Optimizing Embryo Collection for Application of CRISPR/Cas9 System and Generation of Fukutin Knockout Rat Using This Method.

Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological characteristics. However, their use is limited because of difficulties in establishing embryonic stem cells and performing genetic modifications, and insufficient embryological research. In this study, we established optimal superovulation and fertilized-egg transfer conditions, including optimal hormone injection concentration (≥150 IU/kg of PMSG and hCG) and culture medium (mR1ECM), to obtain high-quality zygotes and establish in vitro fertilization conditions for rats. Next, sgRNA with optimal targeting activity was selected by performing PCR analysis and the T7E1 assay, and the CRISPR/Cas9 system was used to construct a rat model for muscular dystrophy by inducing a deficiency in the fukutin gene without any off-target effect detected. The production of fukutin knockout rats was phenotypically confirmed by observing a drop-in body weight to one-third of that of the control group. In summary, we succeeded in constructing the first muscular dystrophy disease rat model using the CRISPR/CAS9 system for increasing future prospects of producing various animal disease models and encouraging disease research using rats.

Fasudil alleviates the vascular endothelial dysfunction and several phenotypes of Fabry disease.

Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.

Hyaluronidase 6 Does Not Affect Cumulus-Oocyte Complex Dispersal and Male Mice Fertility.

Glycosylphosphatidylinositol-anchored sperm hyaluronidases (HYAL) assist sperm penetration through the cumulus-oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we demonstrated that sperm from HYAL 5 and 7 double-knockout (dKO) mice produced significantly less offspring than sperm from wild-type mice due to defective COC dispersal. However, the HYAL6 gene remained active in the sperm from the dKO mice, indicating that they were not entirely infertile. This study explored the role of HYAL6 in fertilization by analyzing HYAL6-mutant mice. In this mouse model, HYAL5 and HYAL7 were present in the HYAL6-knockout sperm, and they could disperse hyaluronic acid. We found that HYAL6 was present on the surface of sperm. However, male mice lacking the HYAL6 gene had normal fertility, testicular integrity, and sperm characteristics. Furthermore, in vitro fertilization assays demonstrated that HYAL6-deficient epididymal sperm functioned normally. Therefore, HYAL6 is dispensable for fertilization.

Sperm hyaluronidase is critical to mammals' fertilization for its ability to disperse cumulus-oocyte complex layer.

Glycosylphosphatidylinositol-anchored sperm hyaluronidases have long been believed to assist in sperm penetration through the cumulus-oocyte complex (COC); however, their role in mammalian fertilization remains unclear. Previously, we have shown that hyaluronidase 5 (Hyal5)/Hyal7 double-knockout (dKO) mice produce significantly fewer offspring than their wild-type (WT) counterparts because of defective COC dispersal. Male infertility is mainly caused by a low sperm count. It can be further exacerbated by the deficiency of sperm hyaluronidase, which disperses the cumulus cells of the outer layer of the COC. In the current study, we evaluated the effects of a low count of Hyal-deficient sperm and conditions of ovulated oocytes on the fertilization rate using a mouse model. Our results demonstrated that a low sperm count further decreases the in vitro fertilization (IVF) rate of Hyal-deficient dKO spermatozoa. In addition, the dKO spermatozoa resulted in a fertilization rate of 12.5% upon fertilizing COCs with a thick cumulus layer, whereas the IVF rate was comparable to that of WT spermatozoa when oocytes with a thin or no cumulus layer were fertilized. Finally, we proved that the IVF rate of dKO spermatozoa could be recovered by adding rat spermatozoa as a source of sperm hyal. Our results suggest that a deficiency of proteins involved in fertilization, such as sperm hyal, has a vital role in fertilization.

Obtusifolin, an Anthraquinone Extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby, Reduces Inflammation in a Mouse Osteoarthritis Model.

Osteoarthritis (OA) is an age-related degenerative disease that causes cartilage dysfunction and inflammation. Obtusifolin, an anthraquinone extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby seeds, has anti-inflammatory functions; it could be used as a drug component to relieve OA symptoms. In this study, we investigated the effects of obtusifolin on OA inflammation. In vitro, interleukin (IL)-1ß (1 ng/mL)-treated mouse chondrocytes were co-treated with obtusifolin at different concentrations. The expression of matrix metalloproteinase (Mmp) 3, Mmp13, cyclooxygenase 2 (Cox2), and signaling proteins was measured by polymerase chain reaction and Western blotting; collagenase activity and the PGE2 level were also determined. In vivo, OA-induced C57BL/6 mice were administered obtusifolin, and their cartilage was stained with Safranin O to observe damage. Obtusifolin inhibited Mmp3, Mmp13, and Cox2 expression to levels similar to or more than those after treatment with celecoxib. Additionally, obtusifolin decreased collagenase activity and the PGE2 level. Furthermore, obtusifolin regulated OA via the NF-κB signaling pathway. In surgically induced OA mouse models, the cartilage destruction decreased when obtusifolin was administered orally. Taken together, our results show that obtusifolin effectively reduces cartilage damage via the regulation of MMPs and Cox2 expression. Hence, we suggest that obtusifolin could be a component of another OA symptom reliever.

V-Set and Immunoglobulin Domain-Containing 1 (VSIG1), Predominantly Expressed in Testicular Germ Cells, Is Dispensable for Spermatogenesis and Male Fertility in Mice.

To elucidate the functional role of V-set and immunoglobulin domain-containing 1 (VSIG1) in spermatogenesis and fertilization, we knocked out (KO) VSIG1 in a mouse embryo using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) -mediated genome editing. Reverse transcription PCR was performed using cDNA synthesized from VSIG1 KO testis RNA. Although Western blot analysis using a specific antibody to VSIG1 confirmed VSIG1 protein defects in the KO mice, hematoxylin-eosin staining analysis was similar in the KO and wild-type mice. Additionally, computer-assisted sperm analysis and in vitro fertilization experiments were conducted to confirm the activity and fertilization ability of sperm derived from the KO mouse. Mice lacking VSIG1 were viable and had no serious developmental defects. As they got older, the KO mice showed slightly higher weight loss, male mice lacking VSIG1 had functional testes, including normal sperm number and motility, and both male and female mice lacking VSIG1 were fertile. Our results from VSIG1 KO mice suggest that VSIG1 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. VSIG1 in sperm is dispensable for spermatogenesis and male fertility in mice. As several genes are known to possess slightly different functions depending on the species, the importance and molecular mechanism of VSIG1 in tissues of other species needs further investigation.

Cryopreserved Human Oocytes and Cord Blood Cells Can Produce Somatic Cell Nuclear Transfer-Derived Pluripotent Stem Cells with a Homozygous HLA Type.

Human pluripotent stem cells (PSCs) through somatic cell nuclear transfer (SCNT) may be an important source for regenerative medicine. The low derivation efficiency of stem cells and the accessibility of human oocytes are the main obstacles to their application. We previously reported that the efficiency of SCNT was increased by overexpression of H3K9me3 demethylase. Here, we applied a modified derivation method to the PSC line and first obtained human SCNT-PSC lines derived from both donated cryopreserved oocytes and cord blood cells with a homozygous human leukocyte antigen (HLA) type. The SCNT-PSCs have very similar characteristics with embryonic stem cells (ESCs) and additionally have shown immunocompatibility in an in vitro and in vivo humanized mouse with a matching HLA type. Our study demonstrates that SCNT technology using donated cryopreserved oocytes and cord blood cells with a known HLA type provides a promising method for establishing a human HLA-matched SCNT-PSC bank for regenerative medicine.

Peroxisome Proliferator-Activated Receptor Gamma Modulator Promotes Neonatal Mouse Primordial Follicle Activation In Vitro.

Peroxisome proliferator-activated receptor gamma (PPARγ) is known as a regulator of cellular functions, including adipogenesis and immune cell activation. The objectives of this study were to investigate the expression of PPARγ and identify the mechanism of primordial follicle activation via PPARγ modulators in mouse ovaries. We first measured the gene expression of PPARγ and determined its relationship with phosphatase and tensin homolog (PTEN), protein kinase B (AKT1), and forkhead box O3a (FOXO3a) expression in neonatal mouse ovaries. We then incubated neonatal mouse ovaries with PPARγ modulators, including rosiglitazone (a synthetic agonist of PPARγ), GW9662 (a synthetic antagonist of PPARγ), and cyclic phosphatidic acid (cPA, a physiological inhibitor of PPARγ), followed by transplantation into adult ovariectomized mice. After the maturation of the transplanted ovaries, primordial follicle growth activation, follicle growth, and embryonic development were evaluated. Finally, the delivery of live pups after embryo transfer into recipient mice was assessed. While PPARγ was expressed in ovaries from mice of all ages, its levels were significantly increased in ovaries from 20-day-old mice. In GW9662-treated ovaries in vitro, PTEN levels were decreased, AKT was activated, and FOXO3a was excluded from the nuclei of primordial follicles. After 1 month, cPA-pretreated, transplanted ovaries produced the highest numbers of oocytes and polar bodies, exhibited the most advanced embryonic development, and had the greatest blastocyst formation rate compared to the rosiglitazone- and GW9662-pretreated groups. Additionally, the successful delivery of live pups after embryo transfer into the recipient mice transplanted with cPA-pretreated ovaries was confirmed. Our study demonstrates that PPARγ participates in primordial follicle activation and development, possibly mediated in part by the PI3K/AKT signaling pathway. Although more studies are required, adapting these findings for the activation of human primordial follicles may lead to treatments for infertility that originates from poor ovarian reserves.

Anti-apoptotic Regulation Contributes to the Successful Nuclear Reprogramming Using Cryopreserved Oocytes.

Cryopreservation has a negative effect on the quality of oocytes and may be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. The purpose of the present study was to evaluate the detrimental effects on the developmental competence of somatic cell nuclear transferred (SCNT) mouse embryos using vitrified (cryopreserved) oocytes and to evaluate the recovery effects of melatonin on cryo-damage in cloned embryos. Development of SCNT embryos using cryopreserved oocyte cytoplasm (SCNT-CROC) was inferior to those using fresh cytoplasm (SCNT-FOC). Using RNA-sequencing analysis, we found upregulation of eight pro-apoptotic-related genes (Cyct, Dapk2, Dffb, Gadd45g, Hint2, Mien1, P2rx7, and Pmaip) in the SCNT-CROC group. Furthermore, the addition of melatonin, an agent that reduces apoptosis and ROS production, enhanced blastocyst formation rates in the SCNT-CROP group when compared with the melatonin-untreated group. Additionally, melatonin treatment increased the derivation efficiency of pluripotent stem cells from cloned embryos using cryopreserved oocyte.

The effect of cell penetrating peptide-conjugated coactivator-associated arginine methyltransferase 1 (CPP-CARM1) on the cloned mouse embryonic development.

Abnormalities in gene expression that negatively affect embryonic development are frequently observed in cloned embryos generated by somatic cell nuclear transfer (SCNT). In the present study, we successfully produced a cell-penetrating peptide (CPP)-conjugated with coactivator-associated arginine methyltransferase 1 (CARM1) protein from mammalian cells and confirmed introduction into donor somatic cells and cloned 8-cell embryos within 3 hours after addition to culture medium. In addition, H3R17 dimethylation and embryonic development up to the blastocyst stage were increased in the group treated with exogenous CPP-CARM1 protein compared with the untreated group (control). Interestingly, the number of total cells and trophectoderm in blastocysts as well as implantation rate were significantly increased in the CPP-CARM1 protein-treated group. However, the cell number of inner cell mass (ICM) was not changed compared with the control group; similarly, expression of pluripotency-related genes Oct4 and Nanog (ICM markers) was not significantly different between groups. On the other hand, expression of the implantation-related gene Cdx2 (trophectoderm marker) was transiently increased after treatment with CPP-CARM1 protein. On the basis of these results, we conclude that supplementation with exogenous CPP-CARM1 protein improves embryonic development of cloned embryos through regulation of histone methylation and gene expression. In addition, our results suggest that CPP-CARM1 protein may be a useful tool for strengthening implantation of mammalian embryos.

In Vitro Derivation of Functional Sertoli-Like Cells from Mouse Embryonic Stem Cells.

Sertoli cells (SCs) in the mammalian testes are well known as supporting cells of spermatogenesis, but have recently become an attractive source of cell therapy because of their capacity for immune modulation and trophic effects. In order to increase their applicable efficacy, we demonstrate a novel differentiation method for mouse embryonic stem cell (ESC)-derived Sertoli-like cells (SLCs) via the intermediate mesoderm (IM). We show that IM derived from an induction of 6 days expressed markers such as Wt1, Lhx1, Pax2 and Osr1, and that a sequential induction of 6 days resulted in ESC-SLCs. The SLCs expressed their marker genes ( Sf1, Sox9, Gata4, Wt1, Fshr and Scf), but the pluripotency-marker gene Oct4 was decreased. After sorting by FSHR expression, high-purity (> 90%) SLCs were collected that showed distinct characteristics of SCs such as high phagocytic and immune modulation activities as well as the expression of immune-related genes. In addition, when transplanted into the seminiferous tubule of busulfan-treated mice, SLCs re-located and were maintained in the basal region of the tubule. These results demonstrated that our robust sequential differentiation system produced functional SLCs from mouse ESCs in vitro.

Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

The harmonized actions of ovarian E2 and progesterone (P4) regulate the proliferation and differentiation of uterine cells in a spatiotemporal manner. Imbalances between these hormones often lead to infertility and gynecologic diseases. Whereas numerous factors that are involved in P4 signaling have been identified, few local factors that mediate E2 actions in the uterus have been revealed. Here, we demonstrate that estrogen induces the transcription factor, early growth response 1 ( Egr1), to fine-tune its actions in uterine epithelial cells (ECs) that are responsible for uterine receptivity for embryo implantation. In the presence of exogenous gonadotrophins, ovulation, fertilization, and embryonic development normally occur in Egr1-/- mice, but these animals experience the complete failure of embryo implantation with reduced artificial decidualization. Although serum levels of E2 and P4 were comparable between Egr1+/+ and Egr1-/- mice on d 4 of pregnancy, aberrantly reduced levels of progesterone receptor in Egr1-/- uterine ECs caused enhanced E2 activity and impaired P4 response. Ultrastructural analyses revealed that Egr1-/- ECs are not fully able to provide proper uterine receptivity. Uterine mRNA landscapes in Egr1-/- mice revealed that EGR1 controls the expression of a subset of E2-regulated genes. In addition, P4 signaling was unable to modulate estrogen actions, including those that are involved in cell-cycle progression, in ECs that were deficient in EGR1. Furthermore, primary coculture of Egr1-/- ECs with Egr1+/+ stromal cells, and vice versa, supported the notion that Egr1 is required to modulate E2 actions on ECs to prepare the uterine environment for embryo implantation. In contrast to its role in ECs, loss of Egr1 in stroma significantly reduced stromal cell proliferation. Collectively, our results demonstrate that E2 induces EGR1 to streamline its actions for the preparation of uterine receptivity for embryo implantation in mice.-Kim, H.-R., Kim, Y. S., Yoon, J. A., Yang, S. C., Park, M., Seol, D.-W., Lyu, S. W., Jun, J. H., Lim, H. J., Lee, D. R., Song, H. Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

Maintained MPF Level after Oocyte Vitrification Improves Embryonic Development after IVF, but not after Somatic Cell Nuclear Transfer.

Levels of maturation-promoting factor (MPF) in oocytes decline after vitrification, and this decline has been suggested as one of the main causes of low developmental competence resulting from cryoinjury. Here, we evaluated MPF activity in vitrified mouse eggs following treatment with caffeine, a known stimulator of MPF activity, and/or the proteasome inhibitor MG132. Collected MII oocytes were vitrified and divided into four groups: untreated, 10 mM caffeine (CA), 10 µM MG132 (MG), and 10 mM caffeine +10 µM MG132 (CA+MG). After warming, the MPF activity of oocytes and their blastocyst formation and implantation rates in the CA, MG, and CA+MG groups were much higher than those in the untreated group. However, the cell numbers in blastocysts did not differ among groups. Analysis of the effectiveness of caffeine and MG132 for improving somatic cell nuclear transfer (SCNT) technology using cryopreserved eggs showed that supplementation did not improve the blastocyst formation rate of cloned mouse eggs. These results suggest that maintaining MPF activity after cryopreservation may have a positive effect on further embryonic development, but is unable to fully overcome cryoinjury. Thus, intrinsic factors governing the developmental potential that diminish during oocyte cryopreservation should be explored.

Expression of Egr3 in mouse gonads and its localization and function in oocytes.

OBJECTIVE: The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. METHODS: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (ΔEgr3) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. RESULTS: Egr3 mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5'untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-ΔEgr3-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-ΔEgr3-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes. CONCLUSION: The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice.

Supplementation With Cell-Penetrating Peptide-Conjugated Estrogen-Related Receptor ß Improves the Formation of the Inner Cell Mass and the Development of Vitrified/Warmed Mouse Embryos.

Estrogen-related receptor ß (ESRRB), which is a member of the nuclear orphan receptor family, regulates the messenger RNA (mRNA) expression levels of the transcription factors, Oct4 and Nanog, in early embryos and germ cells, thereby maintaining the undifferentiated state and pluripotency of the relevant cells. The present study was designed to determine whether the upregulation of pluripotency-related genes by direct delivery of ESRRB protein may affect on the commitment into inner cell mass (ICM) or the development of vitrified/warmed mouse embryos. Recombinant cell-penetrating peptide (CPP) ESRRB protein was synthesized and then added into a culture medium for cryopreserved mouse embryos. Vitrified/warmed 8-cell embryos were cultured in KSOM with/without 2 µg/mL CPP-ESRRB for 48 hours and then analyzed or transferred to the uteri of foster mothers. The mRNA expression of Oct4 and Nanog was higher in CPP-ESRRB-treated blastocysts compared to the untreated controls. No difference was observed in embryonic development, but ICM:trophectoderm ratio was increased in the CPP-ESRRB-treated group compared to the untreated group, and after embryo transfer, a higher implantation rate was obtained in the CPP-ESRRB-treated group compared to the untreated group. This study shows for the first time that recombinant CPP-ESRRB can be easily integrated into vitrified/warmed mouse embryos and that it increases Oct4 expression (via a pluripotency-related gene pathway), ICM formation, and the further embryonic and full-term development of vitrified/warmed mouse embryos. This CPP-conjugated protein delivery system could therefore be a useful tool for improving assisted reproductive technology.

Effect of cell-penetrating peptide-conjugated estrogen-related receptor ß on the development of mouse embryos cultured in vitro.

OBJECTIVE: Estrogen related receptor ß (Esrrb) is a member of the orphan nuclear receptors and may regulate the expression of pluripotency-related genes, such as Oct4 and Nanog. Therefore, in the present study, we have developed a method for delivering exogenous ESRRB recombinant protein into embryos by using cell-penetrating peptide (CPP) conjugation and have analyzed their effect on embryonic development. METHODS: Mouse oocytes and embryos were obtained from superovulated mice. The expression of Oct4 mRNA and the cell number of inner cell mass (ICM) in the in vitro-derived and in vivo-derived blastocysts were first analyzed by real time-reverse transcription-polymerase chain reaction and differential staining. Then 8-cell embryos were cultured in KSOM media with or without 2 µg/mL CPP-ESRRB protein for 24 to 48 hours, followed by checking their integration into embryos during in vitro culture by Western blot and immunocytochemistry. RESULTS: Expression of Oct4 and the cell number of ICM were lower in the in vitro-derived blastocysts than in the in vivo-derived ones (p<0.05). In the blastocysts derived from the CPP-ESRRB-treated group, expression of Oct4 was greater than in the non-treated groups (p<0.05). Although no difference in embryonic development was observed between the treated and non-treated groups, the cell number of ICM was greater in the CPP-ESRRB-treated group. CONCLUSION: Treatment of CPP-ESRRB during cultivation could increase embryos' expression of Oct4 and the formation rate of the ICM in the blastocyst. Additionally, an exogenous delivery system of CPP-conjugated protein would be a useful tool for improving embryo culture systems.

Regulation of Pluripotency-related Genes and Differentiation in Mouse Embryonic Stem Cells by Direct Delivery of Cell-penetrating Peptide-conjugated CARM1 Recombinant Protein.

Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.