A cell-free system based upon the egg extracts from gynogenetic gibel carp (Carassius auratus gibelio)or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmentalbehaviors of th...A cell-free system based upon the egg extracts from gynogenetic gibel carp (Carassius auratus gibelio)or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmentalbehaviors of the demembranated sperm nuclei. Both red common carp and gibel carp sperm nuclei coulddecondense fully and form pronuclei in the red common carp egg extracts. Gibel carp sperm nuclei couldalso decondense fully and form pronuclei in the gibel carp egg extracts, but red common carp sperm nucleicould not decondense sufficiently in the same extracts. The significant differences of morphological changeswere further confirmed by ultrastructural observation of transmission electron microscopy. The data furtheroffer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel carp. Inaddition, the sperm nuclei in vitro decondensation is dependent on the pH in the extracts, and the decon-densed efficiency is optimal at pH 7. However, no DNA replication was observed in the two kinds of eggextracts during the incubation period of the sperm nuclei. It is suggested that the egg extracts preparedfrom the gynogenetic gibel carp should be a valid in vitro system for studying molecular mechanism ongynogenesis and reproduction mode diversity in fish.展开更多
After the third prezygotic division during conjugation of Paramecium caudatum,migratory and stationary pronuclei are produced.The migratory pronuclei remain in the paroral region tightly against the conjugating bounda...After the third prezygotic division during conjugation of Paramecium caudatum,migratory and stationary pronuclei are produced.The migratory pronuclei remain in the paroral region tightly against the conjugating boundaries;while the stationary pronuclei are located beside the migratory pronuclei.To date,however,it is not clear what causes this close side-by-side localization between migratory and stationary pronuclei.In the current study,immunofluorescence staining with monoclonal antibody of anti-α tubulin indicated that ＂U＂ or ＂V＂ shaped spindles connected the migratory and stationary pronuclei during the third prezygotic division.This observation accounts for the close localization between these two types of pronuclei.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
This study was conducted to investi- gate the pattern of DNA methylation in pronuclearstage mouse embryos derived from vitrified-warmed oocytes. Mouse oocytes at metaphase II （MII） stage of meiosis were allocated ra...This study was conducted to investi- gate the pattern of DNA methylation in pronuclearstage mouse embryos derived from vitrified-warmed oocytes. Mouse oocytes at metaphase II （MII） stage of meiosis were allocated randomly into three groups： 1） untreated （control）; 2 ） exposed to vitrification solution without being plunged into liquid nitrogen （toxicity）; and 3 ） vitrified by open-pulled straw （OPS） method （vitrification）. Oocytes were fertilized in vitro （IVF）. The level of DNA methylation was examined at 8 hpf （hours post-fertilization） by immunofluorescence using an anti-5-methylcytosine （5-MeC） monoclonal antibody and fluorescein isothiocyanate （FITC）-conjugated goat anti-mouse IgG. After IVF, rates of 2-cell embryos （ 51.39% ） and blastocysts （35.82%） for vitrified-warmed oocytes were lower （P〈0.01） than that for control （70.83%, 47.82% ） or vitrification solution treated （64.80%, 46.29% ） oocytes. At 8 hpf, there were more （P 〈 0.05） pronuclear-stage oocytes in which syngamy of pronuclei had not occurred in the vitrifica- tion group. In addition, 5-MeC fluorescent intensities for the female pronucleus and zygote were lower in the vitrification group （P 〈0.01 ） compared to pronuclear-stage embryos in the control and toxicity groups. In conclusion, oocyte vitrification causes a reduction in global genomic methylation in the female pronucleus and zygote, resulting in delayed fusion of pronuclei and compromising the developmental potential of mouse zygotes and embryos.展开更多
Background： In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically acti...Background： In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up（SU） or swim up ＋ zona pellucida（SU ＋ ZP） binding.Results： Experiment 1, 4–20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation（precense of one PN）. Treatments showed similar results（54, 47, 42 %, respectively） but statistically differents（P 0.05）.Conclusions： Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU ＋ ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU ＋ ZP were significantly different than frozen-thawed sperm,but between sperm treatments no significant differences were obtained.展开更多
Objective To determine the function of, in vivo, renin and its role in the pathogenesis of hypertension. Methods A renin 2 gene restriction map was constructed by endonuclease digest ion and Southern blotting hy...Objective To determine the function of, in vivo, renin and its role in the pathogenesis of hypertension. Methods A renin 2 gene restriction map was constructed by endonuclease digest ion and Southern blotting hybridization. Transgenic rats were produced via micro injection method.Results The 24 kb fragments containing mouse full length ren 2 and it s flanking sequence were cleaved by single enzymes (EcoRⅠ, KpnⅠ and BamHⅠ) an d combined enzymes (EcoRⅠ/KpnⅠ, KpnⅠ/BamHⅠ and BamHⅠ/EcoRⅠ), respectively. The digests were electrophoresed in 0.8% agarose plates and transferred onto NC membranes. Radioactive 735 bp and 1400 bp probes obtained from half and full l ength renin 1 cDNA were used in southern blotting hybridization. According to t he electrophoresis and hybridization patterns, a ren 2 restriction map was cons tructed. 1603 fertilized rat ova after injection with purified 24 kb renin 2 ge ne were implanted into the oviducts of 81 pseudopregnant recipients in about 2 0 ova per female rat. 306 progenies were obtained from 50 foster mothers (averag e of pregnancies was 56.6%). 248 survived pups were identified by PCR analys is and Southern hybridization, and eight positive rats were found to be the transgenic rats (founder, F). All of them carried long fragments (24 kb) of renin 2 gene with normal blood pressure. Preliminary breeding and screening were carried out in the founder. Total survival pups (17.8%) and overall efficiencies (1%) were h arvested as the same as those reported in the literatures. A systemic observatio n and the problems occurred during production of transgenic rats were also descr ibed besides the technique procedure used in this study.Conclusions Mapping of full length murine ren 2 can be used in invest igation of the structure and function of the gene. The results denoted that the ren 2 tran sgenic rats were successfully established in this study and the technique used i n the production of transgenic rats was proved to be valid in leading to wide s pread application of transgenic technique to many other related researches.展开更多
基金supported by grants from the National Natural Science Foundation of China(No.30130240 and 30070379)the Project of Chinese Academy of Sciences(KSCX2-SW-303)the Frontier Science Projects Program of the Institute of Hydrobiology,Chinese Academy of Sciences(No.220309).
文摘A cell-free system based upon the egg extracts from gynogenetic gibel carp (Carassius auratus gibelio)or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmentalbehaviors of the demembranated sperm nuclei. Both red common carp and gibel carp sperm nuclei coulddecondense fully and form pronuclei in the red common carp egg extracts. Gibel carp sperm nuclei couldalso decondense fully and form pronuclei in the gibel carp egg extracts, but red common carp sperm nucleicould not decondense sufficiently in the same extracts. The significant differences of morphological changeswere further confirmed by ultrastructural observation of transmission electron microscopy. The data furtheroffer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel carp. Inaddition, the sperm nuclei in vitro decondensation is dependent on the pH in the extracts, and the decon-densed efficiency is optimal at pH 7. However, no DNA replication was observed in the two kinds of eggextracts during the incubation period of the sperm nuclei. It is suggested that the egg extracts preparedfrom the gynogenetic gibel carp should be a valid in vitro system for studying molecular mechanism ongynogenesis and reproduction mode diversity in fish.
文摘After the third prezygotic division during conjugation of Paramecium caudatum,migratory and stationary pronuclei are produced.The migratory pronuclei remain in the paroral region tightly against the conjugating boundaries;while the stationary pronuclei are located beside the migratory pronuclei.To date,however,it is not clear what causes this close side-by-side localization between migratory and stationary pronuclei.In the current study,immunofluorescence staining with monoclonal antibody of anti-α tubulin indicated that ＂U＂ or ＂V＂ shaped spindles connected the migratory and stationary pronuclei during the third prezygotic division.This observation accounts for the close localization between these two types of pronuclei.
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.
文摘This study was conducted to investi- gate the pattern of DNA methylation in pronuclearstage mouse embryos derived from vitrified-warmed oocytes. Mouse oocytes at metaphase II （MII） stage of meiosis were allocated randomly into three groups： 1） untreated （control）; 2 ） exposed to vitrification solution without being plunged into liquid nitrogen （toxicity）; and 3 ） vitrified by open-pulled straw （OPS） method （vitrification）. Oocytes were fertilized in vitro （IVF）. The level of DNA methylation was examined at 8 hpf （hours post-fertilization） by immunofluorescence using an anti-5-methylcytosine （5-MeC） monoclonal antibody and fluorescein isothiocyanate （FITC）-conjugated goat anti-mouse IgG. After IVF, rates of 2-cell embryos （ 51.39% ） and blastocysts （35.82%） for vitrified-warmed oocytes were lower （P〈0.01） than that for control （70.83%, 47.82% ） or vitrification solution treated （64.80%, 46.29% ） oocytes. At 8 hpf, there were more （P 〈 0.05） pronuclear-stage oocytes in which syngamy of pronuclei had not occurred in the vitrifica- tion group. In addition, 5-MeC fluorescent intensities for the female pronucleus and zygote were lower in the vitrification group （P 〈0.01 ） compared to pronuclear-stage embryos in the control and toxicity groups. In conclusion, oocyte vitrification causes a reduction in global genomic methylation in the female pronucleus and zygote, resulting in delayed fusion of pronuclei and compromising the developmental potential of mouse zygotes and embryos.
文摘Background： In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up（SU） or swim up ＋ zona pellucida（SU ＋ ZP） binding.Results： Experiment 1, 4–20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation（precense of one PN）. Treatments showed similar results（54, 47, 42 %, respectively） but statistically differents（P 0.05）.Conclusions： Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU ＋ ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU ＋ ZP were significantly different than frozen-thawed sperm,but between sperm treatments no significant differences were obtained.
文摘Objective To determine the function of, in vivo, renin and its role in the pathogenesis of hypertension. Methods A renin 2 gene restriction map was constructed by endonuclease digest ion and Southern blotting hybridization. Transgenic rats were produced via micro injection method.Results The 24 kb fragments containing mouse full length ren 2 and it s flanking sequence were cleaved by single enzymes (EcoRⅠ, KpnⅠ and BamHⅠ) an d combined enzymes (EcoRⅠ/KpnⅠ, KpnⅠ/BamHⅠ and BamHⅠ/EcoRⅠ), respectively. The digests were electrophoresed in 0.8% agarose plates and transferred onto NC membranes. Radioactive 735 bp and 1400 bp probes obtained from half and full l ength renin 1 cDNA were used in southern blotting hybridization. According to t he electrophoresis and hybridization patterns, a ren 2 restriction map was cons tructed. 1603 fertilized rat ova after injection with purified 24 kb renin 2 ge ne were implanted into the oviducts of 81 pseudopregnant recipients in about 2 0 ova per female rat. 306 progenies were obtained from 50 foster mothers (averag e of pregnancies was 56.6%). 248 survived pups were identified by PCR analys is and Southern hybridization, and eight positive rats were found to be the transgenic rats (founder, F). All of them carried long fragments (24 kb) of renin 2 gene with normal blood pressure. Preliminary breeding and screening were carried out in the founder. Total survival pups (17.8%) and overall efficiencies (1%) were h arvested as the same as those reported in the literatures. A systemic observatio n and the problems occurred during production of transgenic rats were also descr ibed besides the technique procedure used in this study.Conclusions Mapping of full length murine ren 2 can be used in invest igation of the structure and function of the gene. The results denoted that the ren 2 tran sgenic rats were successfully established in this study and the technique used i n the production of transgenic rats was proved to be valid in leading to wide s pread application of transgenic technique to many other related researches.
