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ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows

Open AccessPublished:November 15, 2022DOI:https://doi.org/10.3168/jds.2022-22427

      ABSTRACT

      Aberrant epigenetic nuclear reprogramming, especially imprinting pattern disorders, is one of the major causes of failure of clone development from somatic cell nuclear transfer (SCNT). Previous studies showed that ZFP57 is a key protein required for imprint maintenance after fertilization. In this study, we found that imprinting control regions in several imprinted genes were significantly hypomethylated in cloned embryos compared with in vitro fertilization embryos, indicating a loss of imprinted gene methylation. The ZFP57 expression was capable of maintaining the correct degree of methylation at several imprinting control regions and correcting abnormal hypomethylation. Moreover, we successfully obtained bovine fetal fibroblasts overexpressing ZFP57, which were used as donors for SCNT. Our results demonstrated that overexpression of ZFP57 increased total and trophectoderm cell numbers and the ratio of inner cell mass to total cells, reduced the apoptosis rate and significantly enhanced the development of SCNT blastocysts in vitro, ultimately achieving a degree of methylation similar to that in in vitro fertilization embryos. We concluded that overexpression of ZFP57 in donor cells provided an effective method for enhancing nuclear reprogramming and developmental potential in SCNT embryos. The ZFP57 protein played a key role in maintaining the methylation of imprinted genes during early embryonic development, which may be effective for enhanced SCNT in cattle.

      Key words

      INTRODUCTION

      Somatic cell nuclear transfer (SCNT) has been tested for numerous applications, such as breeding transgenic animals, preserving endangered species, studying reprogramming, and researching therapeutic cloning (
      • Kato Y.
      • Tani T.
      • Sotomaru Y.
      • Kurokawa K.
      • Kato J.
      • Doguchi H.
      • Yasue H.
      • Tsunoda Y.
      Eight calves cloned from somatic cells of a single adult.
      ,
      • Gouveia C.
      • Huyser C.
      • Egli D.
      • Pepper M.S.
      Lessons learned from somatic cell nuclear transfer.
      ). However, the low efficiency of cloning has hindered the further development of these applications. Abnormal reprogramming of bovine SCNT embryos results from DNA methylation, histone modification, abnormal X chromosome inactivation, abnormal expression of imprinted genes, and aberrations in the number and phase of gene expressions (
      • Shen C.J.
      • Lin C.C.
      • Shen P.C.
      • Cheng W.T.
      • Chen H.L.
      • Chang T.C.
      • Liu S.S.
      • Chen C.M.
      Imprinted genes and satellite loci are differentially methylated in bovine somatic cell nuclear transfer clones.
      ;
      • Niemann H.
      Epigenetic reprogramming in mammalian species after SCNT-based cloning.
      ). Recent studies have found that the low efficiency and dysplasia of somatic cloning were partly a consequence of aberrant expression of imprinted genes (
      • Urrego R.
      • Rodriguez-Osorio N.
      • Niemann H.
      Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle.
      ;
      • Niemann H.
      Epigenetic reprogramming in mammalian species after SCNT-based cloning.
      ;
      • Wang Y.
      • Liu Q.
      • Kang J.
      • Zhang Y.
      • Quan F.
      Overexpression of PGC7 in donor cells maintains the DNA methylation status of imprinted genes in goat embryos derived from somatic cell nuclear transfer technology.
      ), which play a crucial role in the growth of embryos, fetuses, and placentas after SCNT.
      ZFP57 is a zinc-finger protein with a KRAB domain that exhibits a high affinity for methylated imprinting control regions (ICR;
      • Jiang W.
      • Shi J.
      • Zhao J.
      • Wang Q.
      • Cong D.
      • Chen F.
      • Zhang Y.
      • Liu Y.
      • Zhao J.
      • Chen Q.
      • Gu L.
      • Zhou W.
      • Wang C.
      • Fang Z.
      • Geng S.
      • Xie W.
      • Chen L.N.
      • Yang Y.
      • Bai Y.
      • Lin H.
      • Li X.
      ZFP57 dictates allelic expression switch of target imprinted genes.
      ). The protein ZFP57 contains several conserved C2H2-type zinc fingers that can recognize the TGCCGC motifs present at ICR (
      • Quenneville S.
      • Verde G.
      • Corsinotti A.
      • Kapopoulou A.
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      • Baglivo I.
      • Pedone P.V.
      • Grimaldi G.
      • Riccio A.
      • Trono D.
      In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA Methylation of imprinting control regions.
      ;
      • Voon H.P.J.
      • Gibbons R.J.
      Maintaining memory of silencing at imprinted differentially methylated regions.
      ;
      • Jiang W.
      • Shi J.
      • Zhao J.
      • Wang Q.
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      • Fang Z.
      • Geng S.
      • Xie W.
      • Chen L.N.
      • Yang Y.
      • Bai Y.
      • Lin H.
      • Li X.
      ZFP57 dictates allelic expression switch of target imprinted genes.
      ). In mouse embryos, deletion of the maternal-zygotic Zfp57 gene results in abnormal DNA methylation patterns and the death of mid-gestation embryo (
      • Zuo X.
      • Sheng J.
      • Lau H.T.
      • McDonald C.M.
      • Andrade M.
      • Cullen D.E.
      • Bell F.T.
      • Iacovino M.
      • Kyba M.
      • Xu G.
      • Li X.
      Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain.
      ;
      • Xu Z.
      • Shi J.
      • Zhang Y.
      • Liu Y.
      • Zhao J.
      • Chen Q.
      • Song C.
      • Geng S.
      • Xie W.
      • Wu F.
      • Bai Y.
      • Yang Y.
      • Li X.
      Zfp57 exerts maternal and sexually dimorphic effects on genomic imprinting.
      ). Similarly, when Zfp57 was deleted in embryonic stem cells, multiple ICR were all demethylated (
      • Quenneville S.
      • Verde G.
      • Corsinotti A.
      • Kapopoulou A.
      • Jakobsson J.
      • Offner S.
      • Baglivo I.
      • Pedone P.V.
      • Grimaldi G.
      • Riccio A.
      • Trono D.
      In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA Methylation of imprinting control regions.
      ;
      • Acurzio B.
      • Verma A.
      • Polito A.
      • Giaccari C.
      • Cecere F.
      • Fioriniello S.
      • Della Ragione F.
      • Fico A.
      • Cerrato F.
      • Angelini C.
      • Feil R.
      • Riccio A.
      Zfp57 inactivation illustrates the role of ICR methylation in imprinted gene expression during neural differentiation of mouse ESCs. Sci. Rep. 11:13802.
      ;
      • Zhang H.
      • Li Y.
      • Ma Y.
      • Lai C.
      • Yu Q.
      • Shi G.
      • Li J.
      Epigenetic integrity of paternal imprints enhances the developmental potential of androgenetic haploid embryonic stem cells.
      ). Mutations in ZFP57 reduced methylation of several imprinted gene sequences and were linked to transient neonatal diabetes and other disorders (
      • Takikawa S.
      • Wang X.
      • Ray C.
      • Vakulenko M.
      • Bell F.T.
      • Li X.J.
      Human and mouse ZFP57 proteins are functionally interchangeable in maintaining genomic imprinting at multiple imprinted regions in mouse ES cells.
      ).
      We found several imprinted genes with abnormal demethylation in cloned bovine embryos that contained the specific recognition site, TGCCGC, recognized by ZFP57 (
      • Mackay D.J.G.
      • Callaway J.L.A.
      • Marks S.M.
      • White H.E.
      • Acerini C.L.
      • Boonen S.E.
      • Dayanikli P.
      • Firth H.V.
      • Goodship J.A.
      • Haemers A.P.
      • Hahnemann J.M.D.
      • Kordonouri O.
      • Masoud A.F.
      • Oestergaard E.
      • Storr J.
      • Ellard S.
      • Hattersley A.T.
      • Robinson D.O.
      • Temple I.K.
      Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57.
      ;
      • Jiang W.
      • Shi J.
      • Zhao J.
      • Wang Q.
      • Cong D.
      • Chen F.
      • Zhang Y.
      • Liu Y.
      • Zhao J.
      • Chen Q.
      • Gu L.
      • Zhou W.
      • Wang C.
      • Fang Z.
      • Geng S.
      • Xie W.
      • Chen L.N.
      • Yang Y.
      • Bai Y.
      • Lin H.
      • Li X.
      ZFP57 dictates allelic expression switch of target imprinted genes.
      ), which is involved in maintaining normal methylation levels. Considering the important role of maintaining methylation levels in cloned embryos, we hypothesized that ZFP57 regulated the development of cloned bovine embryos by maintaining DNA methylation at ICR of imprinted genes. Therefore, we inferred that overexpression of ZFP57 could improve the production and quality of bovine SCNT embryos. Our findings showed that overexpression of ZFP57 improved the development of cloned bovine embryos by preserving the methylation of imprinted genes.
      Abnormal demethylation of imprinted genes during reprogramming of somatic cell cloned embryos is an important cause of the abnormal development of cloned animals and the low efficiency of somatic cell cloning. Although several studies have shown ZFP57 regulates embryonic development, the mechanism of ZFP57 action during SCNT reprogramming in mammals remains uncertain, especially with regard to how this could be employed to improve nuclear reprogramming in cattle. Here, we investigated the mechanism of abnormal methylation imprinting during the reprogramming of cloned embryos and elucidated the causes for the low efficiency of somatic cell cloning.

      MATERIALS AND METHODS

      Animals and Ethics Statement

      All animal experiments were carried out in accord with the Guide for the Care and Use of Laboratory Animals (
      • Ministry of Science and Technology of China
      Guidance Suggestions for the Care and Use of Laboratory Animals.
      ) and approved by the Animal Care and Use Committee of Northwest A&F University. We also followed the international guidelines for animal studies. As previously described (
      • Chang H.Y.
      • Xie R.X.
      • Zhang L.
      • Fu L.Z.
      • Zhang C.T.
      • Chen H.H.
      • Wang Z.Q.
      • Zhang Y.
      • Quan F.S.
      Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos.
      ), bovine ovaries were collected from a local abattoir in Xi'an, China. Bovine frozen-thawed semen was purchased from Bright Farming.

      Materials

      All chemicals and reagents, unless otherwise stated, were purchased from Sigma-Aldrich. Disposable, sterile plastic ware was from Corning.

      Experiment Design

      This section gives a brief outline of the experiments performed; particularly, the details of each procedure will be described in the following sections. In experiment 1, we obtained bovine donor cells overexpressing ZFP57 and verified their efficiency. Experiment 2 aimed to determine the effect of ZFP57 overexpression on embryonic development. We tested ZFP57-positive bovine fetal fibroblasts [BFF; nuclear transfer (NT) ZFP57 overexpression group] compared with normal cell lines (NT group) as donors for bovine SCNT embryos. In addition, we used the RNAi technique to knock down ZFP57 expression in metaphase II-stage oocytes. Early embryos from in vitro fertilization (IVF) were compared with metaphase II-stage oocytes injected with ZFP57-siRNA-1580 (IVF-ZFP57-1580 group), or with scrambled small interfering RNA (siRNA) injected in the IVF embryos group (IVF group). The same evaluation was performed on the relevant data from the 4 groups of embryos. The in vitro development of 2-cell stage embryos and blastocysts was determined at 48 and 168 h of culture [embryos transferred to G1.5 (Vitrolife AB) were recorded as 0 h]. In experiment 3, we measured the effects of ZFP57 overexpression on the quality of IVF and SCNT embryos. We measured the total number of blastocyst cells, trophectoderm (TE) cells, inner cell mass (ICM) cells, and the ratio ICM/total in the IVF group, the IVF-ZFP57-1580 group, the NT group, and the NT ZFP57 overexpression group (
      • Bock C.
      • Reither S.
      • Mikeska T.
      • Paulsen M.
      • Walter J.
      • Lengauer T.
      BiQ analyzer: Visualization and quality control for DNA methylation data from bisulfite sequencing.
      ). Experiment 4 showed whether the increase in embryonic development rate in the NT ZFP57 overexpression group was reflected in the quality of blastocysts and the apoptosis rate as measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Experiment 5 was designed to determine the effects of ZFP57 overexpression and knockdown on the DNA methylation level in cattle gametes and early embryonic imprinting genes H19/IGF2, XIST, and IGF2R.

      Cell Culture and Transfection

      Bovine fetal fibroblasts were isolated from ears of newborn Holstein calves as previously described (
      • Wang Y.
      • Su J.
      • Wang L.
      • Xu W.
      • Quan F.
      • Liu J.
      • Zhang Y.
      The effects of 5-aza-2′- deoxycytidine and trichostatin A on gene expression and DNA methylation status in cloned bovine blastocysts.
      ). Briefly, the hair was shaved off, the ear skin was washed with PBS, and cut into 1-mm3 pieces. The tissue fragments were cultured in DMEM (Gibco) plus 10% FBS (Gibco), 100 IU/mL penicillin, and 100 mg/mL streptomycin for 1 to 2 wk. When they reached 90% confluency, the fibroblasts were detached with 0.25% trypsin and then passaged at a ratio of 1:3. Cultured cells at passage 2 to 4 were used for SCNT. Before SCNT, nuclear donor cells were cultured in medium under serum-starved conditions (0.5% FBS) for 2 d.
      A eukaryotic expression system (Clontech) was used to construct a eukaryotic ZFP57 expression vector, composed of pEF1α-Tet3G and pTRE3G-BI with pEF1α-Tet3G tetracycline repressor protein (TetR) and pTRE3G-BI containing the tetracycline manipulation gene (TetO). Subsequently, we transfected HEK293 cells with the ZFP57 vector and cultured them in the presence of tetracycline to produce transient transfectants, and the ZFP57 expression was verified as normal. HEK293 cells were transfected with Lipofectamine 3000 (Invitrogen) and obtained from our laboratory depository. Electroporation was performed using the BTX Electro-cell manipulator ECM2001 (BTX) at 510 V (2-ms) and was used to transfer the ZFP57 expression vector into BFF, and positive cloned cells were selected by geneticin (G418; 800 μg/mL) resistance screening; additionally, tetracycline was added to induce continuous expression of the target and resistance genes.
      ZFP57 siRNA and a scrambled siRNA control were designed and synthesized by GenePharma (GenePharma). The siRNA sequences (si-ZFP57-252, si-ZFP57-282, si-ZFP57-1580) are listed in Supplemental Table S1 ( https://doi.org/10.17605/OSF.IO/4UZ2J ;
      • Yu T.
      ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows—Supplementary figures and tables. OSF. Aug. 24, 2022.
      ). Electroporation was used to transfer ZFP57 siRNA into BFF, and 24 h after transfection, the transfectants were used for experiments. Runs were done in triplicate and assays were performed with at least 3 independent experiments, unless otherwise indicated.

      RNA Isolation, Reverse-Transcriptase PCR, and Quantitative Real Time PCR

      Total RNA was extracted from embryos and reverse transcription was carried out as previously described (
      • 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.
      ;
      • Wang Y.
      • Liu Q.
      • Kang J.
      • Zhang Y.
      • Quan F.
      Overexpression of PGC7 in donor cells maintains the DNA methylation status of imprinted genes in goat embryos derived from somatic cell nuclear transfer technology.
      ). Total RNA was isolated from embryos (n = 15 embryos per pool) with the Cells-to-Signal kit (Ambion Co.) and TRIzol reagent (Takara). The PrimeScript II First-strand cDNA synthesis kit (Takara) was used for reverse transcription. Quantitative real-time PCR (qRT-PCR) was performed on an ABI StepOnePlus PCR system (Applied Biosystems). All reactions were run in triplicate, and the results were normalized to the GAPDH mRNA levels. The primer sequences used for qRT-PCR are given in Supplemental Table S2 ( https://doi.org/10.17605/OSF.IO/4UZ2J ;
      • Yu T.
      ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows—Supplementary figures and tables. OSF. Aug. 24, 2022.
      ).

      Oocyte Harvesting and in Vitro Maturation

      The collection of oocytes and in vitro maturation were performed as described (
      • Su J.M.
      • Yang B.
      • Wang Y.S.
      • Li Y.Y.
      • Xiong X.R.
      • Wang L.J.
      • Guo Z.K.
      • Zhang Y.
      Expression and methylation status of imprinted genes in placentas of deceased and live cloned transgenic calves.
      ;
      • 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.
      ;
      • Zhou C.
      • Zhang J.C.
      • Zhang M.
      • Wang D.B.
      • Ma Y.
      • Wang Y.
      • Wang Y.Z.
      • Huang Y.M.
      • Zhang Y.
      Transcriptional memory inherited from donor cells is a developmental defect of bovine cloned embryos.
      ). Oocytes were collected and placed in a dewar flask with sterile saline at 21 to 24°C for transportation to the laboratory within 5 h. Cumulus-oocyte complexes were aspirated from 2- to 8-mm antral follicles with a 10-mL syringe. The cumulus-oocyte complexes surrounded by >3 layers of cumulus cells and with a homogeneous cytoplasm were selected, washed with Brackett and Oliphant (BO) wash (IVF Bioscience) and BO-IVC (IVF Bioscience) medium, and cultured in BO-IVC medium at 38.5°C in a humidified incubator with 5% CO2 for 20 h.

      SCNT, Activation, and Culture of SCNT Embryos

      The SCNT embryos were produced as described previously (
      • Su J.M.
      • Yang B.
      • Wang Y.S.
      • Li Y.Y.
      • Xiong X.R.
      • Wang L.J.
      • Guo Z.K.
      • Zhang Y.
      Expression and methylation status of imprinted genes in placentas of deceased and live cloned transgenic calves.
      ;
      • Wang Y.
      • Su J.
      • Wang L.
      • Xu W.
      • Quan F.
      • Liu J.
      • Zhang Y.
      The effects of 5-aza-2′- deoxycytidine and trichostatin A on gene expression and DNA methylation status in cloned bovine blastocysts.
      ;
      • 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, after 22 h of maturation, the cumulus-oocyte complexes were stripped of their cumulus cells by pipetting. Oocytes with a first polar body and uniform cytoplasm were collected. The oocytes were enucleated and then treated with PBS containing 0.25% hyaluronidase for 3 min. The first polar body was aspirated with a glass pipette, and injected into the perivitelline space using a FemtoJet 4i (Eppendorf). The oocytes and donor material were given a dual electrical pulse of 35 V for 10 ms. Before activation, reconstructed embryos were incubated in modified synthetic oviductal fluid at least 2 h. We have previously published the formula for modified synthetic oviductal fluid medium (
      • 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.
      ).
      Embryos were chemically activated by treatment with 5 mM ionomycin for 4 min followed by 3 h incubation in synthetic oviductal fluid containing 2 mM 6-dimethynopyridine. Embryos (20–30) were cultured under mineral oil in 100-uL droplets of G1.5 (Vitrolife AB) in a 60-mm culture dish. Embryos were added to G2.5 on d 3 of culture (the day of SCNT was designated as d 0).

      IVF

      In vitro fertilization was performed as described (
      • Wang Y.
      • Su J.
      • Wang L.
      • Xu W.
      • Quan F.
      • Liu J.
      • Zhang Y.
      The effects of 5-aza-2′- deoxycytidine and trichostatin A on gene expression and DNA methylation status in cloned bovine blastocysts.
      ). Cumulus-oocyte complexes were rinsed in BO medium with 6 mg/mL BSA and 50 ng/mL heparin. Cumulus-oocyte complexes (25–30) were added to 100-μL microdrops of BO medium under mineral oil. Upon thawing, bull semen was separated by swim-up in BO medium, then centrifuged 2 times at 500 × g for 10 min at room temperature. About 20 μL (1 × 106 sperm/mL) of resuspended sperm was added to each microdrop. After 20 h, the cumulus cells were completely dispersed in PBS by repeated pipetting and then treated with 0.25% hyaluronidase in PBS for 4 min. After twice washing in synthetic oviductal fluid supplemented with 8 mg/mL BSA, zygotes were cultured in G1.5/G2.5 sequential media (Vitrolife) as described previously (
      • 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.
      ).

      Immunofluorescence Staining of Embryos

      Embryos were fixed overnight at 4°C in immunostaining fixing solution (Beyotime, P0098). Unless otherwise stated, all staining steps were performed at room temperature. Embryos were permeabilized with immunostaining solution containing Triton X-100 (Beyotime, P0096) for 0.5 h. After washing 5 times, they were blocked at 4°C for 12 h in the immunostaining blocking solution (Beyotime, P0260) and then incubated with primary antibody at 4°C for 12 h. Anti-CDX2 antibody (BioGenex) was diluted 1:50 using QuickBlock dilution buffer for immunostaining (Beyotime, P0262). After washing 5 times, embryos were incubated with secondary antibody Alexa Fluor 555-labeled goat anti-mouse IgG (Beyotime, A0459) for 2 h, washed, and then sealed with 4,6-diamidino-2-phenylindole (DAPI; Beyotime, C1005), 10 min, sealing tablets (
      • Su J.
      • Wang Y.
      • Li R.
      • Peng H.
      • Hua S.
      • Li Q.
      • Quan F.
      • Guo Z.
      • Zhang Y.
      Oocytes selected using BCB staining enhance nuclear reprogramming and the in vivo development of SCNT embryos in cattle.
      ). Finally, the embryos were photographed with a LSM 700 fluorescence microscope (Carl Zeiss). All experiments were replicated at least 3 times with a group of 10 to 15 embryos in each replicate. For fluorescence quantification, the intensity levels of the NT group and ZFP57 overexpression/knockdown embryos were expressed relative to the average intensity level of IVF embryos. Intensities were measured as described previously (
      • Zhang Y.
      • Yang Y.
      • Qiao P.
      • Wang X.
      • Yu R.
      • Sun H.
      • Xing X.
      • Zhang Y.
      • Su J.
      CHAF1b, chromatin assembly factor-1 subunit b, is essential for mouse preimplantation embryos.
      ).

      Apoptosis Assays

      To determine the number of apoptotic cells in d 7 blastocysts, the DeadEnd Fluorometric TUNEL System (Promega) was used (
      • 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.
      ). The blastocysts were examined by LSM 700 fluorescence microscopy (Carl Zeiss). All experiments were replicated at least 3 times with a group of 15 to 20 embryos in each replicate.

      Determination of DNA Methylation by Bisulfite Sequencing Analysis

      Single sperm, oocytes, 4-cell stage embryos, and d 7 blastocysts were collected and analyzed for methylation using the bisulfite method in the following 4 groups: IVF, IVF-ZFP57-1580, NT and NT ZFP57 overexpression, respectively. Samples were treated with NaHSO3 using the EZ-DNA methylation direct kit (Zymo Research;
      • Wang Y.
      • Su J.
      • Wang L.
      • Xu W.
      • Quan F.
      • Liu J.
      • Zhang Y.
      The effects of 5-aza-2′- deoxycytidine and trichostatin A on gene expression and DNA methylation status in cloned bovine blastocysts.
      ). The primers for H19/IGF2, IGF2R, and XIST (Supplemental Table S3, https://doi.org/10.17605/OSF.IO/4UZ2J ;
      • Yu T.
      ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows—Supplementary figures and tables. OSF. Aug. 24, 2022.
      ) were synthesized as described previously (
      • Wang Y.
      • Liu Q.
      • Kang J.
      • Zhang Y.
      • Quan F.
      Overexpression of PGC7 in donor cells maintains the DNA methylation status of imprinted genes in goat embryos derived from somatic cell nuclear transfer technology.
      ). Polymerase chain reaction products were purified and cloned into the pMD18-T vector (Takara) and 3 to 5 clones from each independent series of amplifications were sequenced. For each sample, 3 independent amplification experiments were performed. The DNA methylation levels were expressed as the ratio of the number of methylated CpG sites to the total number of CpG sites using BiQ Analyzer software (
      • Bock C.
      • Reither S.
      • Mikeska T.
      • Paulsen M.
      • Walter J.
      • Lengauer T.
      BiQ analyzer: Visualization and quality control for DNA methylation data from bisulfite sequencing.
      ;
      • Ono T.
      • Li C.
      • Mizutani E.
      • Terashita Y.
      • Yamagata K.
      • Wakayama T.
      Inhibition of class IIb histone deacetylase significantly improves cloning efficiency in mice.
      ).

      Statistical Analysis

      All the experiments were repeated in triplicate and data are mean ± standard deviation. Significant differences in the level of gene expression in the experimental groups was determined by 1-way ANOVA and least significant difference test using SPSS (v18.0; SPSS Inc.). Significance was defined as P < 0.05.

      RESULTS AND DISCUSSION

      Selection of Bovine Donor Cells Overexpressing ZFP57 and Effective Interference Fragments

      Using cDNA from BFF as a template for gene specific primers, we amplified the ZFP57 gene (1,713 bp) by PCR (Supplemental Figure S1, https://doi.org/10.17605/OSF.IO/4UZ2J ;
      • Yu T.
      ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows—Supplementary figures and tables. OSF. Aug. 24, 2022.
      ). Geneticin resistance was used to selectively culture positive donor cells overexpressing ZFP57. Afterward, we labeled the target gene by double digests (Figure 1A) and the cells with green fluorescence were selected as donors (Figure 1B, C). It was confirmed by qRT-PCR and western blotting that the expression of ZFP57 was significantly higher in BFF-ZFP57 cells compared with BFF-control cells (Figure 1D, E). The qRT-PCR results showed that ZFP57-1580 effectively downregulated the expression of ZFP57 mRNA compared with the scrambled siRNA-transfected control group. Therefore, ZFP57-1580 was used for subsequent experiments (Figure 1F).
      Figure thumbnail gr1
      Figure 1Successful harvesting of bovine donor cells overexpressing ZFP57 and effective interference fragments. (A) Results of double digests. 1, 2, and M represent the pTRE3G-BI vector carrying ZFP57, the double digests of vector, and the 5k plus II marker (Transgen Biotech), respectively. (B, C) Winnowing positive donor cells. B shows cells in bright field, whereas the green fluorescence in C represents cells overexpressing ZFP57. (D) Relative expression level of ZFP57 mRNA in stable clones. Means with different letters are significantly different (P < 0.05). (E) Western blot of ZFP57 expression in no. 6 monoclonal cells. a, b, 3, and 4 represent flag, β-actin, ZFP57 overexpression vector, and empty overexpression vector, respectively. (F) Relative mRNA expression of ZFP57 following small interfering RNA (siRNA) interference. Means with different letters are significantly different (P < 0.05). NC = bovine fetal fibroblasts transfected with empty vectors; siNC = negative control siRNA. Data are shown as mean ± SEM from 3 experimental replicates.
      • Chang H.Y.
      • Xie R.X.
      • Zhang L.
      • Fu L.Z.
      • Zhang C.T.
      • Chen H.H.
      • Wang Z.Q.
      • Zhang Y.
      • Quan F.S.
      Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos.
      indicated that overexpression of miR-101-2 in donor cells could upregulate miR-101-2 expression in cloned embryos. In other studies, we found similar results that overexpression of the maternal factor primordial germ cell protein 7 (PGC7) in goat fetal fibroblast donor cells (
      • Wang Y.
      • Liu Q.
      • Kang J.
      • Zhang Y.
      • Quan F.
      Overexpression of PGC7 in donor cells maintains the DNA methylation status of imprinted genes in goat embryos derived from somatic cell nuclear transfer technology.
      ) or knock down of histone-lysine N-methyltransferase SUV39H1 (SUV39H1) in 1-cell stage embryos improved development efficiency of cloned embryos (
      • 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 results are in good agreement with our findings.

      Effects of Overexpression and Knockdown of ZFP57 on Embryonic Development

      To assess whether overexpressing ZFP57 improved early development of bovine SCNT embryos, we determined the in vitro developmental rates from the 2-cell stage embryos to the blastocyst stage among the following 4 groups: IVF, IVF-ZFP57-1580, NT, and NT ZFP57 overexpression group (Figure 2; Table 1). We found that the cleavage rate of embryos in the IVF-ZFP57-1580 group was significantly lower than that in the other groups (P < 0.05), but the differences in embryo cleavage rates among the other 3 groups were not significant. However, the blastocyst rate in the IVF-ZFP57-1580 group was significantly lower than that in the IVF group on d 7 (24.1% ± 1.3 vs. 37.7% ± 1.1, P < 0.05). Compared with the NT group, the NT ZFP57 overexpression group conspicuously increased the rate of blastocyst formation on d 7 (38.9% ± 1.3 vs. 27.9% ± 1.1, P < 0.05); moreover, the difference between the IVF group and the NT ZFP57 overexpression group was not significant. Previous SCNT studies verified the use of blastocyst rate as an accurate index of the quality of blastocysts (
      • 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.
      ;
      • 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.
      ;
      • Chang H.Y.
      • Xie R.X.
      • Zhang L.
      • Fu L.Z.
      • Zhang C.T.
      • Chen H.H.
      • Wang Z.Q.
      • Zhang Y.
      • Quan F.S.
      Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos.
      ), so we first examined blastocyst development rate in 4 groups of embryos. ZFP57 is a key protein required for imprint maintenance after fertilization. In mouse embryos, deletion of the maternal-zygotic Zfp57 gene results in abnormal DNA methylation patterns and the death of mid-gestation embryos (
      • Zuo X.
      • Sheng J.
      • Lau H.T.
      • McDonald C.M.
      • Andrade M.
      • Cullen D.E.
      • Bell F.T.
      • Iacovino M.
      • Kyba M.
      • Xu G.
      • Li X.
      Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain.
      ;
      • Xu Z.
      • Shi J.
      • Zhang Y.
      • Liu Y.
      • Zhao J.
      • Chen Q.
      • Song C.
      • Geng S.
      • Xie W.
      • Wu F.
      • Bai Y.
      • Yang Y.
      • Li X.
      Zfp57 exerts maternal and sexually dimorphic effects on genomic imprinting.
      ). Our results suggest that overexpression of ZFP57 improves early development of SCNT embryos and knockdown of ZFP57 perturbed the normal embryonic development in Holstein cows.
      Figure thumbnail gr2
      Figure 2Representative images of bovine IVF and cloned blastocysts. A, B, C, D represent the in vitro fertilization (IVF), IVF-ZFP57-1580, nuclear transfer (NT), and NT ZFP57 overexpression groups, respectively. Original magnification was 400×. Scale bar = 100 µm.
      Table 1Effect of ZFP57 overexpression/knockdown on the development of bovine embryos in vitro
      Five replicates were performed. The data are shown as mean ± SEM.
      Group
      IVF = in vitro fertilization; NT = nuclear transfer.
      MII oocytes
      MII = metaphase II.
      (n, %)
      Cleaved embryos (n, %)Blastocysts on 7 d (n, %) per 2-cell
      IVF486341 (70.2 ± 0.9)
      Values with different superscripts within a column are significantly different (P < 0.05).
      183 (37.7 ± 1.1)
      Values with different superscripts within a column are significantly different (P < 0.05).
      IVF-ZFP57-1580420232 (55.3 ± 1.2)
      Values with different superscripts within a column are significantly different (P < 0.05).
      101 (24.1 ± 1.3)
      Values with different superscripts within a column are significantly different (P < 0.05).
      NT456315 (69.1 ± 0.8)
      Values with different superscripts within a column are significantly different (P < 0.05).
      127 (27.9 ± 1.1)
      Values with different superscripts within a column are significantly different (P < 0.05).
      NT-ZFP57 overexpression460334 (72.6 ± 1.0)
      Values with different superscripts within a column are significantly different (P < 0.05).
      174 (38.9 ± 1.3)
      Values with different superscripts within a column are significantly different (P < 0.05).
      a,b Values with different superscripts within a column are significantly different (P < 0.05).
      1 Five replicates were performed. The data are shown as mean ± SEM.
      2 IVF = in vitro fertilization; NT = nuclear transfer.
      3 MII = metaphase II.

      ZFP57 Overexpression Increased Total Cell Numbers, Numbers of TE and ICM Cells, and the Ratio of ICM to Total Cells in SCNT Blastocysts

      Total cell numbers and especially the ratio of ICM to total cells are the critical assessments of blastocyst quality (
      • Yu Y.
      • Ding C.H.
      • Wang E.Y.
      • Chen X.J.
      • Li X.M.
      • Zhao C.L.
      • Fan Y.
      • Wang L.
      • Beaujean N.
      • Zhou Q.
      • Jouneau A.
      • Ji W.
      Piezo-assisted nuclear transfer affects cloning efficiency and may cause apoptosis.
      ). Abnormal distribution of ICM and TE cells in cloned embryos at the preimplantation stages may lead to placental abnormalities and early fetal loss (
      • Im G.S.
      • Seo J.S.
      • Hwang I.S.
      • Kim D.H.
      • Kim S.W.
      • Yang B.C.
      • Yang B.S.
      • Lai L.
      • Prather R.S.
      Development and apoptosis of pre-implantation porcine nuclear transfer embryos activated with different combination of chemicals.
      ). To further detail the mechanism of the development of SCNT embryos in the NT ZFP57 overexpression group, we made several noteworthy changes as follows: the total number of cells was determined by DAPI staining, the number of TE cells by CDX2 staining, and the number of ICM cells in blastocysts of the 4 groups was estimated as total cells minus TE cells. The numbers of total blastomeres, TE cells, and ICM cells, and the ratio of ICM to total cells in cloned blastocysts were significantly higher in the NT ZFP57 overexpression group than in the NT group (P < 0.05), which were similar to that of the IVF group (Table 2). Various indices were significantly reduced in the IVF-ZFP57-1580 group (P < 0.05) compared with the IVF group. The above results suggest that overexpression of ZFP57 significantly increased the quality of cloned blastocysts. In addition, knockdown of ZFP57 reduces the quality of normal blastocysts.
      Table 2Characterization of d 7 bovine blastocysts in different groups
      The data are shown as mean ± SEM.
      Group
      IVF = in vitro fertilization; NT = nuclear transfer.
      n
      Refers to the total number of blastocysts analyzed. The 4,6-diamidino-2-phenylindole (DAPI) staining was used to estimate the total number of cells in blastocysts. The CDX2 staining was used to analyze the number of trophectoderm (TE) cells. The number of inner cell mass (ICM) cells was calculated as the total cell number minus the TE cell number.
      Total cells (n)TE cells (n)ICM cells (n)ICM total (%)
      IVF23110.12 ± 5.33
      Values with different superscripts within a column are significantly different (P < 0.05).
      76.63 ± 4.31
      Values with different superscripts within a column are significantly different (P < 0.05).
      33.32 ± 2.86
      Values with different superscripts within a column are significantly different (P < 0.05).
      30.27 ± 1.61
      Values with different superscripts within a column are significantly different (P < 0.05).
      IVF-ZFP57-15802883.81 ± 6.87
      Values with different superscripts within a column are significantly different (P < 0.05).
      64.17 ± 4.82
      Values with different superscripts within a column are significantly different (P < 0.05).
      19.61 ± 3.41
      Values with different superscripts within a column are significantly different (P < 0.05).
      23.40 ± 1.81
      Values with different superscripts within a column are significantly different (P < 0.05).
      NT2387.76 ± 6.14
      Values with different superscripts within a column are significantly different (P < 0.05).
      65.81 ± 4.45
      Values with different superscripts within a column are significantly different (P < 0.05).
      21.51 ± 3.39
      Values with different superscripts within a column are significantly different (P < 0.05).
      24.57 ± 1.21
      Values with different superscripts within a column are significantly different (P < 0.05).
      NT-ZFP57 overexpression27113.18 ± 5.10
      Values with different superscripts within a column are significantly different (P < 0.05).
      77.19 ± 4.92
      Values with different superscripts within a column are significantly different (P < 0.05).
      35.91 ± 3.01
      Values with different superscripts within a column are significantly different (P < 0.05).
      31.78 ± 1.88
      Values with different superscripts within a column are significantly different (P < 0.05).
      a,b Values with different superscripts within a column are significantly different (P < 0.05).
      1 The data are shown as mean ± SEM.
      2 IVF = in vitro fertilization; NT = nuclear transfer.
      3 Refers to the total number of blastocysts analyzed. The 4,6-diamidino-2-phenylindole (DAPI) staining was used to estimate the total number of cells in blastocysts. The CDX2 staining was used to analyze the number of trophectoderm (TE) cells. The number of inner cell mass (ICM) cells was calculated as the total cell number minus the TE cell number.

      Percentage of Apoptotic Cells Decreased in Blastocysts from the NT ZFP57 Overexpression Group

      Apoptosis is a significant factor in assessments of blastocyst quality (
      • Hardy K.
      Cell death in the mammalian blastocyst.
      ). In bovine embryos, apoptosis can be detected after the 8-cell stage (
      • Fahrudin M.
      • Otoi T.
      • Karja N.W.K.
      • Mori M.
      • Murakami M.
      • Suzuki T.
      Analysis of DNA fragmentation in bovine somatic nuclear transfer embryos using TUNEL.
      ). A high rate of apoptosis in SCNT blastocysts was related to a reduction in total cell numbers (
      • Yu Y.
      • Ding C.H.
      • Wang E.Y.
      • Chen X.J.
      • Li X.M.
      • Zhao C.L.
      • Fan Y.
      • Wang L.
      • Beaujean N.
      • Zhou Q.
      • Jouneau A.
      • Ji W.
      Piezo-assisted nuclear transfer affects cloning efficiency and may cause apoptosis.
      ). In our study, the number of apoptotic cells was determined by TUNEL assay on blastocysts in each group. As shown in Figure 3, the apoptosis rate in blastocysts from the IVF-ZFP57-1580 group and the NT group was significantly higher on d 7 than in the IVF and NT ZFP57 overexpression groups. This strongly supports our argument that overexpression of ZFP57 improves the quality of early SCNT embryos.
      Figure thumbnail gr3
      Figure 3Level of apoptosis in blastocysts. (A) Cystic cell apoptosis staining [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), green] and nuclear DNA (4,6-diamidino-2-phenylindole; DAPI) co-staining. Magnification = 400×, and the red scale bar = 100 µm. (B) Counts of apoptotic cells from each blastocyst of each group. Different letters indicate samples that differ significantly (P < 0.05). IVF = in vitro fertilization; NT = nuclear transfer. Data are shown as mean ± SEM from 3 experimental replicates.
      Poor embryo quality is a common cause of SCNT pregnancy failure (
      • Choi I.
      • Zhu J.
      • Campbell K.H.S.
      The combined treatment of calcium ionophore with strontium improves the quality of ovine SCNT embryo development.
      ). Enormous efforts have been focused on improving the developmental capacity of SCNT embryos (
      • Meissner A.
      • Jaenisch R.
      Mammalian nuclear transfer.
      ), and studies confirmed that the better-quality cloned blastocysts with lower apoptotic rate, greater total cell number, and so on, produced higher pregnancy and birth rates after 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, further research should be conducted to investigate whether ZFP57 overexpression would improve the in vivo development of bovine SCNT embryos.

      Overexpressing ZFP57 Protected ICR of Some Imprinted Genes from Aberrant Demethylation

      We found several imprinted genes with abnormal demethylation in cloned bovine embryos that contained the specific recognition site, TGCCGC, recognized by ZFP57 (Supplemental Figure S2, https://doi.org/10.17605/OSF.IO/4UZ2J ;
      • Yu T.
      ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows—Supplementary figures and tables. OSF. Aug. 24, 2022.
      ), which is involved in maintaining normal methylation levels. Changes in the DNA methylation status of 3 different imprinted genes were determined in sperm, oocytes, 4-cell stage embryos, and blastocysts from the 4 groups by using bisulfite sequencing (Figures 46). Sequence analysis showed that H19/IGF2 ICR was completely methylated in sperm (100%), but only slightly methylated in oocytes (4.79%). At the blastocyst stage, the same loci had different degrees of aberrant demethylation in embryos after SCNT (36.51%), compared with IVF embryos (52.92%). The H19/IGF2 ICR methylation level of blastocyst embryos in the IVF-ZFP57-1580 group (15.07%) was significantly lower than that in the IVF group (52.92%, P < 0.05). The DNA methylation level in the NT ZFP57 overexpression group at the blastocyst stage reached 52.08%, which was significantly higher than that of the NT group (36.51%, P < 0.05); however, such changes were not seen in the IVF group (52.92%; Figure 4).
      Figure thumbnail gr4
      Figure 4CpG methylation profiles at the H19/IGF2 locus detected by bisulfite sequencing. Circles on each horizontal chain represent CpG sites in the study sequence, whereas white and black circles represent nonmethylated and methylated CpG, respectively. Each horizontal strand represents a sequenced clone. The circles in the bisulfite sequencing (BS-)PCR results indicate DNA methylation levels automatically generated by the BiQ Analyzer. In each sample, the number of methylation sites divided by the total number of sites indicates the level of gene methylation. BS-PCR was repeated 3 times for each sample. IVF = in vitro fertilization; NT = nuclear transfer.
      Figure thumbnail gr6
      Figure 6CpG methylation profiles at the IGF2R locus detected by bisulfite sequencing. Circles on each horizontal chain represent CpG sites in the study sequence. The circles in the bisulfite sequencing (BS-)PCR results indicate DNA methylation levels automatically generated by the BiQ Analyzer. BS-PCR was repeated 3 times for each sample. IVF = in vitro fertilization; NT = nuclear transfer.
      With regard to the XIST ICR, we found almost no methylation modification on sperm (15%), although we detected hypermethylation on oocytes (95%). As shown in Figure 5, in the 4-cell and blastocyst stage, the DNA methylation levels of embryos after SCNT (7.27%, 28.75%) were lower than IVF embryos (54.29%, 54.19%). Similarly, the XIST methylation level of the IVF-ZFP57-1580 group at the 4-cell (12.73%) and blastocyst stage (29.09%) was significantly lower than that of the IVF group. The XIST methylation level at the 4-cell and blastocyst stage (46.25%, 46.92%) of the NT ZFP57 overexpression group was significantly higher than that of the NT group, which was not significantly different from the IVF group.
      Figure thumbnail gr5
      Figure 5CpG methylation profiles at the XIST locus detected by bisulfite sequencing. Circles on each horizontal chain represent CpG sites in the study sequence. The circles in the bisulfite sequencing (BS-)PCR results indicate DNA methylation levels automatically generated by the BiQ Analyzer. BS-PCR was repeated 3 times for each sample. IVF = in vitro fertilization; NT = nuclear transfer.
      Similar to XIST, the IGF2R ICR showed almost no methylation modification on sperm (13.14%), whereas hypermethylation occurred on oocytes (94.03%). In the 4-cell stage, when compared with IVF embryos (55.07%), the DNA methylation level of embryos after SCNT (28.32%) was lower, indicating aberrant methylation of IGF2R ICR in SCNT embryos. The DNA methylation level of the IGF2R ICR in the IVF-ZFP57-1580 group (25.93%) was significantly lower than that of the IVF group (P < 0.05), and significantly higher in the NT ZFP57 overexpression group (50.57%) compared with NT (P < 0.05); however, we found no difference compared with the IVF group (Figure 6).
      Correct gene imprinting is integral to normal in mammalian embryogenesis, and previous research has shown severe perturbation of the imprinting status as a result of SCNT (
      • Wei Y.
      • Zhu J.
      • Huan Y.
      • Liu Z.
      • Yang C.
      • Zhang X.
      • Mu Y.
      • Xia P.
      • Liu Z.
      Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets.
      ;
      • Ruan D.
      • Peng J.
      • Wang X.
      • Ouyang Z.
      • Zou Q.
      • Yang Y.
      • Chen F.
      • Ge W.
      • Wu H.
      • Liu Z.
      • Zhao Y.
      • Zhao B.
      • Zhang Q.
      • Lai C.
      • Fan N.
      • Zhou Z.
      • Liu Q.
      • Li N.
      • Jin Q.
      • Shi H.
      • Xie J.
      • Song H.
      • Yang X.
      • Chen J.
      • Wang K.
      • Li X.
      • Lai L.
      XIST derepression in active X chromosome hinders pig somatic cell nuclear transfer.
      ). Low efficiency and developmental abnormalities in cloned animals are related to faulty gene imprinting (
      • Su J.
      • Wang Y.
      • Xing X.
      • Liu J.
      • Zhang Y.
      Genome-wide analysis of DNA methylation in bovine placentas.
      ). IGF2 and IGF2R are imprinted genes that are essential for preimplantation development (
      • Barlow D.P.
      • Bartolomei M.S.
      Genomic imprinting in mammals.
      ). The expression profiles of the imprinted genes IGF2, IGF2R, and H19 were aberrant in organs from cloned calves (
      • Moore K.
      • Kramer J.M.
      • Rodriguez-Sallaberry C.J.
      • Yelich J.V.
      • Drost M.
      Insulin-like growth factor (IGF) family genes are aberrantly expressed in bovine conceptuses produced in vitro or by nuclear transfer.
      ). Previous studies have identified Xist as necessary for post-implantation development of mouse SCNT embryos (
      • Inoue K.
      • Kohda T.
      • Sugimoto M.
      • Sado T.
      • Ogonuki N.
      • Matoba S.
      • Shiura H.
      • Ikeda R.
      • Mochida K.
      • Fujii T.
      • Sawai K.
      • Otte A.P.
      • Tian X.C.
      • Yang X.
      • Ishino F.
      • Abe K.
      • Ogura A.
      Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.
      ;
      • Matoba S.
      • Inoue K.
      • Kohda T.
      • Sugimoto M.
      • Mizutani E.
      • Ogonuki N.
      • Nakamura T.
      • Abe K.
      • Nakano T.
      • Ishino F.
      • Ogura A.
      RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos.
      ,
      • Matoba S.
      • Wang H.H.
      • Jiang L.
      • Lu F.L.
      • Iwabuchi K.A.
      • Wu X.J.
      • Inoue K.
      • Yang L.
      • Press W.
      • Lee J.T.
      • Ogura A.
      • Shen L.
      • Zhang Y.
      Loss of H3K27me3 imprinting in somatic cell nuclear transfer embryos disrupts post-implantation development.
      ), and somatic cloning or IVF nearly always results in perturbation of XIST expression in bovine blastocysts (
      • Shen C.J.
      • Lin C.C.
      • Shen P.C.
      • Cheng W.T.K.
      • Chen H.L.
      • Chang T.C.
      • Liu S.S.
      • Chen C.M.
      Imprinted genes and satellite loci are differentially methylated in bovine somatic cell nuclear transfer clones.
      ;
      • Niemann H.
      Epigenetic reprogramming in mammalian species after SCNT-based cloning.
      ). Xist is an X-linked noncoding RNA that is expressed only by the paternal X chromosome in IVF embryos, but is aberrantly expressed after SCNT (
      • Inoue K.
      • Kohda T.
      • Sugimoto M.
      • Sado T.
      • Ogonuki N.
      • Matoba S.
      • Shiura H.
      • Ikeda R.
      • Mochida K.
      • Fujii T.
      • Sawai K.
      • Otte A.P.
      • Tian X.C.
      • Yang X.
      • Ishino F.
      • Abe K.
      • Ogura A.
      Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.
      ;
      • Matoba S.
      • Wang H.H.
      • Jiang L.
      • Lu F.L.
      • Iwabuchi K.A.
      • Wu X.J.
      • Inoue K.
      • Yang L.
      • Press W.
      • Lee J.T.
      • Ogura A.
      • Shen L.
      • Zhang Y.
      Loss of H3K27me3 imprinting in somatic cell nuclear transfer embryos disrupts post-implantation development.
      ). Aberrant Xist expression from the maternal X chromosome results in ectopic X chromosome inactivation and global transcriptional alterations in preimplantation embryos, which caused failure of SCNT embryonic development (
      • Inoue K.
      • Kohda T.
      • Sugimoto M.
      • Sado T.
      • Ogonuki N.
      • Matoba S.
      • Shiura H.
      • Ikeda R.
      • Mochida K.
      • Fujii T.
      • Sawai K.
      • Otte A.P.
      • Tian X.C.
      • Yang X.
      • Ishino F.
      • Abe K.
      • Ogura A.
      Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.
      ;
      • Matoba S.
      • Wang H.H.
      • Jiang L.
      • Lu F.L.
      • Iwabuchi K.A.
      • Wu X.J.
      • Inoue K.
      • Yang L.
      • Press W.
      • Lee J.T.
      • Ogura A.
      • Shen L.
      • Zhang Y.
      Loss of H3K27me3 imprinting in somatic cell nuclear transfer embryos disrupts post-implantation development.
      ).
      Reconstructing the DNA methylation of imprinted genes will likely increase the developmental rate of SCNT embryos. Previous studies have shown that interference with the expression of XIST could significantly improve the developmental rate of SCNT embryos, suggesting that abnormal Xist activation at the preimplantation stage had a long-term effect on the developmental capacity of SCNT embryos (
      • Inoue K.
      • Kohda T.
      • Sugimoto M.
      • Sado T.
      • Ogonuki N.
      • Matoba S.
      • Shiura H.
      • Ikeda R.
      • Mochida K.
      • Fujii T.
      • Sawai K.
      • Otte A.P.
      • Tian X.C.
      • Yang X.
      • Ishino F.
      • Abe K.
      • Ogura A.
      Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.
      ;
      • Matoba S.
      • Inoue K.
      • Kohda T.
      • Sugimoto M.
      • Mizutani E.
      • Ogonuki N.
      • Nakamura T.
      • Abe K.
      • Nakano T.
      • Ishino F.
      • Ogura A.
      RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos.
      ).
      Our bisulfite sequencing results here showed that H19/IGF2 ICR was completely methylated in sperm but not in oocytes. For the XIST ICR and IGF2R ICR, we detected almost no methylation modification on sperm, although there was hypermethylation on oocytes, similar to prior results (
      • Ideraabdullah F.Y.
      • Vigneau S.
      • Bartolomei M.S.
      Genomic imprinting mechanisms in mammals.
      ;
      • Liu J.H.
      • Yin S.
      • Xiong B.
      • Hou Y.
      • Chen D.Y.
      • Sun Q.Y.
      Aberrant DNA methylation imprints in aborted bovine clones.
      ;
      • Niemann H.
      Epigenetic reprogramming in mammalian species after SCNT-based cloning.
      ). ZFP57 maintained imprinted expression in the target imprinted genes (
      • Monteagudo-Sánchez A.
      • Hernandez Mora J.R.
      • Simon C.
      • Burton A.
      • Tenorio J.
      • Lapunzina P.
      • Clark S.
      • Esteller M.
      • Kelsey G.
      • López-Siguero J.P.
      • de Nanclares G.P.
      • Torres-Padilla M.-E.
      • Monk D.
      The role of ZFP57 and additional KRAB-zinc finger proteins in the maintenance of human imprinted methylation and multi-locus imprinting disturbances.
      ;
      • Jiang W.
      • Shi J.
      • Zhao J.
      • Wang Q.
      • Cong D.
      • Chen F.
      • Zhang Y.
      • Liu Y.
      • Zhao J.
      • Chen Q.
      • Gu L.
      • Zhou W.
      • Wang C.
      • Fang Z.
      • Geng S.
      • Xie W.
      • Chen L.N.
      • Yang Y.
      • Bai Y.
      • Lin H.
      • Li X.
      ZFP57 dictates allelic expression switch of target imprinted genes.
      ) and is important in ICR maintaining of DNA methylation in humans and mice, and the mechanism of underlying ZFP57 imprint maintenance in bovine clone embryos remains uncertain. We have now confirmed that the methylation levels in the 4-cell and blastocyst stages further confirmed that overexpression of ZFP57 protected imprinted regions from demethylation and knockdown of ZFP57 reduced methylation of several imprinted genes sequences. We hypothesize that aberrant nuclear reprogramming after SCNT, especially faulty expression of imprinted genes like H19/IGF2, XIST and IGF2R, should be effectively controlled by overexpression of ZFP57, achieving levels similar to those in IVF.

      CONCLUSIONS

      In summary, ZFP57 overexpression in somatic donor cells increases the developmental rate and the quality of cloned blastocysts. More importantly, ZFP57 significantly enhances the efficiency of SCNT by maintaining the methylation of a set of imprinted genes during early embryonic development in cattle. Knockdown of ZFP57 reduced methylation of several imprinted gene sequences and were linked to abnormal blastocysts development rate and quality.

      ACKNOWLEDGMENTS

      This work was supported by the National Natural Science Foundation of China (32172812, 31302046) and Natural Science Foundation of Qinghai Province, China (2020-ZJ-917). Author contributions are as follows: J.S. and Y. Z. designed the research; T.Y., R.M., W.S., H.S., Q.A., and C.Z. performed the experiments; T.Y. data analysis, writing—original draft; T.Y. and R.M. contributed equally to this work. The authors have not stated any conflicts of interest.