Enhancing Oocyte Competence With Milrinone as a Phosphodiesterase 3A Inhibitor to Improve the Development of Porcine Cloned Embryos.

Pantu Kumar Roy,Ghangyong Kim,Xun Fang,Seonggyu Bang,Bereket Molla Tanga,Ahmad Yar Qamar,Jongki Cho

doi:10.3389/fcell.2021.647616

Abstract

The objective of this study was to investigate the effect of milrinone supplementation as a phosphodiesterase 3A inhibitor during in vitro maturation (IVM) to coordinate the cytoplasmic and nuclear maturation of porcine oocytes and subsequent development of porcine cloned embryos. Brilliant cresyl blue (BCB)-stained (BCB +) oocytes, classified as well-developed, and BCB− oocytes were used in parthenogenesis (PA) and cloning, and their preimplantation development was compared. In PA embryos, BCB + oocytes had significantly higher rates of development than BCB− oocytes in terms of maturation (87.5 vs. 71.3%), cleavage (88.6 vs. 76.3%), and blastocyst development (34.3 vs. 25.3%) and also had higher cell numbers (46.9 vs. 38.9%), respectively (p < 0.05). In cloned embryos, the BCB + group also had a significantly higher blastocyst formation rate than the BCB− group (30.6 vs. 20.1%; p < 0.05). Supplementation with 75 μM milrinone during IVM of BCB− oocytes showed improvement in maturation and blastocyst development rates, which may be due to the coordinated maturation of the cytoplasm with the nucleus as an effect of milrinone. Moreover, the analysis of nuclear reprogramming via the examination of the expression levels of the reprogramming-related genes POU5F1, DPPA2, and NDP52IL in milrinone-supplemented BCB− oocytes showed higher expression levels than that in non-treated BCB− oocytes. These findings demonstrate that milrinone is useful in improving developmental competence in less competent oocytes during IVM and for proper nuclear reprogramming in the production of porcine cloned embryos by coordinating cytoplasmic and nucleus maturation.

Highlights

In vitro production (IVP) of embryos has low efficiency, for cloned embryos, which is mainly attributed to poor competence of oocytes during in vitro maturation (IVM) (Manjunatha et al, 2007)
We found that cytoplasmic maturation along with nuclear maturation is a critical factor in determining the developmental competence of oocytes
Most of the Brilliant cresyl blue (BCB) + oocytes showed significantly higher developmental competence at the IVM level regarding embryo development and expression of genes related to nuclear reprogramming in cloned embryos

Summary

Introduction

In vitro production (IVP) of embryos has low efficiency, for cloned embryos, which is mainly attributed to poor competence of oocytes during in vitro maturation (IVM) (Manjunatha et al, 2007). The importance of selecting competent oocytes that have high development rates and can result in the successful establishment of pregnancy has been reported in previous studies (Mehlmann et al, 2002; Gilchrist, 2011). Many factors, such as limitations in culturing. Enhancing Porcine Oocyte Competence With Milrinone conditions and the stage of the donor cell cycle, have been identified as affecting the efficiency of embryo production, the ability to attain competent oocytes in IVM remains the main obstacle (Hunter, 2000). Developmental competence of an IVM oocyte refers to the coordinated maturation of the cytoplasm and nucleus in a controlled manner, attaining the full development of the oocyte, similar to that in the in vivo state, which supports the success of subsequent fertilization and embryo development (Nogueira et al, 2006)

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