Prevention of quality decline and delivery of siRNA using exogenous TCTP translocation across the zona pellucida in mouse oocytes

Hyuk-Joon Jeon,Jeong Su Oh,Jae-Sung Kim,Guang-Yu Bai,Yuram Park

doi:10.1038/s41598-019-55449-4

Abstract

The delivery of exogenous molecules into mammalian oocytes or embryos has been a challenge because of the existence of the protective zona pellucida (ZP) surrounding the oocyte membrane. Here we show that exogenous translationally controlled tumor protein (TCTP) is able to translocate into oocytes across the ZP and prevents quality deterioration during in vitro culture. Recombinant TCTP-mCherry added to culture media were incorporated into oocytes after passing through the ZP. After internalization, recombinant TCTP-mCherry were enriched at the cortex with wide distribution within the cytoplasm. This translocation capacity of TCTP is dependent on its N-terminal protein transduction domain (PTD). Moreover, translocated recombinant TCTP-mCherry reduced quality deterioration of oocytes during prolonged in vitro culture, which in turn improved fertilization and early embryo development. Furthermore, conjugates between PTD of TCTP and cyclin B1 siRNAs internalized into the cytoplasm of oocytes and downregulated cyclin B1 level. Therefore, our results are the first to show that TCTP has the ability to translocate into oocyte cytoplasm penetrating through the ZP, providing the possibility for preserving oocyte quality during extended in vitro culture and for delivering siRNAs into mouse oocytes.

Highlights

Mammalian oocytes are arrested at the metaphase of the second meiosis (MII) until fertilization
Because the translationally controlled tumor protein (TCTP)-mCherry signal was localized to oocyte surfaces with enrichment at the polar body, it was of interest to determine whether TCTP was internalized into the cytoplasm of oocytes or if it coated oocyte surfaces after penetrating the zona pellucida (ZP)
We found that the fluorescence signal remained detectable at the surface of oocytes treated with TCTP-mCherry proteins, while no signal was detected in control oocytes treated with mCherry proteins (Fig. 1D)

Summary

Introduction

Mammalian oocytes are arrested at the metaphase of the second meiosis (MII) until fertilization. We previously found that translationally controlled tumor protein (TCTP) has properties to relieve deterioration in oocyte quality and delay the onset of apoptosis during in vitro culture[4]. This suggested that TCTP could be a promising approach to decrease quality decline in oocytes cultured for a prolonged time during ART procedures. In this study, we tested whether TCTP can penetrate the ZP of oocytes and prevent deterioration of quality during in vitro culture. We found that exogenous TCTP is translocated into the cytoplasm of oocytes across the ZP during in vitro culture and prevents time-dependent decline in oocyte quality, which in turn improves fertilization capacity and subsequent embryo development. Our results demonstrate that non-invasive delivery of recombinant TCTP could be used as an anti-aging factor to prevent deterioration in oocyte quality during in vitro culture and a novel delivery system for siRNAs into oocytes

Methods

Results

Conclusion