Effect of cell-penetrating peptide-conjugated estrogen-related receptor β on the development of mouse embryos culturedin vitro

Abstract

ObjectiveEstrogen related receptor β (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.MethodsMouse 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.ResultsExpression 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.ConclusionTreatment 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.

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

“…This concentration was previously determined in experiments testing the efficacy of the protein on unfrozen mouse embryos. 27 Our results revealed that CPP-ESRRB penetrated into both the cytoplasm and the nuclei of embryos by 24 hours posttreatment and that this protein was maintained by 48 hours (Figure 3A). Furthermore, the total ESRRB protein level was much higher in blastocysts of the CPP-ESRRB-treated group compared to the untreated group (Figure 3B).…”

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

“…Support for this strategy is provided by our previous report showing that CPP-conjugated ESRRB, a member of the orphan nuclear receptor families, improved the developmental potential of cryopreserved mouse embryos and increased expression of the transcription factors Oct4 and Nanog 25 . In addition, supplementation of culture medium with CPP-ESRRB protein has been shown to enhance the function of ICM in fresh embryos increasing Oct4 gene expression 24 . Consistent with these observations, we here found that treatment of embryos with CARM1 protein improved the TE development and enhanced the quality of cloned embryos through epigenetic modification.…”

“…Immunofluorescence staining was as described previously 24 with slight modifications. Briefly, the zona-pellucida of the embryo was removed using acidic Tyrode’s solution (Millipore).…”

“…cDNA was synthesized from 1 μg of total RNA using a TOPscript cDNA synthesis Kit (Enzynomics, Seoul, Korea) according to the manufacturer’s instructions. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses of cDNA samples were performed using a CFX98 instrument (Bio-Rad) according to a previously published report 24 with slight modifications. Briefly, all cDNA samples were analyzed by qRT-PCR using primers designed to amplify H2A histone family member Z (H2afz) to test for variations in the expression of this internal control gene.…”

“…Lim and colleagues also identified a new form of CPP, obtained from human papillomavirus L1 capsid protein (LDP12), and confirmed its ability to deliver a fusion protein of enhanced green fluorescence protein and MAP1LC3 (EGFP-LC3) into mouse blastocysts 23 . In our previously reports, the cell number of inner cell mass (ICM) and expression of the Oct4 gene were positively regulated in fresh and cryopreserved embryos after treatment with CPP-conjugated estrogen-related receptor β (CPP-ESRRB) during in vitro cultivation 24 , 25 . In the present study, we provide the first demonstration that exogenous supplementation with a novel CPP-conjugated CARM1 improves the normally poor embryonic development of cloned mouse embryos through regulation of gene expression.…”