In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. T...In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. The possible effective targets of icariin promoting preimplantation embryo development in vitro and anti-apoptosis were determined. The embryos were cultured in modified CZB medium （mCZB） as control group. The experimental group （Ica group） was supplemented with 0.6 μg mL-1 icariin. Mouse pronuclear embryos were cultured in vitro until blastocysts. The development rates of preimplantation embryos were observed. The total cell number, apoptotic cell number and the rate of apoptotic cells in blastocysts were analysed by the staining of Hoechst33342 and labeling of TUNEL and detected under a laser confocal scanning microscope. The miRNA-21 expression, the mRNA levels of pro-apoptotic Caspase3, and the target genes of miRNA-21： pro-apoptotic PTEN, anti-apoptotic Bcl-2 were detected by real-time RT-PCR. The results showed that percentages of morulaes and blastocysts in Ica group were both extremely higher than control group （（85.14±6.57）% vs. （72.04±11.58）%; （82.50±7.11）% vs. （66.80±11.70）%, respectively, P〈0.01）. The total cell number ofblastocysts had extreme difference between Ica group and control group （（61.40±9.64） vs. （46.23±4.50）, P〈0.01）. The apoptotic cell number and rate of apoptotic cells of blastocysts were both reduced in Ica group （（1.47±0.51） vs. （2.94±0.66）; （2.40±0.27）% vs. （6.25±0.62）%, respectively, P〈0.01）. Compared to control group, addition of icariin into mCZB extremely increased the expression of anti-apoptotic miRNA-21 （P〈0.01）, down-regulated pro-apoptotic Caspase3 （P〈0.05） and PTEN （P〈0.01）, up-regulated anti-apoptotic Bcl-2 （P〈0.01）. In conclusion, icariin could reduce the apoptosis, promote the embryo development in vitro by enhancing miRNA-21 expression to up-regulated anti-展开更多
Background Human embryonic stem cells can propagate indefinitely in vitro and are able to differentiate into derivatives of all three embryonic germ layers. The excitement surrounding human embryonic stem cells lies l...Background Human embryonic stem cells can propagate indefinitely in vitro and are able to differentiate into derivatives of all three embryonic germ layers. The excitement surrounding human embryonic stem cells lies largely in their potential to produce specialized cells that can be used for transplant therapies. However, further investigation requires additional cell lines with varying genetic background. Therefore, efforts to derive and establish more human embryonic stern cell lines are highly warranted. Methods Surplus embryos （blastocysts） from donors were used to isolate the inner cell mass by immunosurgery. All cells were cultured continuously on irradiated murine embryonic fibroblasts feed layer and likely human embryonic stem cell colonies were subsequently characterized by cell surface marker staining, karyotyping and teratoma formation. Results Two human embryonic stern cell lines （SYSU-1 and SYSU-2） were established from surplus embryos. The two lines express several pluripotency markers including alkaline phosphatase, SSEA- 4, Tra-1-60, Oct-4, Nanog and Rex-1. They remain in undifferentiated state with normal karyotype after prolonged passages and can form embryoid bodies in vitro and teratoma in vivo. Conclusion Two new human embryonic stem cell lines have been established from surplus embryos. They can be used to understand selfrenewal and differentiating mechanisms and provide more choices for regenerative medicine.展开更多
To our knowledge,no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization （IVF）,intracytoplasmic sperm injection （ICSI） and somatic cell nuc...To our knowledge,no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization （IVF）,intracytoplasmic sperm injection （ICSI） and somatic cell nuclear transfer （SCNT） by controlled freezing and vitrification.This experiment,therefore,was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification.Adenosine-5’-triphosphate （ATP） content and reactive oxygen species （ROS） level in blastocysts were also analyzed.Firstly,for each type of blastocyst （IVF,ICSI or SCNT）,significant differences were observed between the survival rates of the controlled freezing （（81.56±2.33）,（68.18±4.72） or （47.89±5.83）%） and OPS vitrification groups （（92.24±4.54）,（82.40±3.76） or （78.71±5.91）%;P〈0.05）.Secondly,for each type of blastocyst （IVF,ICSI or SCNT）,ATP content was significantly decreased after controlled freezing or vitrification,and the ATP content in the controlled freezing group （0.43±0.06）,（0.35±0.05） or （0.21±0.02） pmol） was significantly lower than that found in the OPS vitrification group （0.62±0.04）,（0.46±0.03） or （0.30±0.01） pmol;P〈0.05）.Thirdly,ROS level in fresh IVF （（47.33±3.56） c.p.s （counted photons per second）,ICSI （（36.51±2.58） c.p.s） or SCNT blastocysts （（26.44±1.49） c.p.s） was significantly lower than that found in the OPS vitrification group （（72.14±4.31）,（58.89±3.89） or （40.11±5.73） c.p.s;P〈0.05）,but higher than that of the controlled freezing group （34.41±3.32）,（23.13±1.26） or （15.46±2.45） c.p.s;P〈0.05）.The present study indicated that vitrification is more efficient in the cryopreservation of bovine blastocysts derived from IVF,ICSI or SCNT than controlled freezing.Furthermore,both vitrification and controlled freezing significantly altered the ATP content and ROS level in those blastocysts.展开更多
Background:To advance the use of embryo vitrification in veterinary practice,we developed a system in which embryo vitrification,warming and dilution can be performed within a straw.Ovine in vitro produced embryos(IVE...Background:To advance the use of embryo vitrification in veterinary practice,we developed a system in which embryo vitrification,warming and dilution can be performed within a straw.Ovine in vitro produced embryos(IVEP)were vitrified at either early(EBs:n=74)or fully expanded blastocyst stage(FEBs:n=195),using a new device named"E.Vit",composed by a 0.25-m L straw with a 50-μm pore polycarbonate grid at one end.Embryos at each stage(EBs and FEBs)were vitrified by either Two-step(TS)or Multi-step(MS;6 different concentrations of vitrification solutions)protocol.Non-vitrified embryos(n=102)were maintained in in vitro culture as a control.Warming consisted of placing the straws directly into 1.5 m L tubes containing a TCM-199 solution with three decreasing concentrations of sucrose.Blastocyst re-expansion,embryo survival and hatching rate were evaluated at2,24 and 48 h post warming.The number of apoptotic cells was determined by TUNEL assay.Results:Blastocyst re-expansion(2 h)after warming was higher(P<0.05)in FEBs group,vitrified with the MS and TS methods(77.90%and 71.25%,respectively)compared with the EBs group(MS:59.38%and TS:48.50%,respectively).Survival rates of vitrified FEBs after 24 h IVC were higher(P<0.001)in both methods(MS and TS)than vitrified EBs(MS:56.25%;TS:42.42%)and was higher(P<0.05)in the MS method(94.19%)compared with those in TS(83.75%).After 48 h of culture the hatching rate for FEBs vitrified in MS system(91.86%)was similar to control(91.89%),but higher than FEB TS(77.5%)and EBs vitrified in MS(37.5%)and TS(33.33%).Number of apoptotic cells were higher in EBs,irrespective of the system used,compared to FEBs.The number of apoptotic cells in FEBs vitrified with MS was comparable to the control.Conclusions:A high survival rate of IVP embryos can be achieved by the new"E.Vit"device with hatching rates in vitro comparable with control fresh embryos.This method has the potential for use in direct embryo transfer in field conditions.展开更多
Kunming mouse strain is widely used in China, and the superovulation was administrated with 10 IU PMSG combined with 10 IU hCG. In this study, the effects of the exogenous gonadotropins on superovulation of Kunming mi...Kunming mouse strain is widely used in China, and the superovulation was administrated with 10 IU PMSG combined with 10 IU hCG. In this study, the effects of the exogenous gonadotropins on superovulation of Kunming mice and embryo quality derived from the superovulated mice were assessed. Female mice at 6-8-wk old were superovulated with 0, 5, 7.5 and 10 IU PMSG/hCG and mated with male mice. The embryos were retrieved at 2.5 d post coitum. No statistic difference was observed for the number of 2-cell embryos collected per mouse between control and 5 IU PMSG/hCG treatment group, but the number significantly increased for 7.5 and 10 IU PMSG/hCG treatment group （P〈0.05）. The average number of 4- cell and 8-cell embryos collected from each mouse significantly differed between control and 5, 7.5, 10 IU PMSG/hCG treatment groups （P〈0.05）. When 8-cell embryos derived from mice administrated with 0, 5, 7.5 and 10 IU PMSG/hCG were cultured in KSOM, the blastocyst development rates were 88.1, 94.7, 96.1 and 94.3%, respectively, which were similar to control （P〉0.05）. This indicated that exogenous gonadotropins have no effects on development of Kunming mouse embryos. The quality of blastocyst was assessed by labelling with Hoechst and propidium iodide for inner cell mass and trophectoderm cells, the result showed that ICM/TE ratio significantly decreased for 10 IU PMSG/hCG treatment group compared with control, 5 and 7.5 IU PMSG/hCG treatment group （P〈0.05）. This suggested that the embryo quality of Kunming mouse has been affected by high dose of gonadotropins.展开更多
Background：Due to high neutral lipids accumulation in the cytoplasm,in vitro-produced embryos from Bos primigenius indicus and their crosses are more sensitive to chilling and cryopreservation than those from Bos pri...Background：Due to high neutral lipids accumulation in the cytoplasm,in vitro-produced embryos from Bos primigenius indicus and their crosses are more sensitive to chilling and cryopreservation than those from Bos primigenius taurus.The objective of the present study was to evaluate the effects of trans-10,cis-12 conjugated linoleic acid（CLA） on the development and cryotolerance of crossbred Bos primigenius taurus x Bos primigenius indicus embryos produced in vitro,and cultured in the presence of fetal calf serum.Bovine zygotes（n = 1,692）were randomly assigned to one of the following treatment groups：1） Control,zygotes cultured in Charles Rosenkrans 2 amino acid（CR2aa） medium（n = 815） or 2） CLA,zygotes cultured in CR2 aa medium supplemented with 100 μmol/L of trans-10,cis-12 CLA（n =877）.Embryo development（cleavage and blastocyst rates evaluated at days 3 and 8 of culture,respectively）,lipid content at morula stage（day 5） and blastocyst cryotolerance（re-expansion and hatching rates,evaluated 24 and 72 h post-thawing,respectively） were compared between groups.Additionally,selected mRNA transcripts were measured by Real-Time PCR in blastocyst stage.Results：The CLA treatment had no effect on cleavage and blastocyst rates,or on mRNA levels for genes related to cellular stress and apoptosis.On the other hand,abundance of mRNA for the 1-acylglycerol-3-phosphate0-acyltransferase-encoding gene（AGPAT）,which is involved in triglycerides synthesis,and consequently neutral lipid content,were reduced by CLA treatment.A significant increase was observed in the re-expansion rate of embryos cultured with trans-10,cis-12 CLA when compared to control（56.3 vs.34.4%,respectively,P = 0.002）.However,this difference was not observed in the hatching rate（16.5 vs.14.0%,respectively,P=0.62）.Conclusions：The supplementation with trans-10,ds-12 CLA isomer in culture medium reduced the lipid content of in vitro produced bovine embryos by reducing the gene expression of 1-acylglycerol 3-ph展开更多
Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for d...Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embrvonic stem cell lines with clarified directly differentiation ability展开更多
基金supported by the Beijing Natural Science Foundation of China (6112004)the High Quality Paper Support Project of Beijing University of Agriculture, China (GL2012006)
文摘In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. The possible effective targets of icariin promoting preimplantation embryo development in vitro and anti-apoptosis were determined. The embryos were cultured in modified CZB medium （mCZB） as control group. The experimental group （Ica group） was supplemented with 0.6 μg mL-1 icariin. Mouse pronuclear embryos were cultured in vitro until blastocysts. The development rates of preimplantation embryos were observed. The total cell number, apoptotic cell number and the rate of apoptotic cells in blastocysts were analysed by the staining of Hoechst33342 and labeling of TUNEL and detected under a laser confocal scanning microscope. The miRNA-21 expression, the mRNA levels of pro-apoptotic Caspase3, and the target genes of miRNA-21： pro-apoptotic PTEN, anti-apoptotic Bcl-2 were detected by real-time RT-PCR. The results showed that percentages of morulaes and blastocysts in Ica group were both extremely higher than control group （（85.14±6.57）% vs. （72.04±11.58）%; （82.50±7.11）% vs. （66.80±11.70）%, respectively, P〈0.01）. The total cell number ofblastocysts had extreme difference between Ica group and control group （（61.40±9.64） vs. （46.23±4.50）, P〈0.01）. The apoptotic cell number and rate of apoptotic cells of blastocysts were both reduced in Ica group （（1.47±0.51） vs. （2.94±0.66）; （2.40±0.27）% vs. （6.25±0.62）%, respectively, P〈0.01）. Compared to control group, addition of icariin into mCZB extremely increased the expression of anti-apoptotic miRNA-21 （P〈0.01）, down-regulated pro-apoptotic Caspase3 （P〈0.05） and PTEN （P〈0.01）, up-regulated anti-apoptotic Bcl-2 （P〈0.01）. In conclusion, icariin could reduce the apoptosis, promote the embryo development in vitro by enhancing miRNA-21 expression to up-regulated anti-
基金This work was supported by the National Basic Research Program of China （973 Program） （No. 2001CB509904） the Key Scientific and Technological Projects of Guangdong Province （No. 2003A3020103, 2005A30201001）+1 种基金 the Key Scientific and Technological Projects of Guangzhou City （No. 2002U13E0011） National Natural Science Foundation of China （No 30571891, 30671023）.
文摘Background Human embryonic stem cells can propagate indefinitely in vitro and are able to differentiate into derivatives of all three embryonic germ layers. The excitement surrounding human embryonic stem cells lies largely in their potential to produce specialized cells that can be used for transplant therapies. However, further investigation requires additional cell lines with varying genetic background. Therefore, efforts to derive and establish more human embryonic stern cell lines are highly warranted. Methods Surplus embryos （blastocysts） from donors were used to isolate the inner cell mass by immunosurgery. All cells were cultured continuously on irradiated murine embryonic fibroblasts feed layer and likely human embryonic stem cell colonies were subsequently characterized by cell surface marker staining, karyotyping and teratoma formation. Results Two human embryonic stern cell lines （SYSU-1 and SYSU-2） were established from surplus embryos. The two lines express several pluripotency markers including alkaline phosphatase, SSEA- 4, Tra-1-60, Oct-4, Nanog and Rex-1. They remain in undifferentiated state with normal karyotype after prolonged passages and can form embryoid bodies in vitro and teratoma in vivo. Conclusion Two new human embryonic stem cell lines have been established from surplus embryos. They can be used to understand selfrenewal and differentiating mechanisms and provide more choices for regenerative medicine.
基金supported by the Fund of China Agriculture Research System(CARS-37)the Genetically Modified Organisms Breeding Major Projects of China(2009ZX08011-031B)+1 种基金the Basic Research Fund of IAS,CAAS(2010jc-3-1)the National Natural Science Foundation of China(31001011)
文摘To our knowledge,no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization （IVF）,intracytoplasmic sperm injection （ICSI） and somatic cell nuclear transfer （SCNT） by controlled freezing and vitrification.This experiment,therefore,was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification.Adenosine-5’-triphosphate （ATP） content and reactive oxygen species （ROS） level in blastocysts were also analyzed.Firstly,for each type of blastocyst （IVF,ICSI or SCNT）,significant differences were observed between the survival rates of the controlled freezing （（81.56±2.33）,（68.18±4.72） or （47.89±5.83）%） and OPS vitrification groups （（92.24±4.54）,（82.40±3.76） or （78.71±5.91）%;P〈0.05）.Secondly,for each type of blastocyst （IVF,ICSI or SCNT）,ATP content was significantly decreased after controlled freezing or vitrification,and the ATP content in the controlled freezing group （0.43±0.06）,（0.35±0.05） or （0.21±0.02） pmol） was significantly lower than that found in the OPS vitrification group （0.62±0.04）,（0.46±0.03） or （0.30±0.01） pmol;P〈0.05）.Thirdly,ROS level in fresh IVF （（47.33±3.56） c.p.s （counted photons per second）,ICSI （（36.51±2.58） c.p.s） or SCNT blastocysts （（26.44±1.49） c.p.s） was significantly lower than that found in the OPS vitrification group （（72.14±4.31）,（58.89±3.89） or （40.11±5.73） c.p.s;P〈0.05）,but higher than that of the controlled freezing group （34.41±3.32）,（23.13±1.26） or （15.46±2.45） c.p.s;P〈0.05）.The present study indicated that vitrification is more efficient in the cryopreservation of bovine blastocysts derived from IVF,ICSI or SCNT than controlled freezing.Furthermore,both vitrification and controlled freezing significantly altered the ATP content and ROS level in those blastocysts.
基金supported by Regione Autonoma della Sardegna.-L.R.7-MIGLIOVINGENSAR ProjectBando competitivo Fondazione di Sardegna–2016,CUP J86C18000800005“Progetto FAR2019LEDDAS Una Tantum 2019,University of Sassari”.
文摘Background:To advance the use of embryo vitrification in veterinary practice,we developed a system in which embryo vitrification,warming and dilution can be performed within a straw.Ovine in vitro produced embryos(IVEP)were vitrified at either early(EBs:n=74)or fully expanded blastocyst stage(FEBs:n=195),using a new device named"E.Vit",composed by a 0.25-m L straw with a 50-μm pore polycarbonate grid at one end.Embryos at each stage(EBs and FEBs)were vitrified by either Two-step(TS)or Multi-step(MS;6 different concentrations of vitrification solutions)protocol.Non-vitrified embryos(n=102)were maintained in in vitro culture as a control.Warming consisted of placing the straws directly into 1.5 m L tubes containing a TCM-199 solution with three decreasing concentrations of sucrose.Blastocyst re-expansion,embryo survival and hatching rate were evaluated at2,24 and 48 h post warming.The number of apoptotic cells was determined by TUNEL assay.Results:Blastocyst re-expansion(2 h)after warming was higher(P<0.05)in FEBs group,vitrified with the MS and TS methods(77.90%and 71.25%,respectively)compared with the EBs group(MS:59.38%and TS:48.50%,respectively).Survival rates of vitrified FEBs after 24 h IVC were higher(P<0.001)in both methods(MS and TS)than vitrified EBs(MS:56.25%;TS:42.42%)and was higher(P<0.05)in the MS method(94.19%)compared with those in TS(83.75%).After 48 h of culture the hatching rate for FEBs vitrified in MS system(91.86%)was similar to control(91.89%),but higher than FEB TS(77.5%)and EBs vitrified in MS(37.5%)and TS(33.33%).Number of apoptotic cells were higher in EBs,irrespective of the system used,compared to FEBs.The number of apoptotic cells in FEBs vitrified with MS was comparable to the control.Conclusions:A high survival rate of IVP embryos can be achieved by the new"E.Vit"device with hatching rates in vitro comparable with control fresh embryos.This method has the potential for use in direct embryo transfer in field conditions.
基金supported by the Natural Science Foundation of Inner Mongolia for the Major Special Projects,China(2010ZD05)
文摘Kunming mouse strain is widely used in China, and the superovulation was administrated with 10 IU PMSG combined with 10 IU hCG. In this study, the effects of the exogenous gonadotropins on superovulation of Kunming mice and embryo quality derived from the superovulated mice were assessed. Female mice at 6-8-wk old were superovulated with 0, 5, 7.5 and 10 IU PMSG/hCG and mated with male mice. The embryos were retrieved at 2.5 d post coitum. No statistic difference was observed for the number of 2-cell embryos collected per mouse between control and 5 IU PMSG/hCG treatment group, but the number significantly increased for 7.5 and 10 IU PMSG/hCG treatment group （P〈0.05）. The average number of 4- cell and 8-cell embryos collected from each mouse significantly differed between control and 5, 7.5, 10 IU PMSG/hCG treatment groups （P〈0.05）. When 8-cell embryos derived from mice administrated with 0, 5, 7.5 and 10 IU PMSG/hCG were cultured in KSOM, the blastocyst development rates were 88.1, 94.7, 96.1 and 94.3%, respectively, which were similar to control （P〉0.05）. This indicated that exogenous gonadotropins have no effects on development of Kunming mouse embryos. The quality of blastocyst was assessed by labelling with Hoechst and propidium iodide for inner cell mass and trophectoderm cells, the result showed that ICM/TE ratio significantly decreased for 10 IU PMSG/hCG treatment group compared with control, 5 and 7.5 IU PMSG/hCG treatment group （P〈0.05）. This suggested that the embryo quality of Kunming mouse has been affected by high dose of gonadotropins.
基金supported by the Brazilian National Council for Scientific and Technological Development(CNPq)the Minas Gerais State Research Foundation(FAPEMIG)+1 种基金Embrapa(Project 01.07.01.002)received a grant from FAPEMIG
文摘Background：Due to high neutral lipids accumulation in the cytoplasm,in vitro-produced embryos from Bos primigenius indicus and their crosses are more sensitive to chilling and cryopreservation than those from Bos primigenius taurus.The objective of the present study was to evaluate the effects of trans-10,cis-12 conjugated linoleic acid（CLA） on the development and cryotolerance of crossbred Bos primigenius taurus x Bos primigenius indicus embryos produced in vitro,and cultured in the presence of fetal calf serum.Bovine zygotes（n = 1,692）were randomly assigned to one of the following treatment groups：1） Control,zygotes cultured in Charles Rosenkrans 2 amino acid（CR2aa） medium（n = 815） or 2） CLA,zygotes cultured in CR2 aa medium supplemented with 100 μmol/L of trans-10,cis-12 CLA（n =877）.Embryo development（cleavage and blastocyst rates evaluated at days 3 and 8 of culture,respectively）,lipid content at morula stage（day 5） and blastocyst cryotolerance（re-expansion and hatching rates,evaluated 24 and 72 h post-thawing,respectively） were compared between groups.Additionally,selected mRNA transcripts were measured by Real-Time PCR in blastocyst stage.Results：The CLA treatment had no effect on cleavage and blastocyst rates,or on mRNA levels for genes related to cellular stress and apoptosis.On the other hand,abundance of mRNA for the 1-acylglycerol-3-phosphate0-acyltransferase-encoding gene（AGPAT）,which is involved in triglycerides synthesis,and consequently neutral lipid content,were reduced by CLA treatment.A significant increase was observed in the re-expansion rate of embryos cultured with trans-10,cis-12 CLA when compared to control（56.3 vs.34.4%,respectively,P = 0.002）.However,this difference was not observed in the hatching rate（16.5 vs.14.0%,respectively,P=0.62）.Conclusions：The supplementation with trans-10,ds-12 CLA isomer in culture medium reduced the lipid content of in vitro produced bovine embryos by reducing the gene expression of 1-acylglycerol 3-ph
文摘Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embrvonic stem cell lines with clarified directly differentiation ability
