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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.
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 (
). 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 (
), 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 (
), bovine ovaries were collected from a local abattoir in Xi'an, China. Bovine frozen-thawed semen was purchased from Bright Farming.
All chemicals and reagents, unless otherwise stated, were purchased from Sigma-Aldrich. Disposable, sterile plastic ware was from Corning.
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 (
). 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 (
). 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 ;
). 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 (
). 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 ;
). 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 (
). 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 (
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).
In vitro fertilization was performed as described (
). 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 (
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 (
). 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 (
). 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;
). 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 (
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 ;
). 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).
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 (
). 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 (
), 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 (
). 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
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.
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.
). 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.
Poor embryo quality is a common cause of SCNT pregnancy failure (
). 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 ;
), 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 4–6). 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).
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.
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 (
). 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 (
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 (
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 (
) 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.
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.
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.