Yongan Xin1, Yifei Jin2, Juan Ge1, Zhenyue Huang1, Longsen Han1, Congyang Li1, Danni Wang1, Shuai Zhu1, Qiang Wang1,3. 1. State Key Laboratory of Reproductive Medicine, Suzhou Municipal Hospital, Nanjing Medical University, Nanjing, China. 2. School of Nursing, Nanjing Medical University, Nanjing, China. 3. Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China.
Abstract
OBJECTIVES: It has been widely reported that maternal diabetes impairs oocyte quality. However, the responsible mechanisms remain to be explored. In the present study, we focused on whether SIRT3-GSK3β pathway mediates the meiotic defects in oocytes from diabetic mice. MATERIALS AND METHODS: GSK3β functions in mouse oocyte meiosis were first detected by targeted siRNA knockdown. Spindle assembly and chromosome alignment were visualized by immunostaining and analysed under the confocal microscope. PCR-based site mutation of specific GSK3β lysine residues was used to confirm which lysine residues function in oocyte meiosis. siRNA knockdown coupled with cRNA overexpression was performed to detect SIRT3-GSK3β pathway functions in oocyte meiosis. Immunofluorescence was performed to detect ROS levels. T1DM mouse models were induced by a single intraperitoneal injection of streptozotocin. RESULTS: In the present study, we found that specific depletion of GSK3β disrupts maturational progression and meiotic apparatus in mouse oocytes. By constructing site-specific mutants, we further revealed that acetylation state of lysine (K) 15 on GSK3β is essential for spindle assembly and chromosome alignment during oocyte meiosis. Moreover, non-acetylation-mimetic mutant GSK3β-K15R is capable of partly preventing the spindle/chromosome anomalies in oocytes with SIRT3 knockdown. A significant reduction in SIRT3 protein was detected in oocytes from diabetic mice. Of note, forced expression of GSK3β-K15R ameliorates maternal diabetes-associated meiotic defects in mouse oocytes, with no evident effects on oxidative stress. CONCLUSION: Our data identify GSK3β as a cytoskeletal regulator that is required for the assembly of meiotic apparatus, and discover a beneficial effect of SIRT3-dependent GSK3β deacetylation on oocyte quality from diabetic mice.
OBJECTIVES: It has been widely reported that maternal diabetes impairs oocyte quality. However, the responsible mechanisms remain to be explored. In the present study, we focused on whether SIRT3-GSK3β pathway mediates the meiotic defects in oocytes from diabeticmice. MATERIALS AND METHODS:GSK3β functions in mouse oocyte meiosis were first detected by targeted siRNA knockdown. Spindle assembly and chromosome alignment were visualized by immunostaining and analysed under the confocal microscope. PCR-based site mutation of specific GSK3β lysine residues was used to confirm which lysine residues function in oocyte meiosis. siRNA knockdown coupled with cRNA overexpression was performed to detect SIRT3-GSK3β pathway functions in oocyte meiosis. Immunofluorescence was performed to detect ROS levels. T1DM mouse models were induced by a single intraperitoneal injection of streptozotocin. RESULTS: In the present study, we found that specific depletion of GSK3β disrupts maturational progression and meiotic apparatus in mouse oocytes. By constructing site-specific mutants, we further revealed that acetylation state of lysine (K) 15 on GSK3β is essential for spindle assembly and chromosome alignment during oocyte meiosis. Moreover, non-acetylation-mimetic mutant GSK3β-K15R is capable of partly preventing the spindle/chromosome anomalies in oocytes with SIRT3 knockdown. A significant reduction in SIRT3 protein was detected in oocytes from diabeticmice. Of note, forced expression of GSK3β-K15R ameliorates maternal diabetes-associated meiotic defects in mouse oocytes, with no evident effects on oxidative stress. CONCLUSION: Our data identify GSK3β as a cytoskeletal regulator that is required for the assembly of meiotic apparatus, and discover a beneficial effect of SIRT3-dependent GSK3β deacetylation on oocyte quality from diabeticmice.
Authors: Juan Ge; Na Zhang; Shoubin Tang; Feifei Hu; Xiaojing Hou; Hongzheng Sun; Longsen Han; Qiang Wang Journal: Front Cell Dev Biol Date: 2021-12-20
Authors: Giovanna Di Emidio; Stefano Falone; Paolo Giovanni Artini; Fernanda Amicarelli; Anna Maria D'Alessandro; Carla Tatone Journal: Antioxidants (Basel) Date: 2021-06-29