Siyuan Xie: State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, Jiangsu Province, China.; School of Medicine and Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Yuyi Wang: School of Medicine and Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Chenxi Zhao: State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, Jiangsu Province, China.
Yugui Cui: State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, Jiangsu Province, China.
Chunyan Gu: School of Medicine and Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Wei Wu: State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, Jiangsu Province, China.. Electronic address: weiwu77@163.com.
RESEARCH QUESTION: Does putrescine (PUT) improve oocytes from reproductively old mice by promoting mitochondrial autophagy? DESIGN: Germinal vesicle stage cumulus-oocyte complexes (COCs) were obtained from 9-month old female C57BL/6N mice and divided into control, PUT and difluoromethylornithine, inhibitor (DFMO) groups. These germinal vesicle COCs underwent mouse in-vitro maturation (IVM) culture to observe the extrusion of the first polar body in each group. Using JC-1, dichloro-dihydro-fluorescein diacetate fluorescent probes and a confocal microscope, the mitochondrial membrane potential integrity and reactive oxygen species levels were measured in metaphase II stage oocytes. The expression and cellular localization of the p53 protein were examined by immunofluorescence. Reverse transcription quantitative polymerase chain reaction was used to detect the activation of mitochondrial autophagy pathways. The potential mechanisms through which PUT improves oocytes from reproductively old mice were explored by single-cell transcriptomic analysis. Autophagosomes, autolysosomes and mitochondria in different groups were directly observed using transmission electron microscopy. RESULTS: The addition of exogenous PUT can promote IVM of oocytes from reproductively old mice. It reduces oxidative stress by promoting the autophagy of damaged mitochondria, decreasing the levels of reactive oxygen species and increasing mitochondrial membrane potential. It affects the expression and subcellular localization of the p53 protein, and increases the expression of transcription factor EB, which may be the potential mechanism behind its promotion of autophagy. CONCLUSION: The target and regulatory pathway of PUT in oocytes was clarified. Putrescine is an effective small molecule compound with significant potential for non-invasively improving the fertility of elderly women.
