Zhichao Li1, Ruihuan Gu1, Xiaowei Lu2, Shen Zhao2, Yun Feng3, Yijuan Sun4. 1. Shanghai Ji Ai Genetics & IVF Institute, Obstetrics & Gynecology Hospital, Fudan University, Shanghai, 200011, People's Republic of China. 2. Reproductive Medical Center, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, People's Republic of China. 3. Reproductive Medical Center, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, People's Republic of China. artruijin1@163.com. 4. Shanghai Ji Ai Genetics & IVF Institute, Obstetrics & Gynecology Hospital, Fudan University, Shanghai, 200011, People's Republic of China. sunyijuansub@126.com.
Abstract
PURPOSE: Oocyte vitrification is currently used for human fertility preservation. However, vitrification damage is a problem caused by decreasing ooplasmic levels of glutathione (GSH). The GSH donor glutathione ethyl ester (GSH-OEt) can significantly increase the GSH content in oocytes. However, it is difficult to obtain oocyte from woman. To overcome this, we used mouse oocytes to replace human oocytes as a model of study. METHODS: Oocytes from B6D2F1 mice were preincubated for 30 min with 2.5 mmol/L GSH-OEt (GSH-OEt group), without GSH-OEt preincubation before vitrification (control vitrification group) or in nonvitrified oocytes (fresh group). After thawing, oocytes were fertilized for evaluating the developmental competence of embryos in vitro and in vivo. Immunofluorescence, Polscope equipment and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to analyze damage, including mitochondrial distribution, reactive oxygen species (ROS) levels, spindle morphology, and gene expression levels (Bcl-2, BAX, and MnSOD). RESULTS: The rates of fertilization, 3-4 cell, blastocyst formation and expanded blastocysts were significantly higher (p < 0.05) in the GSH-OEt group (90.4%; 91.1%; 88.9% and 63.0%) than in the control (80.0%; 81.4%; 77.7% and 50.5%). Provided embryos overcame the 2-cell block and developed to the blastocyst stage, birth rates of all groups were similar. Vitrification altered mitochondrial distribution, increased ROS levels, and caused abnormal spindle morphology; GSH-OEt preincubation could improve such damage. RT-qPCR showed that the expression of Bcl-2 was lower in the control group compared with the GSH-OEt group; BAX and MnSoD expression levels were higher in the control group than in the GSH-OEt group (p < 0.05). CONCLUSIONS: The beneficial effect of GSH-OEt preincubation occurred before the 2-cell stage.
PURPOSE: Oocyte vitrification is currently used for human fertility preservation. However, vitrification damage is a problem caused by decreasing ooplasmic levels of glutathione (GSH). The GSHdonorglutathione ethyl ester (GSH-OEt) can significantly increase the GSH content in oocytes. However, it is difficult to obtain oocyte from woman. To overcome this, we used mouse oocytes to replace human oocytes as a model of study. METHODS: Oocytes from B6D2F1 mice were preincubated for 30 min with 2.5 mmol/L GSH-OEt (GSH-OEt group), without GSH-OEt preincubation before vitrification (control vitrification group) or in nonvitrified oocytes (fresh group). After thawing, oocytes were fertilized for evaluating the developmental competence of embryos in vitro and in vivo. Immunofluorescence, Polscope equipment and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to analyze damage, including mitochondrial distribution, reactive oxygen species (ROS) levels, spindle morphology, and gene expression levels (Bcl-2, BAX, and MnSOD). RESULTS: The rates of fertilization, 3-4 cell, blastocyst formation and expanded blastocysts were significantly higher (p < 0.05) in the GSH-OEt group (90.4%; 91.1%; 88.9% and 63.0%) than in the control (80.0%; 81.4%; 77.7% and 50.5%). Provided embryos overcame the 2-cell block and developed to the blastocyst stage, birth rates of all groups were similar. Vitrification altered mitochondrial distribution, increased ROS levels, and caused abnormal spindle morphology; GSH-OEt preincubation could improve such damage. RT-qPCR showed that the expression of Bcl-2 was lower in the control group compared with the GSH-OEt group; BAX and MnSoD expression levels were higher in the control group than in the GSH-OEt group (p < 0.05). CONCLUSIONS: The beneficial effect of GSH-OEt preincubation occurred before the 2-cell stage.
Authors: Rocío Nuñez-Calonge; Susana Cortés; Luis Miguel Gutierrez Gonzalez; Roman Kireev; Elena Vara; Leonor Ortega; Pedro Caballero; Lisa Rancan; Jesús Tresguerres Journal: Reprod Biomed Online Date: 2016-01-14 Impact factor: 3.828