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Mitochondrial cell-free DNA secreted from porcine granulosa cells

Published online by Cambridge University Press:  14 August 2019

Kazuki Kansaku
Affiliation:
Department of Animal Sciences, Tokyo University of Agriculture, Funako 1737, Kanagawa 243-0034, Japan
Yasuhisa Munakata
Affiliation:
Department of Animal Sciences, Tokyo University of Agriculture, Funako 1737, Kanagawa 243-0034, Japan
Koumei Shirasuna
Affiliation:
Department of Animal Sciences, Tokyo University of Agriculture, Funako 1737, Kanagawa 243-0034, Japan
Takehito Kuwayama
Affiliation:
Department of Animal Sciences, Tokyo University of Agriculture, Funako 1737, Kanagawa 243-0034, Japan
Hisataka Iwata*
Affiliation:
Department of Animal Sciences, Tokyo University of Agriculture, Funako 1737, Kanagawa 243-0034, Japan
*
Address for correspondence: Hisataka Iwata. Professor, Department of Animal Science, Tokyo University of Agriculture. Funako 1737, Kanagawa 243–0034, Japan, E-mail: h1iwata@nodai.ac.jp

Summary

Several studies have proposed that cell-free DNA (cfDNA) is a potential biomarker present in follicular fluid (FF) for oocyte quality. Recently we reported that mitochondria-derived cfDNA (mt-cfDNA) closely reflects the amount of cfDNA in FFs. The present study investigated the mechanism regulating mt-cfDNA secretion from porcine granulosa cells. Oocytes and cumulus cell complexes or granulosa cells (GCs) were cultured in maturation medium for 24 or 48 h respectively. Then, nuclear-derived cell-free DNA (n-cfDNA) or mt-cfDNA contents in the spent medium were examined using real-time polymerase chain reaction. When 10 μM of MG132, a proteasome inhibitor, was added to the culture medium, cellular viability of both COCs and GCs decreased and n-cfDNA significantly increased in the culture medium, whereas mt-cfDNA significantly decreased. Supplementation of the culture medium with GW4869, an inhibitor of intracellular vesicle formation, significantly decreased the mt-cfDNA, whereas no effect was observed on n-cfDNA in the medium of both COCs and GCs. Furthermore, the addition of bafilomycin, an inhibitor of autophagy to the culture medium significantly increased mt-cfDNA in the culture medium. After filtration (0.22 μm) and centrifugation (23,000 g), the mt-cfDNA content of the medium decreased significantly. In conclusion, the proteasomal mitochondrial quality control system is upstream of mt-cfDNA secretion and autophagy plays a role in cellular digestion of mitochondrial DNA in the cytoplasm. It is further suggested that dsDNA is enclosed in certain vesicles or associated with small molecules and secreted into the medium.

Type
Research Article
Copyright
© Cambridge University Press 2019 

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Footnotes

*

These authors contributed equally.

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