ABSTRACT
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.
Subject(s)
Blastocyst/drug effects , Blastocyst/metabolism , Cell-Penetrating Peptides/administration & dosage , Embryonic Development , Estrogens, Conjugated (USP)/administration & dosage , Receptors, Estrogen/administration & dosage , Animals , Cell Count , Culture Media , Embryo Implantation/drug effects , Embryo Transfer , Female , Gene Expression Regulation, Developmental , Male , Mice , Nanog Homeobox Protein/metabolism , Octamer Transcription Factor-3/metabolism , RNA, Messenger/metabolism , VitrificationABSTRACT
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.
