[Objective] This study aimed to improve the in vitro maturation quality of denuded porcine oocytes and provide scientific basis for establishing a stable and efficient denuded oocyte culture system. [Method] The first...[Objective] This study aimed to improve the in vitro maturation quality of denuded porcine oocytes and provide scientific basis for establishing a stable and efficient denuded oocyte culture system. [Method] The first polar body extrusion rate, oocyte glutathione （GSH） content, positive rate of brilliant cresyl blue （BCB） staining and development potential of activated oocytes or fertilized oocytes were employed as main indicators to investigate the effects of follicular mural granulosa cell （MGC） coculture on cytoplasmic maturation of cumulus cell-removal oocytes （Denuded Oocyte, DO）. [Result] According to in vitro maturation results, compared with DO group, the first polar body extrusion rate of porcine oocytes in DO＋MGC group was not significantly different, but the nuclear maturation process was improved and was more similar to that in COC （cumulus-oocyte complex） group. Detection of GSH content in mature oocytes showed that there was no significant difference between DO＋ MGC group （optical density of 1 053.67） and COC group （optical density of 1 426.00） or between DO＋MGC group and COC＋GC group （optical density of 1 541.00）, however, GSH content in mature oocytes of DO group （optical density of 724.67） was significantly lower than that of COC group and COC＋GC group （P0.05）. Detection of glucose-6-phosphate dehydrogenase （G6PDH） activity showed that there was no significant difference in BCB positive oocyte rate between DO ＋MGC group （88.26% ） and COC group （92.75%） or between DO＋MGC group and DO group （82.86% ）, however, BCB positive oocyte rate of DO group was significantly lower than that of COC group （P0.05）. Furthermore, the cleavage rate and blastocyst rate of activated mature oocytes derived from DO ＋MGC group （94.98% and 43.67% , respectively） were significantly higher than those from DO group （52.54% and 8.97%, respectively） （P0.05）, and were not significantly different compared with those from COC group （97.11% and 38.30%, respectively）. In addition, the cleavage rate of fertilized oocytes derived from DO＋MGC group （72.65%） showed no significant difference compared with that from DO group （63.59%）, but the blastocyst rate of DO＋MGC group was significantly higher than that of DO group （9.88%） （P0.05）. [Conclusion] MGC coculture can significantly improve the in vitro cytoplasmic maturation quality of denuded porcine oocytes, thereby enhancing the subsequent developmental potential.展开更多
[Objective] This study aimed to investigate the appropriate concentrations of follicle-stimulating hormone（FSH）, luteotropic hormone（LH） and estrodiol（E2） during in vitro maturation of Tan sheep oocytes. [Method...[Objective] This study aimed to investigate the appropriate concentrations of follicle-stimulating hormone（FSH）, luteotropic hormone（LH） and estrodiol（E2） during in vitro maturation of Tan sheep oocytes. [Method] Tan sheep oocytes were divided into five groups for in vitro maturation culture： control group, FSH group（10,50, 100, 200 and 300 μg/ml FSH, respectively）, LH group（5, 10, 20, 50 and 100μg/ml LH, respectively）, E2group（5, 10, 25, 50 and 100 μg/ml E2, respectively）, and FSH ＋ LH group（100 μg/ml FSH ＋ 20 μg/ml LH）. The releasing rate of first polar bodies was analyzed. [Result] The maturation rate of Tan sheep oocytes in 100 μg/ml FSH ＋ 20 μg/ml LH group reached the highest（64.64%）, which was significantly higher than that in other four groups（P〈0.05）; among different FSH concentrations,100 μg/ml FSH was superior to other four concentrations and the control group, exhibiting significant differences（P〈0.05）; among different LH concentrations, 20 μg/ml LH was superior to other four concentrations and the control group, exhibiting significant differences（P〈0.05）; among different E2 concentrations, 50 μg/ml E2 was superior to other four concentrations and the control group, exhibiting significant differences（P〈0.05）. [Conclusion] Under the experimental conditions, 100 μg/ml FSH ＋20 μg/ml LH was the most appropriate hormone combination for in vitro maturation of Tan sheep oocytes.展开更多
The chronological and morphological changes of the nucleus during mouse oocyte maturation and fertilization were systematically studied. Although most oocytes went through GVBD 2-4 hrs after culture, 13.6% remained at...The chronological and morphological changes of the nucleus during mouse oocyte maturation and fertilization were systematically studied. Although most oocytes went through GVBD 2-4 hrs after culture, 13.6% remained at GV stage 8 hrs after culture.TEM observation revealed that nucleoli of oocytes which failed to go through GVBD were composed of fibrillar-granular component,small vacuoles and fibrillar centers or showed small vacuoles on nuclear surface. During GVBD, the nucleoli became smaller and smaller and finally disappeared with the nuclear-associated chromatin dislocated to the periphery. Nuclear membrane with attached chromatin became folded and electronic dense cores appeared in the center of chromatin clumps at the same time.The last event of GVBD was the disruption of nuclear membrane.At the end of the 5th hr after culture, meiosis progressed to prometaphase I.Chromosomes,distributed in the original GV area free of organelles,were surrounded by large quantity of mitochondria and small SER vesicles. At the end of the 12th hr after culture,48. 1% of the oocytes emitted PB1.Decondensing sperm head and early male pronuclcus(mPN)with condensed nucleoli were found 1-2 hrs after insemination.The formation and enlargement of female PN(fPN) occurred a little earlier than that of mPN. 33.3% finished syngamy at 8-9 hrs after insemination.The process of nucleolus formation was reverse to that in GVBD. The oolemma modification caused by cortical reaction could effectively inhibit polyspermy.in contrast,there were sperm binding to the oolemma where CGs failed to be released. In addition, PB2 was emitted 2-5 hrs after insemination. The difference between PB1 and PB2 as well as the abstriction of polar body were also discussed.展开更多
基金Supported by National Natural Science Foundation of China (30871431)Outstanding Youth Fund of Heilongjiang Province (JC200905)~~
文摘[Objective] This study aimed to improve the in vitro maturation quality of denuded porcine oocytes and provide scientific basis for establishing a stable and efficient denuded oocyte culture system. [Method] The first polar body extrusion rate, oocyte glutathione （GSH） content, positive rate of brilliant cresyl blue （BCB） staining and development potential of activated oocytes or fertilized oocytes were employed as main indicators to investigate the effects of follicular mural granulosa cell （MGC） coculture on cytoplasmic maturation of cumulus cell-removal oocytes （Denuded Oocyte, DO）. [Result] According to in vitro maturation results, compared with DO group, the first polar body extrusion rate of porcine oocytes in DO＋MGC group was not significantly different, but the nuclear maturation process was improved and was more similar to that in COC （cumulus-oocyte complex） group. Detection of GSH content in mature oocytes showed that there was no significant difference between DO＋ MGC group （optical density of 1 053.67） and COC group （optical density of 1 426.00） or between DO＋MGC group and COC＋GC group （optical density of 1 541.00）, however, GSH content in mature oocytes of DO group （optical density of 724.67） was significantly lower than that of COC group and COC＋GC group （P0.05）. Detection of glucose-6-phosphate dehydrogenase （G6PDH） activity showed that there was no significant difference in BCB positive oocyte rate between DO ＋MGC group （88.26% ） and COC group （92.75%） or between DO＋MGC group and DO group （82.86% ）, however, BCB positive oocyte rate of DO group was significantly lower than that of COC group （P0.05）. Furthermore, the cleavage rate and blastocyst rate of activated mature oocytes derived from DO ＋MGC group （94.98% and 43.67% , respectively） were significantly higher than those from DO group （52.54% and 8.97%, respectively） （P0.05）, and were not significantly different compared with those from COC group （97.11% and 38.30%, respectively）. In addition, the cleavage rate of fertilized oocytes derived from DO＋MGC group （72.65%） showed no significant difference compared with that from DO group （63.59%）, but the blastocyst rate of DO＋MGC group was significantly higher than that of DO group （9.88%） （P0.05）. [Conclusion] MGC coculture can significantly improve the in vitro cytoplasmic maturation quality of denuded porcine oocytes, thereby enhancing the subsequent developmental potential.
基金Supported by Natural Science Foundation of Ningxia Hui Autonomous Region(NZ12150)~~
文摘[Objective] This study aimed to investigate the appropriate concentrations of follicle-stimulating hormone（FSH）, luteotropic hormone（LH） and estrodiol（E2） during in vitro maturation of Tan sheep oocytes. [Method] Tan sheep oocytes were divided into five groups for in vitro maturation culture： control group, FSH group（10,50, 100, 200 and 300 μg/ml FSH, respectively）, LH group（5, 10, 20, 50 and 100μg/ml LH, respectively）, E2group（5, 10, 25, 50 and 100 μg/ml E2, respectively）, and FSH ＋ LH group（100 μg/ml FSH ＋ 20 μg/ml LH）. The releasing rate of first polar bodies was analyzed. [Result] The maturation rate of Tan sheep oocytes in 100 μg/ml FSH ＋ 20 μg/ml LH group reached the highest（64.64%）, which was significantly higher than that in other four groups（P〈0.05）; among different FSH concentrations,100 μg/ml FSH was superior to other four concentrations and the control group, exhibiting significant differences（P〈0.05）; among different LH concentrations, 20 μg/ml LH was superior to other four concentrations and the control group, exhibiting significant differences（P〈0.05）; among different E2 concentrations, 50 μg/ml E2 was superior to other four concentrations and the control group, exhibiting significant differences（P〈0.05）. [Conclusion] Under the experimental conditions, 100 μg/ml FSH ＋20 μg/ml LH was the most appropriate hormone combination for in vitro maturation of Tan sheep oocytes.
文摘The chronological and morphological changes of the nucleus during mouse oocyte maturation and fertilization were systematically studied. Although most oocytes went through GVBD 2-4 hrs after culture, 13.6% remained at GV stage 8 hrs after culture.TEM observation revealed that nucleoli of oocytes which failed to go through GVBD were composed of fibrillar-granular component,small vacuoles and fibrillar centers or showed small vacuoles on nuclear surface. During GVBD, the nucleoli became smaller and smaller and finally disappeared with the nuclear-associated chromatin dislocated to the periphery. Nuclear membrane with attached chromatin became folded and electronic dense cores appeared in the center of chromatin clumps at the same time.The last event of GVBD was the disruption of nuclear membrane.At the end of the 5th hr after culture, meiosis progressed to prometaphase I.Chromosomes,distributed in the original GV area free of organelles,were surrounded by large quantity of mitochondria and small SER vesicles. At the end of the 12th hr after culture,48. 1% of the oocytes emitted PB1.Decondensing sperm head and early male pronuclcus(mPN)with condensed nucleoli were found 1-2 hrs after insemination.The formation and enlargement of female PN(fPN) occurred a little earlier than that of mPN. 33.3% finished syngamy at 8-9 hrs after insemination.The process of nucleolus formation was reverse to that in GVBD. The oolemma modification caused by cortical reaction could effectively inhibit polyspermy.in contrast,there were sperm binding to the oolemma where CGs failed to be released. In addition, PB2 was emitted 2-5 hrs after insemination. The difference between PB1 and PB2 as well as the abstriction of polar body were also discussed.
