Regulatory effect of apoptosis on morphological changes in cell mass of porcine blastocyst through supplementation of rapamycin during in vitro culture

Dae Kim,Sang Kim,Jong Yoon

doi:10.5455/javar.2020.g459

Dae Kim, Sang Kim + Show 1 more

Open Access

https://doi.org/10.5455/javar.2020.g459

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Abstract

Objective:The study of an in vitro embryosis is crucial in genetics for breed improvement and reproduction in livestock, identifying the causes of infertility, and stem cell application. Meanwhile, the problem of nucleic acid denaturation observed during embryo development is yet to be resolved. This study was set out to analyze the nucleic acid denaturation during the development of in vitro embryos.Materials and Methods:Using an in-vitro fertilization-embryo in porcine, the cell development and apoptosis were evaluated by adding rapamycin by concentration to the TCM-199 containing 10% FBS or 10% porcine follicle fluid (pFF). Real-time PCR, zymography, DNA fragment, Western blot, and immunofluorescence analysis were also carried out to determine the development rate of inner cell mass in the in-vitro fertilization-embryo.Results:The findings indicated that the addition of rapamycin to the 10% pFF group during in vitro maturation led to an increase in the rates of cleavage and blastocyst development and the expression of active matrix metallopeptidase (MMP-9), while nucleic acid denaturation was suppressed. In other words, the addition of rapamycin was found to increase the expression of MMP-2 in the inner cell mass and trophoblast, while it inhibited apoptosis.Conclusion:The addition of rapamycin influences the regulation of apoptosis and MMPs, and based on this, it is presumed to have a positive effect on blastocyst development.

Highlights

Porcine oocytes require a more extended period for in-vitro maturation than other livestock animals, and the easy separation of the cumulus and oocytes, due to the small number of cells connected through microvilli during in-vitro maturation, leading to a fall in gap junctions that obstruct metabolic activities [1]
The findings indicated that the addition of rapamycin to the 10% porcine follicle fluid (pFF) group during in vitro maturation led to an increase in the rates of cleavage and blastocyst development and the expression of active matrix metallopeptidase (MMP-9), while nucleic acid denaturation was suppressed
During the process of active growth of the ovarian follicle, changes occur to the follicle in the components and structures of the extracellular matrix (ECM), collagen, laminin, and fibronectin, which lead to oocyte maturation [16] and drive the primordial oocyte to grow into a mature oocyte based on the reformation of such structures

Summary

Introduction

Porcine oocytes require a more extended period for in-vitro maturation than other livestock animals, and the easy separation of the cumulus and oocytes, due to the small number of cells connected through microvilli during in-vitro maturation, leading to a fall in gap junctions that obstruct metabolic activities [1]. A disturbance is caused in glutathione synthesis in the ooplasm upon maturation [2], leading to incomplete oocyte maturation and a reduced rate of fertilization due to an increased rate of multiple sperm penetration, sperm head swelling, and incomplete pronuclear formation [3]. These are known to reduce the effect of BSA on porcine oocyte maturation and cumulus growth, compared to FBS, during the maturation and culture of oocytes [4].

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