Hong-Cui Zhao1, Ting Ding2, Yun Ren1, Tian-Jie Li1, Rong Li2, Yong Fan3, Jie Yan4, Yue Zhao5, Mo Li2, Yang Yu6, Jie Qiao6. 1. Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China. 2. Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China. 3. Key Laboratory for Major Obstetric Diseases of Guangdong Province, the Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China. 4. Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China. 5. Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China. 6. Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China yuyang5012@hotmail.com jie.qiao@263.net.
Abstract
STUDY QUESTION: Does Sirt3 dysfunction result in poor developmental outcomes for human oocytes after in vitro maturation (IVM)? SUMMARY ANSWER: Inefficient Sirt3 expression induced decreased mitochondrial DNA copy number and biogenesis, and therefore impaired the developmental competence of human IVM oocytes. WHAT IS KNOWN ALREADY: Cytoplasmic immaturity in IVM oocytes may lead to reduced developmental competence. Mitochondrial dysfunction results in the accumulation of free radicals and leads to DNA mutations, protein damage, telomere shortening and apoptosis. SIRT3 (in the Sirtuin protein family) has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species scavenging proteins. STUDY DESIGN, SIZE, DURATION: In vivo matured metaphase II (IVO-MII) oocytes and IVM-MII oocytes were donated by 324 infertile patients undergoing assisted reproductive technology cycles (12 patients for 60 IVO oocytes, and 312 patients for 403 IVM oocytes). Five oocytes each in the germinal vesicle (GV), IVM and IVO groups were compared with respect to mRNA levels for Sirt1-7 mRNA, and five samples at each developmental stage were analysed for Sirt3 mRNA. IVM-MII oocytes were injected with in vitro transcribed mRNA (n = 59) or small interfering RNA (siRNA) (n = 78). In human and mouse, IVM, mRNA-injection IVM, and siRNA-injection IVM groups (n = 5 each) were analysed for mitochondrial DNA copy number and abundance of Sirt3 and Pgc1α (an inducer of mitochondrial biogenesis) mRNAs. Human blastocysts in the IVO (n = 12), IVM (n = 9), mRNA-injection IVM (n = 13) and siRNA-injection IVM (n = 6) groups were used to generate embryonic stem cells (ESCs). In addition, 587 IVO-MII and 1737 IVM-MII oocytes from 83 mice were collected to compare the preliminary human oocyte data with another species. PARTICIPANTS/MATERIALS, SETTING, METHODS: mRNA abundance was analysed by single-cell real-time PCR. Karyotyping of human embryos was performed with an array comparative genomic hybridization method, and that of ESCs by cytogenetic analysis. The function of the Sirt3 gene was investigated using siRNA and in vitro transcribed mRNA injection. Markers of ESCs were identified using immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: A retrospective analysis revealed a higher spontaneous abortion rate (P < 0.01) and decrease in high quality embryo rate (P < 0.01) in patients with IVM versus controlled ovarian stimulation (COS) cycles. A decrease in abundance of Sirt3 mRNA (P < 0.01) and mitochondrial biogenesis (P < 0.05) were identified in human IVM compared with IVO oocytes. The developmental potential of human IVM-MII oocytes to the blastocyst stage was significantly reduced when Sirt3 mRNA was inhibited by siRNA (P < 0.05 versus IVM-MII group) but could be up-regulated by injection of Sirt3 mRNAs. Compared with IVO-MII group, comparable generation efficiency of human ESCs can be obtained using blastocysts from IVM-MII oocytes with Sirt3 mRNA injection. Sirt3 mRNA was significantly increased in mouse zygotes after IVF (P < 0.001 versus MII oocytes) but gradually declined until the blastocyst stage. In mice, lower Sirt3 mRNA levels were observed IVM-MII oocytes and preimplantation embryos compared with in vivo controls, and mitochondrial biogenesis and the developmental efficiency from oocytes to blastocyst were affected by the abundance of Sirt3 mRNA in accordance with human. Therefore a similar role for Sirt3 mRNA in IVM-MII oocytes was observed in mouse and human. LIMITATIONS, REASONS FOR CAUTION: The couples in the study had a variety of different simple and complex factors causing infertility. Additional studies with a larger number of oocytes are required to confirm the present results owing to the limited number of human oocytes in the present study. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study investigating a role of the Sirt3 gene in mitochondrial biogenesis and the developmental competence of human IVM-MII oocytes. The observation may help to improve clinical application of the IVM procedure. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by the National Natural Science Foundation of Key Program (31230047), Ministry of Science and Technology of China Grants (973 program; 2014CB943203), the National Natural Science Foundation of General Program (31371521 and 81571400), Beijing Nova Program (xxjh2015011), and Specialized Research Fund for the Doctoral Program of Higher Education (20120001130008) and the National Natural Science Foundation of Young Scholar (31501201). The authors have declared that no conflict of interest exists.
STUDY QUESTION: Does Sirt3 dysfunction result in poor developmental outcomes for human oocytes after in vitro maturation (IVM)? SUMMARY ANSWER: Inefficient Sirt3 expression induced decreased mitochondrial DNA copy number and biogenesis, and therefore impaired the developmental competence of human IVM oocytes. WHAT IS KNOWN ALREADY: Cytoplasmic immaturity in IVM oocytes may lead to reduced developmental competence. Mitochondrial dysfunction results in the accumulation of free radicals and leads to DNA mutations, protein damage, telomere shortening and apoptosis. SIRT3 (in the Sirtuin protein family) has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species scavenging proteins. STUDY DESIGN, SIZE, DURATION: In vivo matured metaphase II (IVO-MII) oocytes and IVM-MII oocytes were donated by 324 infertilepatients undergoing assisted reproductive technology cycles (12 patients for 60 IVO oocytes, and 312 patients for 403 IVM oocytes). Five oocytes each in the germinal vesicle (GV), IVM and IVO groups were compared with respect to mRNA levels for Sirt1-7 mRNA, and five samples at each developmental stage were analysed for Sirt3 mRNA. IVM-MII oocytes were injected with in vitro transcribed mRNA (n = 59) or small interfering RNA (siRNA) (n = 78). In human and mouse, IVM, mRNA-injection IVM, and siRNA-injection IVM groups (n = 5 each) were analysed for mitochondrial DNA copy number and abundance of Sirt3 and Pgc1α (an inducer of mitochondrial biogenesis) mRNAs. Humanblastocysts in the IVO (n = 12), IVM (n = 9), mRNA-injection IVM (n = 13) and siRNA-injection IVM (n = 6) groups were used to generate embryonic stem cells (ESCs). In addition, 587 IVO-MII and 1737 IVM-MII oocytes from 83 mice were collected to compare the preliminary human oocyte data with another species. PARTICIPANTS/MATERIALS, SETTING, METHODS: mRNA abundance was analysed by single-cell real-time PCR. Karyotyping of human embryos was performed with an array comparative genomic hybridization method, and that of ESCs by cytogenetic analysis. The function of the Sirt3 gene was investigated using siRNA and in vitro transcribed mRNA injection. Markers of ESCs were identified using immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: A retrospective analysis revealed a higher spontaneous abortion rate (P < 0.01) and decrease in high quality embryo rate (P < 0.01) in patients with IVM versus controlled ovarian stimulation (COS) cycles. A decrease in abundance of Sirt3 mRNA (P < 0.01) and mitochondrial biogenesis (P < 0.05) were identified in human IVM compared with IVO oocytes. The developmental potential of human IVM-MII oocytes to the blastocyst stage was significantly reduced when Sirt3 mRNA was inhibited by siRNA (P < 0.05 versus IVM-MII group) but could be up-regulated by injection of Sirt3 mRNAs. Compared with IVO-MII group, comparable generation efficiency of human ESCs can be obtained using blastocysts from IVM-MII oocytes with Sirt3 mRNA injection. Sirt3 mRNA was significantly increased in mouse zygotes after IVF (P < 0.001 versus MII oocytes) but gradually declined until the blastocyst stage. In mice, lower Sirt3 mRNA levels were observed IVM-MII oocytes and preimplantation embryos compared with in vivo controls, and mitochondrial biogenesis and the developmental efficiency from oocytes to blastocyst were affected by the abundance of Sirt3 mRNA in accordance with human. Therefore a similar role for Sirt3 mRNA in IVM-MII oocytes was observed in mouse and human. LIMITATIONS, REASONS FOR CAUTION: The couples in the study had a variety of different simple and complex factors causing infertility. Additional studies with a larger number of oocytes are required to confirm the present results owing to the limited number of human oocytes in the present study. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study investigating a role of the Sirt3 gene in mitochondrial biogenesis and the developmental competence of human IVM-MII oocytes. The observation may help to improve clinical application of the IVM procedure. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by the National Natural Science Foundation of Key Program (31230047), Ministry of Science and Technology of China Grants (973 program; 2014CB943203), the National Natural Science Foundation of General Program (31371521 and 81571400), Beijing Nova Program (xxjh2015011), and Specialized Research Fund for the Doctoral Program of Higher Education (20120001130008) and the National Natural Science Foundation of Young Scholar (31501201). The authors have declared that no conflict of interest exists.