Background： L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In ...Background： L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation. Methods： The freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F 1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion ofdimethyl sulfoxide （DMSO） and ethylene glycol （EG）, comparing with the control group （15% DMSO and 15% EG without L-proline）. The survival rate, 5-methylcytosine （5-mC） expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test. Results： L-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control. Conclusions： It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification.展开更多
To study whether integrins on cell membrane ligate with intracellular cytoskeletal proteins and mediate their reorganization in egg activation, female mice were used for su- perovulation. The zona-free oocytes were in...To study whether integrins on cell membrane ligate with intracellular cytoskeletal proteins and mediate their reorganization in egg activation, female mice were used for su- perovulation. The zona-free oocytes were incubated separately with specific ligand of integrins, an active RGD peptide, in vitro for certain period of time. RGE peptide and mouse capacitated sperm were used as controls. Freshly ovulated oocytes and those treated with different factors were immunostained with FITC-labeled anti-actin antibody, then detected with confocal micro- scope. The results demonstrated that freshly ovulated mouse oocytes, oocytes incubated for 2 h in vitro and those treated with control RGE peptide for 15 min showed hardly visible fluorescene or only thin fluorescence in plasma membrane region. Oocytes coincubated with sperms for 15 min and those treated with active RGD peptide for 10 min, 30 min and 2 hours respectively had strong and thick fluorescence in the plasma membrane and cortical region of oocytes, and some of them showed asymmetrically fluorescent distribution. It is proved that integrins on membrane are ligated directly with cytoskeletal protein. Integrins binding with their ligands regulate reor- ganization of cytoskelal protein, which may be involved in transmembrane signaling in egg acti- vation.展开更多
RanGAP1 is a Ran GTPase-activating protein that plays a pivotal role in the majority of nucleocytoplasmic transport pathways. The protein is limited to somatic cells. In this study, the localization and possible funct...RanGAP1 is a Ran GTPase-activating protein that plays a pivotal role in the majority of nucleocytoplasmic transport pathways. The protein is limited to somatic cells. In this study, the localization and possible functions of RanGAP1 were examined during mouse oocyte fertilization. Immunofluorescence analysis showed that after sperm penetration, RanGAP1 was found to diffuse within the cytoplasm, but concentrated in the microtuble of the reversed spindle and the constriction ring between the oocyte and the second polar body; with the expansion of sperm chromatin, RanGAP1 began to move to the region around the expanding sperm and oocyte chromatin, and gradually concentrated around the growing parents pronuclei. After the male and female pronuclei apposed, the membrane of one pronuclei broke first, numerous concentrated RanGAP1 dots were observed in the chromosome region. With the chromatin condensing into chromosome, the parents chromosomes mixed together and prepared to start the first mitosis, the condensed RanGAP1 was just the shape of the microtuble to assemble the first mitosis spindle. These showed that RanGAP1 played an important role in regulating spindle functions, chromosome alignment, PB2 extrusion and pronuclei nuclear envelope assembly/disassembly in mouse oocyte fertilization.展开更多
Background： This study was conducted to investigate effect of exogenous melatonin on the development of mouse mature oocytes after cryopreservation. Results： First, mouse metaphase II （MII） oocytes were vitrified ...Background： This study was conducted to investigate effect of exogenous melatonin on the development of mouse mature oocytes after cryopreservation. Results： First, mouse metaphase II （MII） oocytes were vitrified in the open-pulled straws （OPS）. After warming, they were cultured for 1 h in M2 medium containing melatonin at different concentrations （0, 10-9, 10-7, 10-s, 10 3 mol/L）. Then the oocytes were used to detect reactive oxygen species （ROS） and glutathione （GSH） levels （fluorescence microscopy）, and the developmental potential after parthenogenetic activation. The experimental results showed that the ROS level and cleavage rate in 10-3 mol/L melatonin group was significantly lower than that in melatonin-free group （control）. The GSH levels and blastocyst rates in all melatonin-treated groups were similar to that in control. Based on the above results, we detected the expression of gene Hsp9Oaal, Hsfl, Hspalb, Nrf2 and Bcl-xl with qRT-PCR in oocytes treated with 10-7, or 10-3 mol/L melatonin and untreated control. After warming and culture for 1 h, the oocytes showed higher Hsp90oal expression in 10-7 mol/L melatonin-treated group than in the control （P 〈 0.05）; the Hsfl, Hsp9Oaal and Bcl-xl expression were significantly decreased in 10-3 mol/L melatonin-treated group when compared to the control. Based on the above results and previous research, we detected the development of vitrified-warmed oocytes treated with either 10-7 or 0 mol/L melatonin by in vitro fertilization. No difference was observed between them. Conclusions： Our results indicate that the supplementation of melatonin （10-9 to 10-3 mol/L） in culture medium and incubation for 1 h did not improve the subsequent developmental potential of vitrified-warmed mouse MII oocytes, even if there were alteration in gene expression.展开更多
基金The work was supported by grants from the National Natural Science Foundation of China （No. 31230047, No. 31429004, No. 81571386, and No. 81471508）, the Interdisciplinary Project of Peking University Third Hospital and Chinese Academy of Sciences, Research Fund of National Health and Family Planning Commission of China （No. 201402004）, the Mega-projects of Science Research for the 12th Five-year Plan （No. 2012ba132b05）, and the Key Research Program of the Chinese Academy of Sciences （No. KJZD-EW-TZ-L03-2）.
文摘Background： L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation. Methods： The freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F 1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion ofdimethyl sulfoxide （DMSO） and ethylene glycol （EG）, comparing with the control group （15% DMSO and 15% EG without L-proline）. The survival rate, 5-methylcytosine （5-mC） expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test. Results： L-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control. Conclusions： It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification.
文摘To study whether integrins on cell membrane ligate with intracellular cytoskeletal proteins and mediate their reorganization in egg activation, female mice were used for su- perovulation. The zona-free oocytes were incubated separately with specific ligand of integrins, an active RGD peptide, in vitro for certain period of time. RGE peptide and mouse capacitated sperm were used as controls. Freshly ovulated oocytes and those treated with different factors were immunostained with FITC-labeled anti-actin antibody, then detected with confocal micro- scope. The results demonstrated that freshly ovulated mouse oocytes, oocytes incubated for 2 h in vitro and those treated with control RGE peptide for 15 min showed hardly visible fluorescene or only thin fluorescence in plasma membrane region. Oocytes coincubated with sperms for 15 min and those treated with active RGD peptide for 10 min, 30 min and 2 hours respectively had strong and thick fluorescence in the plasma membrane and cortical region of oocytes, and some of them showed asymmetrically fluorescent distribution. It is proved that integrins on membrane are ligated directly with cytoskeletal protein. Integrins binding with their ligands regulate reor- ganization of cytoskelal protein, which may be involved in transmembrane signaling in egg acti- vation.
基金Supported by Heilongjiang Provincial Natural Science Foundation(Face Project)(C2016021)the Open Project of Key Laboratory of Feed Science,Northeast Agricultural University,Heilongjiang Province(yy-2012-10)
文摘RanGAP1 is a Ran GTPase-activating protein that plays a pivotal role in the majority of nucleocytoplasmic transport pathways. The protein is limited to somatic cells. In this study, the localization and possible functions of RanGAP1 were examined during mouse oocyte fertilization. Immunofluorescence analysis showed that after sperm penetration, RanGAP1 was found to diffuse within the cytoplasm, but concentrated in the microtuble of the reversed spindle and the constriction ring between the oocyte and the second polar body; with the expansion of sperm chromatin, RanGAP1 began to move to the region around the expanding sperm and oocyte chromatin, and gradually concentrated around the growing parents pronuclei. After the male and female pronuclei apposed, the membrane of one pronuclei broke first, numerous concentrated RanGAP1 dots were observed in the chromosome region. With the chromatin condensing into chromosome, the parents chromosomes mixed together and prepared to start the first mitosis, the condensed RanGAP1 was just the shape of the microtuble to assemble the first mitosis spindle. These showed that RanGAP1 played an important role in regulating spindle functions, chromosome alignment, PB2 extrusion and pronuclei nuclear envelope assembly/disassembly in mouse oocyte fertilization.
基金supported in part by the National genetically modified organisms breeding major projects(Grant No.2014ZX0800802B)the fund for Backup Candidate of Academic Technology Leaders in Sichuan Province
文摘Background： This study was conducted to investigate effect of exogenous melatonin on the development of mouse mature oocytes after cryopreservation. Results： First, mouse metaphase II （MII） oocytes were vitrified in the open-pulled straws （OPS）. After warming, they were cultured for 1 h in M2 medium containing melatonin at different concentrations （0, 10-9, 10-7, 10-s, 10 3 mol/L）. Then the oocytes were used to detect reactive oxygen species （ROS） and glutathione （GSH） levels （fluorescence microscopy）, and the developmental potential after parthenogenetic activation. The experimental results showed that the ROS level and cleavage rate in 10-3 mol/L melatonin group was significantly lower than that in melatonin-free group （control）. The GSH levels and blastocyst rates in all melatonin-treated groups were similar to that in control. Based on the above results, we detected the expression of gene Hsp9Oaal, Hsfl, Hspalb, Nrf2 and Bcl-xl with qRT-PCR in oocytes treated with 10-7, or 10-3 mol/L melatonin and untreated control. After warming and culture for 1 h, the oocytes showed higher Hsp90oal expression in 10-7 mol/L melatonin-treated group than in the control （P 〈 0.05）; the Hsfl, Hsp9Oaal and Bcl-xl expression were significantly decreased in 10-3 mol/L melatonin-treated group when compared to the control. Based on the above results and previous research, we detected the development of vitrified-warmed oocytes treated with either 10-7 or 0 mol/L melatonin by in vitro fertilization. No difference was observed between them. Conclusions： Our results indicate that the supplementation of melatonin （10-9 to 10-3 mol/L） in culture medium and incubation for 1 h did not improve the subsequent developmental potential of vitrified-warmed mouse MII oocytes, even if there were alteration in gene expression.
