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
The somatic cell nuclear transfer （SCNT） technique has been applied successfully in a range of mammalian species giving rise to offspring, however, the efficiency of development to term has remained low. Oocyte meio...The somatic cell nuclear transfer （SCNT） technique has been applied successfully in a range of mammalian species giving rise to offspring, however, the efficiency of development to term has remained low. Oocyte meiotic arrest is an important option to make the SCNT more flexible and increase the number of cloned embryos produced. This paper showed that the use of butyrolactone I to arrest the meiotic division for 24 h prior to in vitro maturation （IVM） provided bovine oocytes capability of supporting development of blastocysts as efficiently as non arrested oocytes. In the SCNT group, cleavage rates were not affected by prematuration when adding butyrolactone I during IVM （57.9% vs. 62.4%; control vs. 10 μmol.L^-1 butyrolactone I）, developmental rates to biastocyst were unaffected （22.0% vs. 20.3%; control vs. 10 μmol.L^-1 butyrolactone I, p〉0.05） by the addition ofbutyrolactone I during IVM in cloned embryos. Moreover, the total cell numbers of blastocyst were not affected by butyrolactone I （total nucleus numbers, 132±16.5 vs. 128±19.4, p〉0.05）. In conclusion, the SCNT embryos from butyrolactone I- prematured bovine oocytes had the same developmental potential as the non treated one. The present study provided a method for laboratories of in vitro embryos production, mainly those working on the SCNT, where prematuration could be used to iiacrease the flexibility of the procedure.展开更多
The aim of this study was to compare the effect of GPAG and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after the fertilization. COCs were aspirated from follicles and cultured ...The aim of this study was to compare the effect of GPAG and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after the fertilization. COCs were aspirated from follicles and cultured for 16, 24, 32, 40 and 48 h in TCM-199 medium either with GPAG or FCS. After 24 h with GPAG, 89.4% of oocytes reached M Ⅰ stage while in the medium supplemented with FCS, only 27.7% of oocytes reached the same stage （P〈0.05）. Prolonged incubation for up to 32 h clearly demonstrated that some of oocytes cultured in GPAG medium were at M Ⅱ stage （35.7%）, few of oocytes from FCS medium were at M Ⅱ stage （7.5%） （P〈0.05）. Both groups of oocytes reached the same stage of maturation within 48 h. After 48 h of culture, the oocytes with extruded polar bodies were inseminated. Fertilized oocytes were cultured in PZM3 medium supplemented with 3 mg.mL of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPAG significantly increased their subsequent developmental ability when compared with FCS supplementation （29.2% ： 18.9% of blastocysts, P〈0.05）. However, differential staining revealed that once blastocysts were formed in either group, they had the same total cell number （39 ： 38） and the ICM/total cell ratio （0.26 ： 0.28）展开更多
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
Insulin-like growth factor-I （IGF-I） plays a key role in female reproduction, because it has the effect of anti-apoptosis improving cell proliferation, transformation and differentiation. This paper reviewed the eff...Insulin-like growth factor-I （IGF-I） plays a key role in female reproduction, because it has the effect of anti-apoptosis improving cell proliferation, transformation and differentiation. This paper reviewed the effects of IGF-I on ovary, follicle growth, acquisition of oocyte competence and preimplantation embryo viability, and then summarized different points about IGF-1 for reproduction system展开更多
Some charaterasics of female fetal reproductive system were studied in different gestation phases of Chinese northeast fine-wool sheep. The results showed that reproductive system consisted of ovary, oviduct, uterus a...Some charaterasics of female fetal reproductive system were studied in different gestation phases of Chinese northeast fine-wool sheep. The results showed that reproductive system consisted of ovary, oviduct, uterus and genitalia. The fetal ovaries were granular in early phase and became elliptic in later phase. The functional formula of ovarian weight, length and volume was obtained. Primary oocyte was encapsulated by monolayer cells in 7-week fetal ovary cortex. Primordial follicles were formed in 8-week fetal ovary, the follicular cells were sporadically arranged and not always regular. Complete organization of primordial follicles appeared until 10-week gestation, most of them scattered in nests in the following weeks of gestation. Two types of follicular complexes were found in fetal. There were continuous and quick mitosis of oogonia in fetal ovaries, large complexes were formed by oogoniaes encapsulated by multilayer of follicular cells, then they differentiated into small complexes, one or more primary oocytes were formed by mitosis of oogonia, primordial follicles which were caused by follicular cell approaching into them developed.展开更多
A series of experiments were conducted to study the major procedures in nuclear transplantation such as oocyte enucleation and activation, electrofusion and developnent of the nuclear transplant embryos in the mouse, ...A series of experiments were conducted to study the major procedures in nuclear transplantation such as oocyte enucleation and activation, electrofusion and developnent of the nuclear transplant embryos in the mouse, rabbits and sheep. The important results are as follows:11. In the mouse, only 35% of the oocytes collected 15～16 h after hCG had a notable first polar body (FPb) and those without FPb were enucleated by removing cytoplasm from the PVS-wider side and the enucleation rate was similar to that in the oocytes with FPb, and the enucleation rate of removing 1/3 cytoplasm was remarkably higher than that of removing 1/4 cytoplasm. 2. Among the three fusion media tested, mannitol and sucrose solutions produced better results than M2 in electrofusion of mouse 2-cell embryos. Under favorable pulse conditions, the osmotic pressure of fusion medium had no motable effect on electrofusion, but as the conditions became so unfavorable that some embryos began to lyse, the fusion rates in hypertonie mannitol solution were significantly higher than those in isotonic or hypotonic solutions. A wide range of pulse strengths (0.31～2.04 by/ cm) and durations(10～1280 μs) were used and 100% of fusion were obtained in many cases. Optimal pulse durations were plotted for field strengths to obtain high fusion rates (96%～ 100%) in mouse2-cell embryos. 3. With one pulse of 0.45 by / cm, satisfactory results of mouse oocyte activation were obtained only when the duration increased to 160 μs or longer. The activation rate increased as the oocytes got older. Some of the oocytes ar. rested at metaphase Ⅲ after electrical stimulation and their proportion to the number of oocytes not activated increased with egg age. 4. 10% and 31% of the nuclear transplant embryos developed to morula or blastocyst stage in sheep and rabbits, respectively, with Chinese-made hormones and chemicals.展开更多
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 by the Scientific Research Fund of Heilongjiang Provincial Education Department (11531028)Heilongjiang Postdoctoral Fund (LBH-Z11245)
文摘The somatic cell nuclear transfer （SCNT） technique has been applied successfully in a range of mammalian species giving rise to offspring, however, the efficiency of development to term has remained low. Oocyte meiotic arrest is an important option to make the SCNT more flexible and increase the number of cloned embryos produced. This paper showed that the use of butyrolactone I to arrest the meiotic division for 24 h prior to in vitro maturation （IVM） provided bovine oocytes capability of supporting development of blastocysts as efficiently as non arrested oocytes. In the SCNT group, cleavage rates were not affected by prematuration when adding butyrolactone I during IVM （57.9% vs. 62.4%; control vs. 10 μmol.L^-1 butyrolactone I）, developmental rates to biastocyst were unaffected （22.0% vs. 20.3%; control vs. 10 μmol.L^-1 butyrolactone I, p〉0.05） by the addition ofbutyrolactone I during IVM in cloned embryos. Moreover, the total cell numbers of blastocyst were not affected by butyrolactone I （total nucleus numbers, 132±16.5 vs. 128±19.4, p〉0.05）. In conclusion, the SCNT embryos from butyrolactone I- prematured bovine oocytes had the same developmental potential as the non treated one. The present study provided a method for laboratories of in vitro embryos production, mainly those working on the SCNT, where prematuration could be used to iiacrease the flexibility of the procedure.
文摘The aim of this study was to compare the effect of GPAG and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after the fertilization. COCs were aspirated from follicles and cultured for 16, 24, 32, 40 and 48 h in TCM-199 medium either with GPAG or FCS. After 24 h with GPAG, 89.4% of oocytes reached M Ⅰ stage while in the medium supplemented with FCS, only 27.7% of oocytes reached the same stage （P〈0.05）. Prolonged incubation for up to 32 h clearly demonstrated that some of oocytes cultured in GPAG medium were at M Ⅱ stage （35.7%）, few of oocytes from FCS medium were at M Ⅱ stage （7.5%） （P〈0.05）. Both groups of oocytes reached the same stage of maturation within 48 h. After 48 h of culture, the oocytes with extruded polar bodies were inseminated. Fertilized oocytes were cultured in PZM3 medium supplemented with 3 mg.mL of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPAG significantly increased their subsequent developmental ability when compared with FCS supplementation （29.2% ： 18.9% of blastocysts, P〈0.05）. However, differential staining revealed that once blastocysts were formed in either group, they had the same total cell number （39 ： 38） and the ICM/total cell ratio （0.26 ： 0.28）
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Insulin-like growth factor-I （IGF-I） plays a key role in female reproduction, because it has the effect of anti-apoptosis improving cell proliferation, transformation and differentiation. This paper reviewed the effects of IGF-I on ovary, follicle growth, acquisition of oocyte competence and preimplantation embryo viability, and then summarized different points about IGF-1 for reproduction system
基金Supported by Heilongjiang Province Nature Science Fund(ZJN04-02)
文摘Some charaterasics of female fetal reproductive system were studied in different gestation phases of Chinese northeast fine-wool sheep. The results showed that reproductive system consisted of ovary, oviduct, uterus and genitalia. The fetal ovaries were granular in early phase and became elliptic in later phase. The functional formula of ovarian weight, length and volume was obtained. Primary oocyte was encapsulated by monolayer cells in 7-week fetal ovary cortex. Primordial follicles were formed in 8-week fetal ovary, the follicular cells were sporadically arranged and not always regular. Complete organization of primordial follicles appeared until 10-week gestation, most of them scattered in nests in the following weeks of gestation. Two types of follicular complexes were found in fetal. There were continuous and quick mitosis of oogonia in fetal ovaries, large complexes were formed by oogoniaes encapsulated by multilayer of follicular cells, then they differentiated into small complexes, one or more primary oocytes were formed by mitosis of oogonia, primordial follicles which were caused by follicular cell approaching into them developed.
文摘A series of experiments were conducted to study the major procedures in nuclear transplantation such as oocyte enucleation and activation, electrofusion and developnent of the nuclear transplant embryos in the mouse, rabbits and sheep. The important results are as follows:11. In the mouse, only 35% of the oocytes collected 15～16 h after hCG had a notable first polar body (FPb) and those without FPb were enucleated by removing cytoplasm from the PVS-wider side and the enucleation rate was similar to that in the oocytes with FPb, and the enucleation rate of removing 1/3 cytoplasm was remarkably higher than that of removing 1/4 cytoplasm. 2. Among the three fusion media tested, mannitol and sucrose solutions produced better results than M2 in electrofusion of mouse 2-cell embryos. Under favorable pulse conditions, the osmotic pressure of fusion medium had no motable effect on electrofusion, but as the conditions became so unfavorable that some embryos began to lyse, the fusion rates in hypertonie mannitol solution were significantly higher than those in isotonic or hypotonic solutions. A wide range of pulse strengths (0.31～2.04 by/ cm) and durations(10～1280 μs) were used and 100% of fusion were obtained in many cases. Optimal pulse durations were plotted for field strengths to obtain high fusion rates (96%～ 100%) in mouse2-cell embryos. 3. With one pulse of 0.45 by / cm, satisfactory results of mouse oocyte activation were obtained only when the duration increased to 160 μs or longer. The activation rate increased as the oocytes got older. Some of the oocytes ar. rested at metaphase Ⅲ after electrical stimulation and their proportion to the number of oocytes not activated increased with egg age. 4. 10% and 31% of the nuclear transplant embryos developed to morula or blastocyst stage in sheep and rabbits, respectively, with Chinese-made hormones and chemicals.
