Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ （M Ⅱ ） oocytes. Oocyte growth differentiation factor-9 （GDF-9） gene expression was measured during different growth stages to ex...Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ （M Ⅱ ） oocytes. Oocyte growth differentiation factor-9 （GDF-9） gene expression was measured during different growth stages to explore the relationship between oocyte maturation and GDF-9 gene expression. Preantral follicles of lO-day old mice were isolated from the ovaries and were cultured for 12 days. Oocytes from day 2 （D2）, D4, D6, D8, DIO, D12 cultured in vitro were named the in vitro group and oocytes of day 12 （D12）, D14, D16, D18, D20, D22 grown in vivo were named the in vivo group. Follicle survival, antrum formation and maturation rate were 89.5%, 51.8% and 56.6% respectively in follicles cultured in vitro. After RT-PCR and agarose gel electrophoresis, relative mRNA abundance of GDF-9 was measured in each group of oocytes. At day 8 - 12, the GDF-9 gene expression level of oocytes in vitro was significantly lower than that in vivo （P 〈 0.05）. We conclude that M Ⅱ oocytes can be obtained from in vitro culture of preantral follicles. The GDF- 9 gene expression of oocytes varies at different growth stages in vivo. The low expression of GDF-9 in oocytes cuhured in vitro may be the cause of their low developmental capacity.展开更多
Comparisons of activation rates and fertilization rates were made among oocytes at different ages. Results showed that oocytes at different ages had different activation and fertilization rates when stimulated by sper...Comparisons of activation rates and fertilization rates were made among oocytes at different ages. Results showed that oocytes at different ages had different activation and fertilization rates when stimulated by sperm or ethanol. Oocytes at 15～24 h after the injection of hCG were readily activated by 8% ethanol. The activation rate of oocytes increased with the age of oocytes, up to the highest average of 81.6%, but decreased after 20 h posthCG. Oocytes at 20 h posthCG exhibited the highest immediate cleavage rate(48.0%) after being stimulated by ethanol. On the other hand, 13～15 h oocytes exhibited higher fertilization rates, and the older oocytes were more difficult to be fertilized by sperm in vitro. These suggest that oocytes can be activated in different ways; the mechanism of fertilization might be different from that of activation; and in vitro fertilization is more dependent on oocyte age.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
Tripchlorolide (TC) is a potent antitumor reagent purified from a Chinese herb Tripterygium Wilfordii Hook. f.. However, its cellular effects and mechanism of action are unknown. We showed here that TC induced apoptos...Tripchlorolide (TC) is a potent antitumor reagent purified from a Chinese herb Tripterygium Wilfordii Hook. f.. However, its cellular effects and mechanism of action are unknown. We showed here that TC induced apoptosis of Chinese Hamster Ovary (CHO) cells in time- and dose-dependent manners. TC resulted in the degradation of Bcl-2, the translocation of Bax from the cytosol to mitochondria, and the release of cytochrome c from mitochondria. Stable overexpression of human Bcl-2 could reduce the apoptosis of TCtreated cells by blocking the translocation of Bax and the release of cytochrome c. These results indicate that TC induces apoptosis of CHO cell by activating the mitochondrion-mediated apoptotic pathway involving the proteins of Bcl-2 family and cytochrome c.展开更多
Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, a chain), and CDlla (LFA-1, a chain) on mouse oocytes, and pre- and peri-implantation stage embryos was examined by quantitative indirect...Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, a chain), and CDlla (LFA-1, a chain) on mouse oocytes, and pre- and peri-implantation stage embryos was examined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at the oocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM, also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On the other hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at the compacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophecto-derm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remained significantly expressed throughout and after blastocyst hatching was expressed on the polar trophecto-derm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression of both VCAM-1 and CDlla was undetectable throughout. The diametrical temporal expression pattern of ICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesion molecules may be important for interaction of the embryo with the maternal cellular environment as well as for continuing development and survival of the early embryo.展开更多
AIM: Hepatitis B is a worldwide public health problem. To explore the feasibility of hepatitis B virus (HBV) vertical transmission via oocytes, the presence and integration of HBV DNA in mouse oocytes were studied. ME...AIM: Hepatitis B is a worldwide public health problem. To explore the feasibility of hepatitis B virus (HBV) vertical transmission via oocytes, the presence and integration of HBV DNA in mouse oocytes were studied. METHODS: Genomic DNA was isolated and metaphases were prepared, respectively from mouse oocytes cocultured with pBR322-HBV DNA plasmids. PCR, Southern blot, dot hybridization and fluorescence in situ hybridization (FISH) were performed to explore the existence and integration of HBV DNA in oocytes.RESULTS: PCR detected positive bands in the tested samples, and then Southern blot revealed clear hybridization signals in PCR products. Final washing solutions were collected for dot hybridization and no signal for HBV DNA was observed, which excluded the possibility that contamination of washing solutions gave rise to positive results of PCR and Southern blot. FISH demonstrated that 36 of 1 000 metaphases presented positive signals. CONCLUSION: HBV DNA sequences are able to pass through the zona and oolemma to enter into oocytes and tointegrate into their chromosomes. HBV DNA sequences might be brought into embryo via oocytes as vectors when they are fertilized with normal spermatozoa.展开更多
Mature eggs (at metaphase Ⅱ stage) produce a series of Ca2+ oscillation at fertilization. To define whether the fertilization-induced Ca2+ oscillation is restrict to the metaphase Ⅱ eggs and cell cycle dependent, mo...Mature eggs (at metaphase Ⅱ stage) produce a series of Ca2+ oscillation at fertilization. To define whether the fertilization-induced Ca2+ oscillation is restrict to the metaphase Ⅱ eggs and cell cycle dependent, mouse oocytes at prophase Ⅰ (arrested at germinal vesicle stage),metaphase Ⅰ, metaphase Ⅱ, as well as the pronuclear embryos at interphase of the first mitotic division derived from fertilization or parthenogenetic activation were inseminated after removal of zona pellucida. The results show that the fertilization-induced Ca2+ oscillation is not specific to metaphase Ⅱ eggs. This is supported by the fact that immature oocytes generated the Ca2+ oscillations at fertilization regardless of their nuclear progression from prophase Ⅰ to metaphase Ⅰ (in vitro matured) stage. More interestingly, it was first found that pronuclear embryos at interphase derived from parthenogenetic activation showed Ca2+ oscillations in response to fertilization while the zygotes at interphase did not after reinsemination or intracytoplasmic injection of sperm extracts which induce Ca2+ oscillations in MII eggs. This suggests that the ability of oocytes to generate Ca2+ oscillation in response to sperm penetration is not regulated in a cell cycle dependent manner but dependent on the cytoplasmic maturation.展开更多
Mouse oocytes undergo polarization during meiotic maturation, and this polarization is essential for asymmetric cell divisions that maximize retention of maternal components required for early development. Without con...Mouse oocytes undergo polarization during meiotic maturation, and this polarization is essential for asymmetric cell divisions that maximize retention of maternal components required for early development. Without conventional centrosomes, the meiotic spindle has less focused poles and is barrel-shaped. The migration of meiotic spindles to the cortex is accompanied by a local reorganization and polarization of the cortex. LGN is a conserved protein involved in cell polarity and regulation of spindle organization. In the present study, we characterized the localization dynamics of LGN during mouse oocyte maturation and analyzed the effects of LGN upregulation and downregulation on meiotic spindle organization. At the germinal vesicle stage, LGN is distributed both cytoplasmically and at the cortex. During maturation, LGN localizes to the meiotic spindle apparatus and cortical LGN becomes less concentrated at the actin cap region. Excessive LGN induces meiotic spindle organization defects by elongating the spindle and enhancing pole focusing, whereas depletion of LGN by RNA interference results in meiotic spindle deformation and chromosome misalignment. Furthermore, the N-terminus of LGN has the ability of full-length LGN to regulate spindle organization, whereas the C-terminus of LGN controls cortical localization and polarization. Our results reveal that LGN is cortically polarized in mouse oocytes and is critical for meiotic spindle organization.展开更多
Although the role of oxidative stress in maternal aging and infertility has been suggested, the underlying mechanisms are not fully understood. The present study is designed to determine the relationship between mitoc...Although the role of oxidative stress in maternal aging and infertility has been suggested, the underlying mechanisms are not fully understood. The present study is designed to determine the relationship between mitochondrial function and spindle stability in metaphase II (MII) oocytes under oxidative stress. MII mouse oocytes were treated with H2O2 in the presence or absence of permeability transition pores (PTPs) blockers cyclosporin A (CsA). In addition, antioxidant N-acetylcysteine (NAC), F0/F1 synthase inhibitor oligomycin A, the mitochondria uncoupler carbonyl cyanide 4-trifluoro- methoxyphenylhydrazone (FCCP) or thapsigargin plus 2.5 mM Ca^2+ (Th+2.5 mM Ca^2+) were used in mechanistic studies. Morphologic analyses of oocyte spindles and chromosomes were performed and mitochondrial membrane potential (AWm), cytoplasmic free calcium concentration ([Ca^2+]c) and cytoplasmic ATP content within oocytes were also assayed. In a time- and H202 dose-dependent manner, disruption of meiotic spindles was found after oocytes were treated with H202, which was prevented by pre-treatment with NAC. Administration of H2O2 led to a dissipation of AWm, an increase in [Ca^2+]c and a decrease in cytoplasmic ATP levels. These detrimental responses of oocytes to H2O2 treatment could be blocked by pre-incubation with CsA. Similar to H2O2, both oligomycin A and FCCP dissipated AWm, decreased cytoplasmic ATP contents and disassembled MII oocyte spindles, while high [Ca^2+]c alone had no effects on spindle morphology. In conclusion, the decrease in mitochondria-derived ATP during oxidative stress may cause a disassembly of mouse MII oocyte spindles, presumably due to the opening of the mitochondrial PTPs.展开更多
To study the effects of XW630 on bone formation in overiectomized(OVX) rats and in human osteoblast like cell line TE85. [WT5”BX]Method.[WT5”BZ] Bone histomorphometric analysis was performed with undecalcified bone ...To study the effects of XW630 on bone formation in overiectomized(OVX) rats and in human osteoblast like cell line TE85. [WT5”BX]Method.[WT5”BZ] Bone histomorphometric analysis was performed with undecalcified bone sections and tetracycline intraperitoneally labeling. [WT5”BX]Results.[WT5”BZ] Compared with that of OVX rats, the static data of trabecular bone volume(TBV)/ total tissue volume(TTV), TBV/sponge bone volume(SBV) and mean trabecular plate density (MTPD) were enhanced while mean trabecular plate spacing(MTPS) decreased after treated with XW630 for 13w. The dynamic data of single labeled surface [Sfract(s)], double labeled surface[Sfract(d)],Sfract(d+1/2s),trabecular osteoid surface(TOS), and bone formation rate in tissue level (Svf) were increased and osteoid maturation period (OMP) shortened in XW630 group. In osteoblast like cells, both 3H thymidine incorporation and cell count increased after treated with XW630 for 48. Treated with XW630 for 12～18h,inducible nitric oxide synthase(iNOS) activity and cGMP content increased in time dependent manners. [WT5”BX]Conclusions.[WT5”BZ] XW630 enhanced bone activation frequency and increased trabecular connectivity, stability, and strength. The cellular mechanism related to effects of XW630 on bone formation in ovariectomized rats.展开更多
Objective To acquire oval cells （progenitor stem cells ） from adult rat liver of different models including diabetic rats. Methods Thirty Sprague-Dawley （ SD ） rats were divided into 5 groups randomly： control, 2...Objective To acquire oval cells （progenitor stem cells ） from adult rat liver of different models including diabetic rats. Methods Thirty Sprague-Dawley （ SD ） rats were divided into 5 groups randomly： control, 2-acetylaminofluorene （ 2-AAF ）, 2-AAF ＋ partial hepatectomy （ PH ）, 2-AAF ＋ carbon tetrachloride （ CCl4 ）, and diabetic groups. As two-step collagenase perfusion protocol of Seglen, oval cells were isolated by Percoll density gradient centrifugation. Thy1. 1 positive cells were sorted by flow cytometry, and then cultured in Dulbecco＇s minimum Eagle＇s medium （DMEM）. Immunofluorescence staining was applied to labelling Thyl. 1. Results Different rates of Thy1.1 positive oval cells were found in different rat model groups ： 0. 5 % in 2-AAF, 0. 3% in 2-AAF ＋ PH, 0. 2% in 2-AAF ＋ CCl4, 0. 1% in diabetic, and 0. 0% in control. Isolated cells adhered to plate with fusiform or polygon as epithelial cells. Conclusion Progenitor stem cells exist in injured liver tissue including those from diabetic rats.展开更多
An expressive plasmid pSMTPCH was constructed from porcine growth hormone gene,sheep metallothionein promoter (MT-011)and the vector,pUC19. The linear pSMTPGH and circular pSV2-dhfr were cotransfected into CHO-dhfr ce...An expressive plasmid pSMTPCH was constructed from porcine growth hormone gene,sheep metallothionein promoter (MT-011)and the vector,pUC19. The linear pSMTPGH and circular pSV2-dhfr were cotransfected into CHO-dhfr cell by calcium phosphate coprecipitation. Positive clones made up 74% of total clones, which were identified with ELISA. The expression of pSMTPGH was induced by 0.5 μM of Cd￣++. The clone 1-C-3 was found to secrete hGH at the level of 3800 μg/10￣6 cells/24 hrs in media containing 10 μMTX. After 20 generations in culture, the clone was still stable with hGH expression.The molecular weight of secreted protein was the same as that of the natural pGH, 22KD;the identity was further supported by Western blot.展开更多
Objective : To study the antitoxic role of vesicular monoamine transporter 2 (VMAT2) in transgenic Chinese Hamster ovary (CHO) cell. Methods :With the technology of trans-gene from PC 12 to CHO, MTT reduction assay wa...Objective : To study the antitoxic role of vesicular monoamine transporter 2 (VMAT2) in transgenic Chinese Hamster ovary (CHO) cell. Methods :With the technology of trans-gene from PC 12 to CHO, MTT reduction assay was used to detect MPP+ toxic effect on wild type CHO (wtCHO) and transgenic CHO. Meanwhile, the role of reserpine was also observed in MPP+ toxic effects. Results :The sensitivity of transgenic CHO to MPP+ was much less than that of wtCHO with 0. 5 mmol/L MPP+. Transgenic CHO had the same sensitivity as wtCHO if rotenone was given. WtCHO, by given reserpine alone, didn't change its sensitivity to MPP+. Conclusions :VMAT2 has protective effect on transgenic CHO by transporting MPP+ to vesicles.展开更多
Guava leaf tea has been used as a folk medicine for treating hyperglycemic conditions in Asia and Africa. The hypoglycemic efficacy of guava leaf has been documented by many scientists in these regions, but the hypogl...Guava leaf tea has been used as a folk medicine for treating hyperglycemic conditions in Asia and Africa. The hypoglycemic efficacy of guava leaf has been documented by many scientists in these regions, but the hypoglycemic mechanism is poorly understood. Guava leaves were extracted with methanol and the crude extract was partitioned against hexane, ethyl acetate, and butanol in sequence. The leftover in water is defined as the aqueous partition. A second smaller batch was extracted with hot water directly. Oral glucose tolerance test was carried out on healthy mice instead of diabetic mice that lack endogenous insulin. Glucose uptake was examined with 3T3-L1 adipocytes. Oxidative effect on PTP1B （protein tyrosine phosphatase 1b） was carried out with real-time PTP1B enzymatic assay. The aqueous partition of guava leaf extract possesses a potent inhibitory effect on PTP1B enzymatic activity and this PTP1B inhibition is through a slow oxidative but reversible inactivation on the enzyme. The reversible inactivation would suggest guava leaf extract may augment PTP1B inhibition alongside the endogenous H2O2 which itself is induced by insulin. In addition, our study confirmed the hypoglycemic efficacy being associated with guava leaf and found the most effective molecules reside in the aqueous partition which is also less cytotoxic to Chinese hamster ovary cells when compared to other less polar partitions. The guava leaf extract can modulate insulin activity through a redox regulation on PP1B enzymatic activity. It is speculated that a compound similar to gallocatechin in the aqueous partition can reduce an oxygen molecule to hydrogen peroxide which in turn oxidizes the catalytic residue Cys in PTP1B. Therefore, the guava leaf tea can serve as a functional hypoglycemic drink that is suitable for either healthy or diabetic subjects.展开更多
On Nov 17th,a team of researchers from the Shanghai Institute of Biochemistry and Cell Biology（SIBCB）,Shanghai Institutes for Biological Sciences,CAS led by Prof.LI Jinsong reports online in Cell Research a novel te...On Nov 17th,a team of researchers from the Shanghai Institute of Biochemistry and Cell Biology（SIBCB）,Shanghai Institutes for Biological Sciences,CAS led by Prof.LI Jinsong reports online in Cell Research a novel technique to induce from mice oocytes haploid embryonic stems cells（haESCs）that can fully replace the reproductive functions of sperms,greatly simplifying the otherwise complicated techniques to produce such stem cells and semi-cloned（SC）mice.It is anticipated that this will further facilitate research in the field of stem cells and embryonic development;展开更多
Objective This study investigated the feasibility of screening residual normal ovarian tissues based on the expression of Bmi-1 and EZH2 in tissues adjacent to orthotopic ovarian carcinomas in nude mice. Methods The h...Objective This study investigated the feasibility of screening residual normal ovarian tissues based on the expression of Bmi-1 and EZH2 in tissues adjacent to orthotopic ovarian carcinomas in nude mice. Methods The human epithelial ovarian cancer cell line OVCAR3 was grown in subcutaneous tissues and the tumor tissues were orthotopically implanted. The expression levels of Bmi-1 and EZH2 were detected by immunohistochemical staining and RT-PCR in cancer tissues, proximal and remote tissues with respect to the cancer tissues, and normal ovarian tissues of nude mice.Results Thirty-five ovarian tissue samples with normal biopsy results were obtained from 40 cases of human epithelial ovarian cancer in the nude mice in which the tumor tissues were orthotopically implanted. Bmi-1 and EZH2 expression levels were lower in proximal paraneoplastic tissue samples than in cancer tissue samples(P < 0.05) and higher than in remote paraneoplastic tissue samples(P < 0.01). No significant difference was found in the expression levels of Bmi-1 and EZH2 using immunohistochemistry among residual normal ovarian tissues obtained from orthotopically implanted models that differed in severity. The expression of Bmi-1 and EZH2 was negative in 20 normal ovarian tissue samples.Conclusion The expression levels of Bmi-1 and EZH2 were reduced with increasing distance from the cancer tissues. Negative expression of these tumor-associated genes can be used as a standard for the screening of normal ovarian tissues adjacent to tumor tissues. Normal ovarian tissues can be obtained from the tissues adjacent to tumors.展开更多
The effects of equine chorionic gonadotropin （eCG） on follicular development and ovulation in cyclic guinea pigs were investigated by histological and immunohistochemical analyses. Three groups of guinea pigs （n=12...The effects of equine chorionic gonadotropin （eCG） on follicular development and ovulation in cyclic guinea pigs were investigated by histological and immunohistochemical analyses. Three groups of guinea pigs （n=12） were administrated subcutaneously with saline, 20 or 50 IU of eCG, respectively, on cyclic Day 12 （Day 1=vaginal openings）. Ovaries were collected at 4 and 8 d after administration （6 animals per group each time）. The eCG administration induced significant and distinct morphological changes in the ovaries, as it promoted the luteinization of granulosa cells, but not follicular development. In addition, proliferating cell nuclear antigen （PCNA） and steroidogenic acute regulatory protein （STAR） were immunolocalized specifically in luteinized follicles. Our experiments together indicate that eCG administration can induce follicular luteinization but not superovulation in guinea pigs. The eCG in cyclic guinea pigs functions similar to that of luteinizing hormone （LH）, but not follicle-stimulating hormone （FSH）.展开更多
文摘Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ （M Ⅱ ） oocytes. Oocyte growth differentiation factor-9 （GDF-9） gene expression was measured during different growth stages to explore the relationship between oocyte maturation and GDF-9 gene expression. Preantral follicles of lO-day old mice were isolated from the ovaries and were cultured for 12 days. Oocytes from day 2 （D2）, D4, D6, D8, DIO, D12 cultured in vitro were named the in vitro group and oocytes of day 12 （D12）, D14, D16, D18, D20, D22 grown in vivo were named the in vivo group. Follicle survival, antrum formation and maturation rate were 89.5%, 51.8% and 56.6% respectively in follicles cultured in vitro. After RT-PCR and agarose gel electrophoresis, relative mRNA abundance of GDF-9 was measured in each group of oocytes. At day 8 - 12, the GDF-9 gene expression level of oocytes in vitro was significantly lower than that in vivo （P 〈 0.05）. We conclude that M Ⅱ oocytes can be obtained from in vitro culture of preantral follicles. The GDF- 9 gene expression of oocytes varies at different growth stages in vivo. The low expression of GDF-9 in oocytes cuhured in vitro may be the cause of their low developmental capacity.
文摘Comparisons of activation rates and fertilization rates were made among oocytes at different ages. Results showed that oocytes at different ages had different activation and fertilization rates when stimulated by sperm or ethanol. Oocytes at 15～24 h after the injection of hCG were readily activated by 8% ethanol. The activation rate of oocytes increased with the age of oocytes, up to the highest average of 81.6%, but decreased after 20 h posthCG. Oocytes at 20 h posthCG exhibited the highest immediate cleavage rate(48.0%) after being stimulated by ethanol. On the other hand, 13～15 h oocytes exhibited higher fertilization rates, and the older oocytes were more difficult to be fertilized by sperm in vitro. These suggest that oocytes can be activated in different ways; the mechanism of fertilization might be different from that of activation; and in vitro fertilization is more dependent on oocyte age.
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.
基金supported by Grant 39825115 of the National Outstanding Young Scientists,a special grant of major state basic research program of China(No.G1999053901)a grant from the Chinese Academy of Sciences KSCX2-SW-203 to JR Wu.
文摘Tripchlorolide (TC) is a potent antitumor reagent purified from a Chinese herb Tripterygium Wilfordii Hook. f.. However, its cellular effects and mechanism of action are unknown. We showed here that TC induced apoptosis of Chinese Hamster Ovary (CHO) cells in time- and dose-dependent manners. TC resulted in the degradation of Bcl-2, the translocation of Bax from the cytosol to mitochondria, and the release of cytochrome c from mitochondria. Stable overexpression of human Bcl-2 could reduce the apoptosis of TCtreated cells by blocking the translocation of Bax and the release of cytochrome c. These results indicate that TC induces apoptosis of CHO cell by activating the mitochondrion-mediated apoptotic pathway involving the proteins of Bcl-2 family and cytochrome c.
文摘Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, a chain), and CDlla (LFA-1, a chain) on mouse oocytes, and pre- and peri-implantation stage embryos was examined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at the oocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM, also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On the other hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at the compacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophecto-derm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remained significantly expressed throughout and after blastocyst hatching was expressed on the polar trophecto-derm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression of both VCAM-1 and CDlla was undetectable throughout. The diametrical temporal expression pattern of ICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesion molecules may be important for interaction of the embryo with the maternal cellular environment as well as for continuing development and survival of the early embryo.
基金Supported by the National Natural Science Foundation of China,No. 39970374
文摘AIM: Hepatitis B is a worldwide public health problem. To explore the feasibility of hepatitis B virus (HBV) vertical transmission via oocytes, the presence and integration of HBV DNA in mouse oocytes were studied. METHODS: Genomic DNA was isolated and metaphases were prepared, respectively from mouse oocytes cocultured with pBR322-HBV DNA plasmids. PCR, Southern blot, dot hybridization and fluorescence in situ hybridization (FISH) were performed to explore the existence and integration of HBV DNA in oocytes.RESULTS: PCR detected positive bands in the tested samples, and then Southern blot revealed clear hybridization signals in PCR products. Final washing solutions were collected for dot hybridization and no signal for HBV DNA was observed, which excluded the possibility that contamination of washing solutions gave rise to positive results of PCR and Southern blot. FISH demonstrated that 36 of 1 000 metaphases presented positive signals. CONCLUSION: HBV DNA sequences are able to pass through the zona and oolemma to enter into oocytes and tointegrate into their chromosomes. HBV DNA sequences might be brought into embryo via oocytes as vectors when they are fertilized with normal spermatozoa.
文摘Mature eggs (at metaphase Ⅱ stage) produce a series of Ca2+ oscillation at fertilization. To define whether the fertilization-induced Ca2+ oscillation is restrict to the metaphase Ⅱ eggs and cell cycle dependent, mouse oocytes at prophase Ⅰ (arrested at germinal vesicle stage),metaphase Ⅰ, metaphase Ⅱ, as well as the pronuclear embryos at interphase of the first mitotic division derived from fertilization or parthenogenetic activation were inseminated after removal of zona pellucida. The results show that the fertilization-induced Ca2+ oscillation is not specific to metaphase Ⅱ eggs. This is supported by the fact that immature oocytes generated the Ca2+ oscillations at fertilization regardless of their nuclear progression from prophase Ⅰ to metaphase Ⅰ (in vitro matured) stage. More interestingly, it was first found that pronuclear embryos at interphase derived from parthenogenetic activation showed Ca2+ oscillations in response to fertilization while the zygotes at interphase did not after reinsemination or intracytoplasmic injection of sperm extracts which induce Ca2+ oscillations in MII eggs. This suggests that the ability of oocytes to generate Ca2+ oscillation in response to sperm penetration is not regulated in a cell cycle dependent manner but dependent on the cytoplasmic maturation.
文摘Mouse oocytes undergo polarization during meiotic maturation, and this polarization is essential for asymmetric cell divisions that maximize retention of maternal components required for early development. Without conventional centrosomes, the meiotic spindle has less focused poles and is barrel-shaped. The migration of meiotic spindles to the cortex is accompanied by a local reorganization and polarization of the cortex. LGN is a conserved protein involved in cell polarity and regulation of spindle organization. In the present study, we characterized the localization dynamics of LGN during mouse oocyte maturation and analyzed the effects of LGN upregulation and downregulation on meiotic spindle organization. At the germinal vesicle stage, LGN is distributed both cytoplasmically and at the cortex. During maturation, LGN localizes to the meiotic spindle apparatus and cortical LGN becomes less concentrated at the actin cap region. Excessive LGN induces meiotic spindle organization defects by elongating the spindle and enhancing pole focusing, whereas depletion of LGN by RNA interference results in meiotic spindle deformation and chromosome misalignment. Furthermore, the N-terminus of LGN has the ability of full-length LGN to regulate spindle organization, whereas the C-terminus of LGN controls cortical localization and polarization. Our results reveal that LGN is cortically polarized in mouse oocytes and is critical for meiotic spindle organization.
文摘Although the role of oxidative stress in maternal aging and infertility has been suggested, the underlying mechanisms are not fully understood. The present study is designed to determine the relationship between mitochondrial function and spindle stability in metaphase II (MII) oocytes under oxidative stress. MII mouse oocytes were treated with H2O2 in the presence or absence of permeability transition pores (PTPs) blockers cyclosporin A (CsA). In addition, antioxidant N-acetylcysteine (NAC), F0/F1 synthase inhibitor oligomycin A, the mitochondria uncoupler carbonyl cyanide 4-trifluoro- methoxyphenylhydrazone (FCCP) or thapsigargin plus 2.5 mM Ca^2+ (Th+2.5 mM Ca^2+) were used in mechanistic studies. Morphologic analyses of oocyte spindles and chromosomes were performed and mitochondrial membrane potential (AWm), cytoplasmic free calcium concentration ([Ca^2+]c) and cytoplasmic ATP content within oocytes were also assayed. In a time- and H202 dose-dependent manner, disruption of meiotic spindles was found after oocytes were treated with H202, which was prevented by pre-treatment with NAC. Administration of H2O2 led to a dissipation of AWm, an increase in [Ca^2+]c and a decrease in cytoplasmic ATP levels. These detrimental responses of oocytes to H2O2 treatment could be blocked by pre-incubation with CsA. Similar to H2O2, both oligomycin A and FCCP dissipated AWm, decreased cytoplasmic ATP contents and disassembled MII oocyte spindles, while high [Ca^2+]c alone had no effects on spindle morphology. In conclusion, the decrease in mitochondria-derived ATP during oxidative stress may cause a disassembly of mouse MII oocyte spindles, presumably due to the opening of the mitochondrial PTPs.
基金This project was supported by the National Natural ScienceFoundation of ChinaNo.39430 120
文摘To study the effects of XW630 on bone formation in overiectomized(OVX) rats and in human osteoblast like cell line TE85. [WT5”BX]Method.[WT5”BZ] Bone histomorphometric analysis was performed with undecalcified bone sections and tetracycline intraperitoneally labeling. [WT5”BX]Results.[WT5”BZ] Compared with that of OVX rats, the static data of trabecular bone volume(TBV)/ total tissue volume(TTV), TBV/sponge bone volume(SBV) and mean trabecular plate density (MTPD) were enhanced while mean trabecular plate spacing(MTPS) decreased after treated with XW630 for 13w. The dynamic data of single labeled surface [Sfract(s)], double labeled surface[Sfract(d)],Sfract(d+1/2s),trabecular osteoid surface(TOS), and bone formation rate in tissue level (Svf) were increased and osteoid maturation period (OMP) shortened in XW630 group. In osteoblast like cells, both 3H thymidine incorporation and cell count increased after treated with XW630 for 48. Treated with XW630 for 12～18h,inducible nitric oxide synthase(iNOS) activity and cGMP content increased in time dependent manners. [WT5”BX]Conclusions.[WT5”BZ] XW630 enhanced bone activation frequency and increased trabecular connectivity, stability, and strength. The cellular mechanism related to effects of XW630 on bone formation in ovariectomized rats.
基金Supported by Shanghai Municipal Education Commission Key Project (07ZZ35)
文摘Objective To acquire oval cells （progenitor stem cells ） from adult rat liver of different models including diabetic rats. Methods Thirty Sprague-Dawley （ SD ） rats were divided into 5 groups randomly： control, 2-acetylaminofluorene （ 2-AAF ）, 2-AAF ＋ partial hepatectomy （ PH ）, 2-AAF ＋ carbon tetrachloride （ CCl4 ）, and diabetic groups. As two-step collagenase perfusion protocol of Seglen, oval cells were isolated by Percoll density gradient centrifugation. Thy1. 1 positive cells were sorted by flow cytometry, and then cultured in Dulbecco＇s minimum Eagle＇s medium （DMEM）. Immunofluorescence staining was applied to labelling Thyl. 1. Results Different rates of Thy1.1 positive oval cells were found in different rat model groups ： 0. 5 % in 2-AAF, 0. 3% in 2-AAF ＋ PH, 0. 2% in 2-AAF ＋ CCl4, 0. 1% in diabetic, and 0. 0% in control. Isolated cells adhered to plate with fusiform or polygon as epithelial cells. Conclusion Progenitor stem cells exist in injured liver tissue including those from diabetic rats.
文摘An expressive plasmid pSMTPCH was constructed from porcine growth hormone gene,sheep metallothionein promoter (MT-011)and the vector,pUC19. The linear pSMTPGH and circular pSV2-dhfr were cotransfected into CHO-dhfr cell by calcium phosphate coprecipitation. Positive clones made up 74% of total clones, which were identified with ELISA. The expression of pSMTPGH was induced by 0.5 μM of Cd￣++. The clone 1-C-3 was found to secrete hGH at the level of 3800 μg/10￣6 cells/24 hrs in media containing 10 μMTX. After 20 generations in culture, the clone was still stable with hGH expression.The molecular weight of secreted protein was the same as that of the natural pGH, 22KD;the identity was further supported by Western blot.
基金Supported by grant from Innovation Foundation of Nanjing Medical University(MC9901)
文摘Objective : To study the antitoxic role of vesicular monoamine transporter 2 (VMAT2) in transgenic Chinese Hamster ovary (CHO) cell. Methods :With the technology of trans-gene from PC 12 to CHO, MTT reduction assay was used to detect MPP+ toxic effect on wild type CHO (wtCHO) and transgenic CHO. Meanwhile, the role of reserpine was also observed in MPP+ toxic effects. Results :The sensitivity of transgenic CHO to MPP+ was much less than that of wtCHO with 0. 5 mmol/L MPP+. Transgenic CHO had the same sensitivity as wtCHO if rotenone was given. WtCHO, by given reserpine alone, didn't change its sensitivity to MPP+. Conclusions :VMAT2 has protective effect on transgenic CHO by transporting MPP+ to vesicles.
文摘Guava leaf tea has been used as a folk medicine for treating hyperglycemic conditions in Asia and Africa. The hypoglycemic efficacy of guava leaf has been documented by many scientists in these regions, but the hypoglycemic mechanism is poorly understood. Guava leaves were extracted with methanol and the crude extract was partitioned against hexane, ethyl acetate, and butanol in sequence. The leftover in water is defined as the aqueous partition. A second smaller batch was extracted with hot water directly. Oral glucose tolerance test was carried out on healthy mice instead of diabetic mice that lack endogenous insulin. Glucose uptake was examined with 3T3-L1 adipocytes. Oxidative effect on PTP1B （protein tyrosine phosphatase 1b） was carried out with real-time PTP1B enzymatic assay. The aqueous partition of guava leaf extract possesses a potent inhibitory effect on PTP1B enzymatic activity and this PTP1B inhibition is through a slow oxidative but reversible inactivation on the enzyme. The reversible inactivation would suggest guava leaf extract may augment PTP1B inhibition alongside the endogenous H2O2 which itself is induced by insulin. In addition, our study confirmed the hypoglycemic efficacy being associated with guava leaf and found the most effective molecules reside in the aqueous partition which is also less cytotoxic to Chinese hamster ovary cells when compared to other less polar partitions. The guava leaf extract can modulate insulin activity through a redox regulation on PP1B enzymatic activity. It is speculated that a compound similar to gallocatechin in the aqueous partition can reduce an oxygen molecule to hydrogen peroxide which in turn oxidizes the catalytic residue Cys in PTP1B. Therefore, the guava leaf tea can serve as a functional hypoglycemic drink that is suitable for either healthy or diabetic subjects.
文摘On Nov 17th,a team of researchers from the Shanghai Institute of Biochemistry and Cell Biology（SIBCB）,Shanghai Institutes for Biological Sciences,CAS led by Prof.LI Jinsong reports online in Cell Research a novel technique to induce from mice oocytes haploid embryonic stems cells（haESCs）that can fully replace the reproductive functions of sperms,greatly simplifying the otherwise complicated techniques to produce such stem cells and semi-cloned（SC）mice.It is anticipated that this will further facilitate research in the field of stem cells and embryonic development;
基金Supported by a grant from the Health Department of Hainan Province(No.QW2013-040)
文摘Objective This study investigated the feasibility of screening residual normal ovarian tissues based on the expression of Bmi-1 and EZH2 in tissues adjacent to orthotopic ovarian carcinomas in nude mice. Methods The human epithelial ovarian cancer cell line OVCAR3 was grown in subcutaneous tissues and the tumor tissues were orthotopically implanted. The expression levels of Bmi-1 and EZH2 were detected by immunohistochemical staining and RT-PCR in cancer tissues, proximal and remote tissues with respect to the cancer tissues, and normal ovarian tissues of nude mice.Results Thirty-five ovarian tissue samples with normal biopsy results were obtained from 40 cases of human epithelial ovarian cancer in the nude mice in which the tumor tissues were orthotopically implanted. Bmi-1 and EZH2 expression levels were lower in proximal paraneoplastic tissue samples than in cancer tissue samples(P < 0.05) and higher than in remote paraneoplastic tissue samples(P < 0.01). No significant difference was found in the expression levels of Bmi-1 and EZH2 using immunohistochemistry among residual normal ovarian tissues obtained from orthotopically implanted models that differed in severity. The expression of Bmi-1 and EZH2 was negative in 20 normal ovarian tissue samples.Conclusion The expression levels of Bmi-1 and EZH2 were reduced with increasing distance from the cancer tissues. Negative expression of these tumor-associated genes can be used as a standard for the screening of normal ovarian tissues adjacent to tumor tissues. Normal ovarian tissues can be obtained from the tissues adjacent to tumors.
基金supported by the National Natural Science Foundation of China(No.31172206)
文摘The effects of equine chorionic gonadotropin （eCG） on follicular development and ovulation in cyclic guinea pigs were investigated by histological and immunohistochemical analyses. Three groups of guinea pigs （n=12） were administrated subcutaneously with saline, 20 or 50 IU of eCG, respectively, on cyclic Day 12 （Day 1=vaginal openings）. Ovaries were collected at 4 and 8 d after administration （6 animals per group each time）. The eCG administration induced significant and distinct morphological changes in the ovaries, as it promoted the luteinization of granulosa cells, but not follicular development. In addition, proliferating cell nuclear antigen （PCNA） and steroidogenic acute regulatory protein （STAR） were immunolocalized specifically in luteinized follicles. Our experiments together indicate that eCG administration can induce follicular luteinization but not superovulation in guinea pigs. The eCG in cyclic guinea pigs functions similar to that of luteinizing hormone （LH）, but not follicle-stimulating hormone （FSH）.
