. Enucleation was carried out by using a 20-μm (internal diameter) glass pipette. The first polar body and a small amount of surrounding cytoplasm were aspirated by the glass pipette in microdrops of PBS supplemented with 7.5 μg/mL of cytochalasin B and 10% FBS. A single donor cell was then injected into the space between the zona pellucida and oocyte membrane. The oocyte-donor cell complex was transferred into a microdrop of Zimmermann's fusion medium, and sandwiched between 2 platinum electrodes, connected to the micromanipulator. Cell-oocyte fusion was performed by using a double electrical pulse of 35 V for 10 μs. Reconstructed embryos were then cultured in modified synthetic oviductal fluid (mSOF) for 2 h, and activated in 5 μM ionomycin for 4 min, followed by a 4-h exposure to 1.9 mM dimethylaminopyridine in mSOF. After activation, the activated embryos were transferred into drops of mSOF medium with 8 mg/mL of BSA and cultured in a humid atmosphere of 5% CO2 at 38.5°C. 513-B1-BFF and miR-101-BFF cells from passage 3–5 were used as donor cells for SCNT. Serum deprivation was used to induce the G0/G1 phase. Embryos were cultured for 8 d to evaluate their developmental rate.

      In Vitro Fertilization

      In vitro fertilization was carried out according to previous descriptions with minor modifications (
      • Zhang J.
      • Qu P.
      • Zhou C.
      • Liu X.
      • Ma X.
      • Wang M.
      • Wang Y.
      • Su J.
      • Liu J.
      • Zhang Y.
      microRNA-125b is a key epigenetic regulatory factor that promotes nuclear transfer reprogramming.
      ). Briefly, frozen-thawed spermatozoa were rinsed and washed in BO-IVF medium (IVF Bioscience, Falmouth, UK) for 30 min. Then, 20 to 25 COC were transferred into a 60-μL microdrop of BO-IVF medium (IVF Bioscience) covered by mineral oil, after which 50 μL of sperm suspension (2 × 106 spermatozoa/mL) was added into it. After incubating in a humid atmosphere of 5% CO2 at 38.5°C for 18 h, they were transferred and washed in PBS with 0.1% bovine testicular hyaluronidase to remove cumulus cells and redundant spermatozoa from the zygotes, and then the zygotes were transferred to be cultured in mSOF with 8 mg/mL of BSA for further use.

      Apoptosis Assay

      An apoptosis assay was carried out using the DeadEnd Fluorometric TUNEL (Promega, Madison, WI) system as previously described with minor modifications (
      • Chen H.
      • Zhang L.
      • Guo Z.
      • Wang Y.
      • He R.
      • Qin Y.
      • Quan F.
      • Zhang Y.
      Improving the development of early bovine somatic-cell nuclear transfer embryos by treating adult donor cells with vitamin C.
      ). All steps were performed at room temperature, unless stated otherwise. After 7 d of culture, IVF blastocysts and in vitro cloned blastocysts from the miR-101-BFF nuclear transfer (miR-101-NT) group and the 513-B1-BFF nuclear transfer (513-B1-NT) group were cleaned for 5 min in PBS containing 0.2% polyvinyl acetate (PVA-PBS) and fixed for 2 h with 4% paraformaldehyde. After washes in PVA-PBS, fixed embryos were permeated by 0.2% Triton X-100 for 20 min, and transferred into E-buffer for 8 min. All the following procedures were carried out in the dark to prevent fluorescence bleaching. Embryos were incubated for 1 h at 37°C in 51 mL of apoptosis staining medium (45 mL of E-buffer, 1 mL of terminal deoxynucleotidyl transferase enzyme, and 5 mL of nucleotide mix), which was followed by terminating with 2 × saline sodium citrate for 15 min. Embryos were washed again and incubated for 5 min in 4,6-diamidino-2-phenylindole (DAPI; 5 mg/mL, C1002, Beyotime, Jiangsu, China). Stained embryos were mounted on a glass slide for observation under a Nikon (Tokyo, Japan) fluorescence microscope. Blastocyst cell numbers were estimated by counting the total number of nuclei using DAPI, and the number of apoptotic cells per blastocyst was determined by counting nuclei positive for green fluorescence.

      CDX2 Staining in Blastocysts

      The DAPI staining was performed to reveal the total cell number in blastocysts. The CDX2 staining was used to analyze trophectoderm (TE) cell number. Then, the inner cell mass (ICM) cell number was assessed by calculating the difference between the total cell number and TE cell number.
      Blastocysts were collected and washed 3 times (5 min each) in PVA-PBS, after which they were fixed with 4% paraformaldehyde for 2 h at room temperature. Immunol Staining Blocking Solution (P0102, Beyotime, Jiangsu, China) was used to block blastocysts for 12 h at 4°C before washing them 3 times (5 min each) in PVA-PBS. Then, blastocysts were washed 3 times (5 min each) in PVA-PBS, which was followed by incubating them for 12 h at 4°C with anti-CDX2 mouse monoclonal antibody (BioGenex Inc., San Ramon, CA) diluted 1:300 in Immunol Staining Primary Antibody Dilution Solution (P0103, Beyotime). After 3 cleanings, samples were incubated for 2 h at room temperature with Alexa Fluor 555-labeled goat anti-mouse IgG secondary antibody (A0459, Beyotime) diluted 1:600 in Immunol Staining Secondary Antibody Dilution Solution (P0108, Beyotime). Then we performed another 3 washes and used 5 mg/mL of DAPI (C1002, Beyotime) to stain the nucleus for 5 min. Samples were then cleaned and mounted on a glass slide with a drop of Antifade Mounting Medium (P0126, Beyotime) for analysis using a Nikon fluorescence microscope. The cell numbers in blastocysts were estimated by counting nuclei stained with DAPI, and the number of TE cells was determined by counting nuclei positive for CDX2. The cell number of ICM was estimated as the total number of nuclei minus the number of TE nuclei.

      Statistical Analysis

      Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA). Data are presented as the mean ± standard deviation or mean ± standard error of the mean from at least 3 independent experiments. All embryos had been systematically produced from the same cohort of oocytes on the same day and were allocated randomly to each treatment group. The development rate, apoptosis rate, ICM/TE ratio, and total cell number were analyzed using one-way ANOVA. The relative gene expressions were established by testing the data for normal and equal variance using the Levene median test, ANOVA, followed by the Tukey's test for multiple pairwise comparisons. The 2-tailed Student's t-test was used for pairwise comparisons of relative luciferase activity. P-values <0.05 were considered statistically significant.

      RESULTS AND DISCUSSION

      ING3 Is a Direct Target of miR-101-2

      Our analysis reveals that ING3, a member of the inhibitor of growth family, was a potential target of miR-101-2, as the 3′-UTR in ING3 mRNA contained a complementary site for the seed region of miR-101-2 (Figure 1A). To confirm that ING3 is a target of miR-101-2, we co-transfected the reporter plasmid psiCHECK2-ING3-3′-UTR WT or Mut and the miR-101-2 expression plasmid PCDH-miR-101-2 into the HEK293T cell line. As predicted, the relative luciferase activity of the WT 3′-UTR reporter significantly decreased in cells co-transfected with pCDH-miR-101-2 compared with that in cells co-transfected with empty PCDH vector. In contrast, the luciferase activity upon the co-transfection of mutant 3′-UTR reporter and pCDH-miR-101-2 was no longer able to be suppressed compared with that in cells co-transfected with empty PCDH vector. These results indicate that miR-101-2 targets the 3′-UTR of ING3, directly suppressing its gene expression (Figure 1B). To test the hypothesis that miR-101-2 could downregulate ING3 protein expression in bovine fibroblasts, we first obtained the clone cells with pCDH-miR-101-2 or PCDH vector. We found that the overexpression of miR-101-2 caused significant decreases in ING3 at both protein and mRNA levels (Figure 1, Figure 2). These findings indicate that the presence of miR-101-2 in bovine fibroblasts suppresses ING3 expression.
      Figure thumbnail gr1
      Figure 1The ING3 gene is direct target of miR-101-2. (A) The part structure of miR-101-2 overexpression vector PCDH-miR-101-2. (B) Dual-luciferase assay of 293T cells co-transfected with the firefly luciferase constructs containing the ING3 wild type (WT) or mutant (MUT) 3′-untranslated region (UTR) as well as miR-101-2 overexpression vector PCDH-miR-101-2 or the empty PCDH-513-B1 as the negative control. (C) Western blot analysis of the protein levels of ING3 in response to miR-101-2 overexpression. β-actin was used as a loading control. Data are shown as mean ± SD from 3 experimental replicates (*P < 0.05).
      Figure thumbnail gr2
      Figure 2The mRNA expression of ING3 and apoptosis-related genes in 513-B1-bovine fetal fibroblasts (BFF) and miR-101-BFF. ING3, TP53, CDKN1A, BAX, and BCL2L1 mRNA expression was assessed by real-time PCR in 513-B1-BFF (BFF transfected with empty vectors) and miR-101-BFF (BFF with miR-101-2 overexpression). Data are shown as mean ± SEM from 3 experimental replicates (*P < 0.05, **P < 0.01).

      miR-101-2 Affects the Expression of ING3 and Related Genes in miR-101-BFF

      First, we tested whether miR-101-2 was successfully overexpressed after transfection and found that miR-101-2 expression was greatly upregulated (about 15-fold) in miR-101-BFF compared with that in 513-B1-BFF and BFF (P < 0.01; Supplemental Figure S1; https://doi.org/10.3168/jds.2018-15072).
      The ING3 activates TP53-transactivated promoters and modulates TP53-mediated transcription and apoptosis. Against this background, we tested a series of apoptosis-related genes to reveal whether these regulators are involved in the apoptosis inhibition of miR-101-2. We checked the mRNA levels of some regulators by qRT-PCR, including ING3, TP53, CDKN1A, BAX, and BCL2L1. The results showed that the levels of ING3, TP53, CDKN1A, and BAX decreased significantly in miR-101-BFF (P < 0.05) compared with that in 513-B1-BFF, whereas BCL2L1 level showed an increase in miR-101-BFF (P < 0.05) compared with that in 513-B1-BFF (Figure 2). These findings indicate that miR-101-2 can affect the expression of some apoptosis-related genes.

      Effects of miR-101-2 Overexpression on Apoptosis, Cell Cycle, and Proliferation

      The nuclear donor cell is a crucial factor affecting SCNT embryo quality and the efficiency of SCNT, and many efforts have been made to improve them by improving donor-cell status (
      • Chen H.
      • Zhang L.
      • Guo Z.
      • Wang Y.
      • He R.
      • Qin Y.
      • Quan F.
      • Zhang Y.
      Improving the development of early bovine somatic-cell nuclear transfer embryos by treating adult donor cells with vitamin C.
      ;
      • Mizutani E.
      • Wakayama S.
      • Wakayama T.
      Treatment of donor cell/embryo with different approaches to improve development after nuclear transfer.
      ). Furthermore, miR-101 can regulate cell proliferation, the cell cycle, and apoptosis in different cell types (
      • Wang L.
      • Li L.
      • Guo R.
      • Li X.
      • Lu Y.
      • Guan X.
      • Gitau S.C.
      • Wang L.
      • Xu C.
      • Yang B.
      miR-101 promotes breast cancer cell apoptosis by targeting Janus kinase 2.
      ;
      • Zhou X.
      • Xia Y.
      • Li L.
      • Zhang G.
      MiR-101 inhibits cell growth and tumorigenesis of Helicobacter pylori related gastric cancer by repression of SOCS2.
      ;
      • Qian L.
      • Zhang W.
      • Lei B.
      • He A.
      • Ye L.
      • Li X.
      • Dong X.
      MicroRNA-101 regulates T-cell acute lymphoblastic leukemia progression and chemotherapeutic sensitivity by targeting Notch1.
      ). Therefore, we detected the apoptosis, cell cycle, and proliferation in donor cells after miR-101-2 overexpression.
      The clone cells miR-101-BFF and 513-B1-BFF were first examined by flow cytometry to test whether miR-101-2 could affect apoptosis and cell cycle distribution of BFF. The miR-101-BFF had lower percentages of apoptotic and dead cells than in 513-B1-BFF (Figure 3A). These findings indicate that miR-101-2 could inhibit apoptosis in BFF.
      Figure thumbnail gr3
      Figure 3Effect of miR-101-2 overexpression on cell status. (A) Flow cytometry results show the effect of miR-101-2 overexpression on cell status. The x-axis represents cell status. Different letters (a,b) indicate P < 0.05. (B) Flow cytometry results show the effect of miR-101-2 overexpression on cell-cycle distribution. The x-axis represents the cell cycle stage. G1 = first gap; S = synthesis; G2/M = second gap/mitotic phase. Different letters (a,b) indicate P < 0.05. (C) CCK8 assays were performed to investigate the effect of miR-101-2 on the proliferation of bovine fetal fibroblasts (BFF) at the indicated time points. Data are shown as mean ± SEM from 3 experimental replicates (*P < 0.05, **P < 0.01).
      Recently, many studies have focused on the association of miR-101 with apoptosis and cell proliferation through interaction with its target genes. The miR-101 inhibits the apoptosis of trophoblast HTR-8/SVneo cells by targeting endoplasmic reticulum protein 44 during preeclampsia (
      • Zou Y.
      • Jiang Z.
      • Yu X.
      • Zhang Y.
      • Sun M.
      • Wang W.
      • Ge Z.
      • De W.
      • Sun L.
      MiR-101 regulates apoptosis of trophoblast HTR-8/SVneo cells by targeting endoplasmic reticulum (ER) protein 44 during preeclampsia.
      ). By contrast, the overexpression of human ING3, a putative homolog of the Caenorhabditis elegans ING3 gene, promotes apoptosis (
      • Nagashima M.
      • Shiseki M.
      • Pedeux R.M.
      • Okamura S.
      • Kitahama-Shiseki M.
      • Miura K.
      • Yokota J.
      • Harris C.C.
      A novel PHD-finger motif protein, p47ING3, modulates p53-mediated transcription, cell cycle control, and apoptosis.
      ;
      • Wang Y.
      • Dai D.L.
      • Martinka M.
      • Li G.
      Prognostic significance of nuclear ING3 expression in human cutaneous melanoma.
      ). In the present study, we found that miR-101-2 downregulated ING3 and inhibited the apoptosis of bovine SCNT donor cells. The miR-101-2 was inversely correlated with ING3 in apoptosis regulation. It has also been suggested that ING3 overexpression increases the apoptotic rate of RKO cells (WT TP53) but not RKO-E6 cells (inactivated TP53), and it was further confirmed that ING3 activates TP53-transactivated promoters, including the promoters of CDKN1A and BAX (
      • Nagashima M.
      • Shiseki M.
      • Pedeux R.M.
      • Okamura S.
      • Kitahama-Shiseki M.
      • Miura K.
      • Yokota J.
      • Harris C.C.
      A novel PHD-finger motif protein, p47ING3, modulates p53-mediated transcription, cell cycle control, and apoptosis.
      ;
      • Jafarnejad S.M.
      • Li G.
      Regulation of p53 by ING family members in suppression of tumor initiation and progression.
      ). The ING3-mediated apoptosis occurs in a TP53-dependent/independent manner or via the inhibition of CASP8 or FAS activation (
      • Garkavtsev I.
      • Grigorian I.A.
      • Ossovskaya V.S.
      • Chernov M.V.
      The candidate tumour suppressor p33ING1 cooperates with p53 in cell growth control.
      ;
      • Wang Y.
      • Dai D.L.
      • Martinka M.
      • Li G.
      Prognostic significance of nuclear ING3 expression in human cutaneous melanoma.
      ;
      • Coles A.H.
      • Jones S.N.
      The ING gene family in the regulation of cell growth and tumorigenesis.
      ). In our study, the mRNA levels of TP53, CDKN1A, and BAX were decreased significantly following miR-101-2 overexpression. Accordingly, we infer that miR-101-2 regulation of apoptosis by targeting ING3 in BFF may occur in a TP53-dependent manner.
      The flow cytometry results demonstrated that the percentage of cells in the G1 phase decreased and the percentage of cells in the S phase increased in the clone cells miR-101-BFF compared with that in the clone cells 513-B1-BFF. This indicates that miR-101-2 may be involved in the G1-to-S phase transition. These findings show that the G1-to-S phase transition of BFF can be expedited by miR-101-2 (Figure 3B). To investigate the possible function of miR-101-2 in BFF, we examined its effect on cell proliferation. The CCK8 assay results showed that the overexpression of miR-101-2 resulted in the promotion of the growth of BFF compared with that of the vector control (Figure 3C).
      The miR-101 could inhibit cell proliferation by targeting certain genes (RAC1, FOS, MITF, and SOCS2;
      • Konno Y.
      • Dong P.
      • Xiong Y.
      • Suzuki F.
      • Lu J.
      • Cai M.
      • Watari H.
      • Mitamura T.
      • Hosaka M.
      • Hanley S.J.
      MicroRNA-101 targets EZH2, MCL-1 and FOS to suppress proliferation, invasion and stem cell-like phenotype of aggressive endometrial cancer cells.
      ;
      • Wang C.
      • Lu S.
      • Jiang J.
      • Jia X.
      • Dong X.
      • Bu P.
      Hsa-microRNA-101 suppresses migration and invasion by targeting Rac1 in thyroid cancer cells.
      ;
      • Zheng H.-B.
      • Zheng X.-G.
      • Liu B.-P.
      miRNA-101 inhibits ovarian cancer cells proliferation and invasion by down-regulating expression of SOCS-2.
      ). However, we found that miR-101-2 promoted the proliferation of bovine SCNT donor cells, which is probably because it downregulates ING3. ING3 is one member of the well-conserved family of ING proteins, and the overexpression of ING3 in RKO cells resulted in a decreased proportion of cells in S phase and an increased proportion in G0/G1 phase, suppressing cell growth (
      • Nagashima M.
      • Shiseki M.
      • Pedeux R.M.
      • Okamura S.
      • Kitahama-Shiseki M.
      • Miura K.
      • Yokota J.
      • Harris C.C.
      A novel PHD-finger motif protein, p47ING3, modulates p53-mediated transcription, cell cycle control, and apoptosis.
      ). We found that the downregulation of ING3 via miR-101-2 overexpression resulted in a decreased proportion of cells in G1 phase and an increase in proportion in S phase in BFF. These findings showed that miR-101-2 could accelerate the proliferation of BFF.

      Characterization of miR-101-2 Expression in IVF and SCNT Embryos

      The qRT-PCR results showed that the expression of miR-101-2 in 513-B1-NT group was lower than that in IVF group at the 2-cell and 8-cell stage (P < 0.05; Figure 4), whereas it reached a similar level in the IVF group when using miR-101-BFF as donor cells (P > 0.05). However, miR-101-2 expression levels at the blastocyst stage showed no significant differences (P > 0.05).
      • Wang P.
      • Li X.
      • Cao L.
      • Huang S.
      • Li H.
      • Zhang Y.
      • Yang T.
      • Jiang J.
      • Shi D.
      MicroRNA-148a overexpression improves the early development of porcine somatic cell nuclear transfer embryos.
      indicated using donor cells with miR-148a overexpression could upregulate its expression in cloned embryo, and similar results were found in another study, with miR-449b being selected (
      • Wang M.
      • Gao Y.
      • Qu P.
      • Qing S.
      • Qiao F.
      • Zhang Y.
      • Mager J.
      • Wang Y.
      Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine.
      ). These results are in line with our findings.
      Figure thumbnail gr4
      Figure 4The expression pattern of miR-101-2 in in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. Relative miR-101-2 expression level of genes in 2-cell, 8-cell, and d-7 blastocysts from IVF, 513-B1-NT [SCNT embryos produced by using bovine fetal fibroblasts (BFF) transfected with empty vectors as donor cells], or miR-101-NT groups (SCNT embryos produced by using BFF with miR-101-2 overexpression as donor cells). Different letters (a,b) indicate P < 0.05. The results from 2-cell IVF samples are set as 1. Data are shown as mean ± SEM from 3 experimental replicates.

      Effects of miR-101-2 Overexpression on the Quality of Cloned Blastocysts

      Primarily, we analyzed the differences in developmental rate among 3 groups. The 2-cell rate and 8-cell rate between the miR-101-BFF NT (miR-101-NT) group and the 513-B1-BFF NT (513-B1-NT) group showed no significant difference (78.42 ± 2.07 vs. 75.76 ± 0.61 and 51.66 ± 1.98 vs. 47.80 ± 1.28, respectively, P > 0.05), which were both lower than that of IVF embryos (P < 0.05). The blastocyst rate has been widely used as an index of blastocyst quality in studies on SCNT (
      • Liu X.
      • Wang Y.
      • Gao Y.
      • Su J.
      • Zhang J.
      • Xing X.
      • Zhou C.
      • Yao K.
      • An Q.
      • Zhang Y.
      H3K9 demethylase KDM4E is an epigenetic regulator for bovine embryonic development and a defective factor for nuclear reprogramming.
      ). Day-7 blastocyst rate, however, was higher in the miR-101-NT group than in the 513-B1-NT group (39.08 ± 1.08 vs. 32.90 ± 2.10, P < 0.05), attaining a rate comparable to that of IVF embryos, and Day-8 blastocyst rate showed similar tendency in miR-101-NT, 513-B1-NT, and IVF group (44.68 ± 1.35 vs. 37.93 ± 1.97 vs. 47.01 ± 1.43, Table 1).
      Table 1Effect of micro RNA miR-101-2 on the development of cloned bovine embryos in vitro
      Three replicates were performed. The data are shown as mean ± SEM.
      Group
      IVF = in vitro fertilization; 513-B1-NT = somatic cell nuclear transfer (SCNT) embryos produced by using bovine fetal fibroblasts (BFF) transfected with empty vectors as donor cells; miR-101-NT = SCNT embryos produced by using BFF with miR-101-2 overexpression as donor cells.
      No. of reconstructedNo. of cleaved embryos (%)No. of 8-cell (%) per 2-cellNo. of blastocysts on 7 d (%) per 2-cellNo. of blastocysts on 8 d (%) per 2-cell
      IVF211178 (84.38 ± 1.16)
      Values with different superscripts within columns differ significantly (P < 0.05).
      101 (56.66 ± 0.89)
      Values with different superscripts within columns differ significantly (P < 0.05).
      77 (42.80 ± 1.78)
      Values with different superscripts within columns differ significantly (P < 0.05).
      83 (47.01 ± 1.43)
      Values with different superscripts within columns differ significantly (P < 0.05).
      513-B1-NT165125 (75.76 ± 0.61)
      Values with different superscripts within columns differ significantly (P < 0.05).
      60 (47.80 ± 1.28)
      Values with different superscripts within columns differ significantly (P < 0.05).
      42 (32.90 ± 2.10)
      Values with different superscripts within columns differ significantly (P < 0.05).
      47 (37.93 ± 1.97)
      Values with different superscripts within columns differ significantly (P < 0.05).
      miR-101-NT193151 (78.42 ± 2.07)
      Values with different superscripts within columns differ significantly (P < 0.05).
      78 (51.66 ± 1.98)
      Values with different superscripts within columns differ significantly (P < 0.05).
      60 (39.08 ± 1.08)
      Values with different superscripts within columns differ significantly (P < 0.05).
      67 (44.68 ± 1.35)
      Values with different superscripts within columns differ significantly (P < 0.05).
      a,b Values with different superscripts within columns differ significantly (P < 0.05).
      1 Three replicates were performed. The data are shown as mean ± SEM.
      2 IVF = in vitro fertilization; 513-B1-NT = somatic cell nuclear transfer (SCNT) embryos produced by using bovine fetal fibroblasts (BFF) transfected with empty vectors as donor cells; miR-101-NT = SCNT embryos produced by using BFF with miR-101-2 overexpression as donor cells.
      We also performed immunostaining of blastocysts for the trophectoderm marker CDX2 and the DNA marker DAPI to compare the numbers of different cell types among the 3 groups (Figures 5A and 5B). Total cell number for individual 513-B1-NT blastocysts was less than that for IVF blastocysts (86.4 vs. 102.5, P < 0.05), but this figure rose significantly when miR-101-BFF was used as donor cells (100.8 vs. 86.4, P < 0.05). The ICM/TE ratio was also significantly higher in the miR-101-NT group than in the 513-B1-NT counterpart (28.8 vs. 23.9%, P < 0.05), at a level comparable to that of the IVF group, whereas ICM cell number between these groups did not show significant difference (Figure 5B). To clarify why a higher ICM/TE ratio was found in the miR-101-NT group, qRT-PCR was performed to reveal the expression levels of 4 development-related genes, namely, POU5F1 NANOG, SOX2, and CDX2; among these, POU5F1 and SOX2 were upregulated in the miR-101-NT group (Figure 6).
      Figure thumbnail gr5
      Figure 5Overexpression miR-101-2 enhance the early development of somatic cell nuclear transfer (SCNT) embryos. (A) Immunostaining of CDX2 (red) and 4,6-diamidino-2-phenylindole (DAPI; blue) in blastocysts from in vitro fertilized (IVF), 513-B1-NT (SCNT embryos produced by using bovine fetal fibroblasts (BFF) transfected with empty vectors as donor cells), and miR-101-NT (SCNT embryos produced by using BFF with miR-101-2 overexpression as donor cells) groups. Scale bar = 50 μm. (B) Histogram shows the total cell number, inner cell mass (ICM) cell number, and ICM:trophectoderm (TE) ratio of each blastocyst from IVF, 513-B1-NT, and miR-101-NT groups (n = 34, 28, and 31, respectively). Different letters (a,b) indicate P < 0.05. (C) Apoptotic cells (green) in the nuclei (blue) of blastocysts in IVF, 513-B1-NT, and miR-101-NT. Scale bar = 50 μm. (D) The apoptotic rate in blastocysts from IVF, 513-B1-NT, and miR-101-NT groups (n = 32, 23, and 28, respectively). Different letters (a,b) indicate P < 0.05. Data are shown as mean ± SEM from 3 experimental replicates. TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
      Figure thumbnail gr6
      Figure 6Relative expression of development-related genes and apoptosis-related genes. Relative expression level of genes in d-7 blastocysts from in vitro fertilization (IVF). 513-B1-NT = somatic cell nuclear transfer (SCNT) embryos produced by using bovine fetal fibroblasts (BFF) transfected with empty vectors as donor cells; miR-101-NT = SCNT embryos produced by using BFF with miR-101-2 overexpression as donor cells. Three experimental replicates were performed. Data are shown as mean ± SEM from 3 experimental replicates (*P < 0.05, **P < 0.01).
      The POU5F1 gene (also known as OCT4), which encodes a factor that maintains ICM fate, plays a key role in embryonic development and stem cell pluripotency. It has been reported that POU5F1 transcript levels were decreased in clones compared with those in IVF embryos at the blastocyst stage (
      • Beyhan Z.
      • Forsberg E.
      • Eilertsen K.
      • Kent-First M.
      • First N.
      Gene expression in bovine nuclear transfer embryos in relation to donor cell efficiency in producing live offspring.
      ). Similar results were obtained in our study. The SOX2 is also involved in maintaining pluripotency. In ICM cells, SOX2 collaborating with POU5F1 and NANOG acts as a circuit to form the pluripotent regulatory network (
      • Chen X.
      • Xu H.
      • Yuan P.
      • Fang F.
      • Huss M.
      • Vega V.B.
      • Wong E.
      • Orlov Y.L.
      • Zhang W.
      • Jiang J.
      Integration of external signaling pathways with the core transcriptional network in embryonic stem cells.
      ;
      • Kim J.
      • Chu J.
      • Shen X.
      • Wang J.
      • Orkin S.H.
      An extended transcriptional network for pluripotency of embryonic stem cells.
      ). The observed rise in the expression of SOX2 and POU5F1, but not in NANOG or CDX2, in the miR-101-NT group is consistent with previous studies indicating the relationship between the expression of related genes and blastocyst quality (
      • Su J.
      • Wang Y.
      • Li Y.
      • Li R.
      • Li Q.
      • Wu Y.
      • Quan F.
      • Liu J.
      • Guo Z.
      • Zhang Y.
      Oxamflatin significantly improves nuclear reprogramming, blastocyst quality, and in vitro development of bovine SCNT embryos.
      ;
      • Chen H.
      • Zhang L.
      • Guo Z.
      • Wang Y.
      • He R.
      • Qin Y.
      • Quan F.
      • Zhang Y.
      Improving the development of early bovine somatic-cell nuclear transfer embryos by treating adult donor cells with vitamin C.
      ).
      Serum starvation treatment is commonly used in SCNT, but it might result in DNA damage and subsequent apoptosis. Therefore, apoptosis was considered as a criterion for the evaluation of blastocyst quality (
      • Hardy K.
      Cell death in the mammalian blastocyst.
      ). Here, results indicated the apoptotic rate in 513-B1-NT embryos was significantly higher than that in IVF embryos. The apoptotic rate was lower in miR-101-NT blastocysts than in the 513-B1-NT group (P < 0.05), at levels comparable to those in IVF embryos (Figures 5C and 5D). Furthermore, the expression of ING3 and several genes associated with apoptosis was assessed by qRT-PCR (Figure 6). The BCL2L1 was upregulated in miR-101-NT blastocysts compared with that in the IVF group and the 513-B1-NT group (P < 0.05). The ING3 expression was higher in 513-B1-NT than in the IVF blastocysts (P < 0.01), but it decreased significantly in miR-101-NT blastocysts compared with the 513-B1-NT group (P < 0.01). The expression of BAX was lower in miR-101-NT blastocysts than in both the IVF and the 513-B1-NT group (P < 0.05).
      The suppression of ING3 was associated with fewer apoptotic germ cells in C. elegans. Also, the embryonic death rate was decreased in the ING3 mutant (
      • Luo J.
      • Shah S.
      • Riabowol K.
      • Mains P.E.
      The Caenorhabditis elegans ing-3 gene regulates ionizing radiation-induced germ-cell apoptosis in a p53-associated pathway.
      ). Here, ING3 expression in SCNT blastocysts was suppressed when using miR-101-BFF as donor cells. The BCL2 family proteins influence programmed cell death or apoptosis, among which BCL2L1 is antiapoptotic, whereas BAX is proapoptotic (
      • Martinou J.-C.
      • Youle R.J.
      Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics.
      ). We measured the transcript abundance of BAX and BCL2L1 among the 3 groups and found that BAX was downregulated and BCL2L1 was upregulated in the miR-101-NT group compared with their levels in the 513-B1-BFF and IVF counterparts. These results are consistent with previous studies indicating the relationship between the expression of related genes and apoptosis (
      • Su J.
      • Wang Y.
      • Li Y.
      • Li R.
      • Li Q.
      • Wu Y.
      • Quan F.
      • Liu J.
      • Guo Z.
      • Zhang Y.
      Oxamflatin significantly improves nuclear reprogramming, blastocyst quality, and in vitro development of bovine SCNT embryos.
      ). Taking these findings together, the changes of expression of ING3, BAX, and BCL2L1 might contribute to the decrease in apoptotic rate in miR-101-NT blastocysts.
      Collectively, these results demonstrated that the quality of cloned blastocysts from miR-101-BFF was better than for those derived from 513-B1-BFF, which was similar to the quality of IVF blastocysts. Poor embryo quality has been a major problem contributing to pregnancy failure in SCNT (
      • Choi I.
      • Zhu J.
      • Campbell K.H.
      The combined treatment of calcium ionophore with strontium improves the quality of ovine SCNT embryo development.
      ). Great effort has been placed into enhancing developmental competence of SCNT embryos, and they found that those cloned blastocysts with better quality (lower apoptotic rate, more total cell number, and so on) tend to be more able to result in development in vivo (higher pregnancy rate, birth rate, and so on) after performing embryo transfer (
      • Su J.
      • Wang Y.
      • Xing X.
      • Zhang L.
      • Sun H.
      • Zhang Y.
      Melatonin significantly improves the developmental competence of bovine somatic cell nuclear transfer embryos.
      ;
      • Liu X.
      • Wang Y.
      • Gao Y.
      • Su J.
      • Zhang J.
      • Xing X.
      • Zhou C.
      • Yao K.
      • An Q.
      • Zhang Y.
      H3K9 demethylase KDM4E is an epigenetic regulator for bovine embryonic development and a defective factor for nuclear reprogramming.
      ). However, whether miR-101-2 would improve the in vivo development of bovine SCNT embryos needs further investigation.

      CONCLUSIONS

      Our results indicate that miR-101-2 can reduce cell apoptosis and expedite the cell cycle. More importantly, it can also enhance the development rate and reduce the apoptotic rate in SCNT embryos and subsequently enhance the early development of bovine SCNT embryos.

      ACKNOWLEDGMENTS

      We thank our laboratory colleagues for their help in this study. This work was supported by the National Science and Technology Major Project (no. 2016ZX08007-002; Yangling, China).

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