Bovine cumulus-oocyte complexes were cultured in the maturation medium containing 4 different concentrations of verapamil and trifluoperazine to testify the necessity of extracellular Ca2+ and Ca2+-calmodulin complex ...Bovine cumulus-oocyte complexes were cultured in the maturation medium containing 4 different concentrations of verapamil and trifluoperazine to testify the necessity of extracellular Ca2+ and Ca2+-calmodulin complex for the resumption and completion of meiosis as well as cumulus expansion.Ultrastructure of the treated oocytes was also observed to investigate the cytoplasm maturation. The results showed that verapamil didn't influence the cumulus expansion,meiosis resumption and completion and cytoplasm maturation significantly.TFP inhibited cumulus expansion in a dose-dependent manner.25 μm trifluoperazine significantly inhibited the GVBD and maturation(p<0.01),wherease 1 μm TFP had no effect.Both oocytes and cumulus cells treated with 25 μm TFP severely degenerated.Our observations suggest that the resumption and completion of meiosis and cumulus expansion are Ca2+ -CaM dependent and blocking membrane Ca2+ channel does not influence oocyte germinal vesicle breakdown,nuclear and cytoplasm maturation significantly in cattle.展开更多
The in vitro maturation, fertilization and development of bovine ovary oocytes in two differentcultural systems A and B were studied under the conditions of 38.5℃,5%CO2,95%air and 100%humidity.The maturation rates we...The in vitro maturation, fertilization and development of bovine ovary oocytes in two differentcultural systems A and B were studied under the conditions of 38.5℃,5%CO2,95%air and 100%humidity.The maturation rates were 94.5%and 91.3%,respectively,and the difference wasextremely significant.Frozen semen were thawed and sperm were capacitated with three kinds ofcapacitation agents for fertilization.The pronucleus rates were 76%,65%-68%and 62%respectively.The rates of embryos developed to morula and blastocyst were 19%,16% and 17%respectively.The developmental rates of embryos cocultured with bovine oviductal epithelium cellsand bovine granulosa cells were 25% and 23.4% respectively,with no significant difference. Freshembryos were transplanted into 15 recipicns,and three of them were pregnant and calves were bornin 1990 and 1991.The pregnant rate was 20%.The emryos developed faster before 8-cell stage andslower after 8-cell stage,in vitro than in vivo.展开更多
It is well established that the decline in female reproductive outcomes is related to postovulatory aging of oocytes and advanced maternal age.Poor oocyte quality is correlated with compromised genetic integrity and e...It is well established that the decline in female reproductive outcomes is related to postovulatory aging of oocytes and advanced maternal age.Poor oocyte quality is correlated with compromised genetic integrity and epigenetic changes during the oocyte aging process.Here,we review the epigenetic alterations,mainly focused on DNA methylation,histone acetylation and methylation associated with postovulatory oocyte aging as well as advanced maternal age.Furthermore,we address the underlying epigenetic mechanisms that contribute to the decline in oocyte quality during oocyte aging.展开更多
A method for DNA isolation from early development of blastocyst and further analysis of nuclear and mitochondrial DNA was developed in present study. Total DNA was prepared from interspecies reconstructed blastocyst a...A method for DNA isolation from early development of blastocyst and further analysis of nuclear and mitochondrial DNA was developed in present study. Total DNA was prepared from interspecies reconstructed blastocyst and a giant panda specific microsatellite locus g010 was successfully amplified. DNA sequencing of the PCR product showed that two sequences of reconstructed blastocysts are the same as that of positive control giant panda. Our results prove that the nucleus of interspecies reconstructed blastocyst comes from somatic nucleus of donor giant panda.展开更多
Interspecies somatic nuclear transfer (NT) may provide a new approach for preservation of the endangered rare species. Previous interspecies cloning studies have shown that a nucleus from a quiescent somatic cell supp...Interspecies somatic nuclear transfer (NT) may provide a new approach for preservation of the endangered rare species. Previous interspecies cloning studies have shown that a nucleus from a quiescent somatic cell supports early development of reconstructed embryos in the ooplasm from another species. In this study, we transferred nonqui-escent somatic cells from a giant panda into the perivitelline space of the enucleated rabbit oocytes. After electrofusion (at the rate of 71.6%) and electrical activation, 4.2% of the panda-rabbit reconstructed embryos developed to blastocyst in vitro. For improving the development rate of reconstructed embryos, we used serial NT in this study, i.e. blas-tomeres from reconstructed morulae were transferred into the perivitelline space of the enucleated rabbit oocytes. The fusion rates in the groups of serial I, serial Ⅱ and serial Ⅲ were 79.5%, 84.1% and 78.0%, respectively, having no difference with that of somatic group. And the blastocyst rates in serial NT groups展开更多
Oocytes collected from prepubertal gilt ovaries were matured in vitro (IVM), and fertilized in vitro (IVF) or electrically activated. Phosphorylation of mitogen-activated protein kinase (MAPK) was detected with SDS-PA...Oocytes collected from prepubertal gilt ovaries were matured in vitro (IVM), and fertilized in vitro (IVF) or electrically activated. Phosphorylation of mitogen-activated protein kinase (MAPK) was detected with SDS-PAGE and Western blotting, and translocation of ERK2 was observed with immunofluorescent cytochemistry. We found that the quantity of MAPK kept unchanged during oocyte maturation. There was no phosphorylated MAPK in porcine oocytes at the germinal vesicle (GV) stage; a little MAPK was phosphorylated at 20 h of IVM; a high level phosphory-lation was detected at 30 h, while MAPK phosphorylation decreased at 36 h; and then MAPK phosphorylation increased again to the peak level from 40 to 60 h. ERK2 translocated from the peripheral cytoplasm to inner cytoplasm and nuclear area during oocyte maturation. There was nearly no phosphorylated MAPK at 18 and 20 h of electrically activated oocytes, but phosphorylation increased at 22 h. There was no phosphorylated MAPK at 12 h of IVF, while展开更多
文摘Bovine cumulus-oocyte complexes were cultured in the maturation medium containing 4 different concentrations of verapamil and trifluoperazine to testify the necessity of extracellular Ca2+ and Ca2+-calmodulin complex for the resumption and completion of meiosis as well as cumulus expansion.Ultrastructure of the treated oocytes was also observed to investigate the cytoplasm maturation. The results showed that verapamil didn't influence the cumulus expansion,meiosis resumption and completion and cytoplasm maturation significantly.TFP inhibited cumulus expansion in a dose-dependent manner.25 μm trifluoperazine significantly inhibited the GVBD and maturation(p<0.01),wherease 1 μm TFP had no effect.Both oocytes and cumulus cells treated with 25 μm TFP severely degenerated.Our observations suggest that the resumption and completion of meiosis and cumulus expansion are Ca2+ -CaM dependent and blocking membrane Ca2+ channel does not influence oocyte germinal vesicle breakdown,nuclear and cytoplasm maturation significantly in cattle.
文摘The in vitro maturation, fertilization and development of bovine ovary oocytes in two differentcultural systems A and B were studied under the conditions of 38.5℃,5%CO2,95%air and 100%humidity.The maturation rates were 94.5%and 91.3%,respectively,and the difference wasextremely significant.Frozen semen were thawed and sperm were capacitated with three kinds ofcapacitation agents for fertilization.The pronucleus rates were 76%,65%-68%and 62%respectively.The rates of embryos developed to morula and blastocyst were 19%,16% and 17%respectively.The developmental rates of embryos cocultured with bovine oviductal epithelium cellsand bovine granulosa cells were 25% and 23.4% respectively,with no significant difference. Freshembryos were transplanted into 15 recipicns,and three of them were pregnant and calves were bornin 1990 and 1991.The pregnant rate was 20%.The emryos developed faster before 8-cell stage andslower after 8-cell stage,in vitro than in vivo.
基金supported in part by the National Natural Science Foundation of China (Grant No. 81100422)National Basic Research Program of China (Grant Nos. 2012CB944404 and 2011CB944501)
文摘It is well established that the decline in female reproductive outcomes is related to postovulatory aging of oocytes and advanced maternal age.Poor oocyte quality is correlated with compromised genetic integrity and epigenetic changes during the oocyte aging process.Here,we review the epigenetic alterations,mainly focused on DNA methylation,histone acetylation and methylation associated with postovulatory oocyte aging as well as advanced maternal age.Furthermore,we address the underlying epigenetic mechanisms that contribute to the decline in oocyte quality during oocyte aging.
文摘A method for DNA isolation from early development of blastocyst and further analysis of nuclear and mitochondrial DNA was developed in present study. Total DNA was prepared from interspecies reconstructed blastocyst and a giant panda specific microsatellite locus g010 was successfully amplified. DNA sequencing of the PCR product showed that two sequences of reconstructed blastocysts are the same as that of positive control giant panda. Our results prove that the nucleus of interspecies reconstructed blastocyst comes from somatic nucleus of donor giant panda.
基金This work was supported by the Climbing Project of the Ministry of Sciences and Technology of China (Grant No.97021109-2)the Important Project of Knowledge Innovation of Chinese Academy of Sciences (Grant No. KSCX1-05-01).
文摘Interspecies somatic nuclear transfer (NT) may provide a new approach for preservation of the endangered rare species. Previous interspecies cloning studies have shown that a nucleus from a quiescent somatic cell supports early development of reconstructed embryos in the ooplasm from another species. In this study, we transferred nonqui-escent somatic cells from a giant panda into the perivitelline space of the enucleated rabbit oocytes. After electrofusion (at the rate of 71.6%) and electrical activation, 4.2% of the panda-rabbit reconstructed embryos developed to blastocyst in vitro. For improving the development rate of reconstructed embryos, we used serial NT in this study, i.e. blas-tomeres from reconstructed morulae were transferred into the perivitelline space of the enucleated rabbit oocytes. The fusion rates in the groups of serial I, serial Ⅱ and serial Ⅲ were 79.5%, 84.1% and 78.0%, respectively, having no difference with that of somatic group. And the blastocyst rates in serial NT groups
基金This work was supported by the Major State Basic Research ("973") Project (Grant No. G1999055902)the Special Project of Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KSCX2-SW-303).
文摘Oocytes collected from prepubertal gilt ovaries were matured in vitro (IVM), and fertilized in vitro (IVF) or electrically activated. Phosphorylation of mitogen-activated protein kinase (MAPK) was detected with SDS-PAGE and Western blotting, and translocation of ERK2 was observed with immunofluorescent cytochemistry. We found that the quantity of MAPK kept unchanged during oocyte maturation. There was no phosphorylated MAPK in porcine oocytes at the germinal vesicle (GV) stage; a little MAPK was phosphorylated at 20 h of IVM; a high level phosphory-lation was detected at 30 h, while MAPK phosphorylation decreased at 36 h; and then MAPK phosphorylation increased again to the peak level from 40 to 60 h. ERK2 translocated from the peripheral cytoplasm to inner cytoplasm and nuclear area during oocyte maturation. There was nearly no phosphorylated MAPK at 18 and 20 h of electrically activated oocytes, but phosphorylation increased at 22 h. There was no phosphorylated MAPK at 12 h of IVF, while
