刘林
干细胞转基因克隆及哺乳动物早期发育的分子机制
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- 姓名:刘林
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学术头衔:
博士生导师
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学科领域:
光学
- 研究兴趣:干细胞转基因克隆及哺乳动物早期发育的分子机制
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【期刊论文】Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice
刘林, Lin Liu, and David L.Keefe,
Human Reproduction Vol. 17, No.10 pp. 2678-2685, 2002,-0001,():
-1年11月30日
The senescence-accelerated mouse (SAM) has been shown to exhibit ageing-associated mitochondrial dysfunction and oxidative stress, and early decline in fertility. METHODS: We compared meiotic progression of germinal vesicle oocytes between young (2-3 months) and old (10-14 months) SAM mice using triple immunostaining and fluorescence microscopy as well as Pol-Scope imaging. RESULTS: At 8-9 h of in-vitro maturation (IVM), most young SAM oocytes (86%, 32/37) were at meiosis I (MI) stage, with chromosomes aligned in the mid-region of MI spindles, whereas disrupted MI spindles and/or chromosome misalignments (45%, 18/40) and a few oocytes (20%, 8/40) with abnormal MII spindles were found in old SAM oocytes. At 15-17h of IVM, old SAM oocytes, despite errors at MI stage, extruded a first polar body at an incidence of 88% (n=85), which did not differ from that (92%, n=106) of young SAM oocytes. However, oocytes from old SAM (64%, 32/50) showed aberrant MII, with chromosome misalignment and dispersal, in contrast to normal MII in most young SAM oocytes (87%, 65/75), showing chromosome alignment at the metaphase plate of MII spindles. Moreover, Pol-Scope imaging non-invasively detected disrupted or absent visible spindles and possibly aberrant chromosome alignment. CONCLUSIONS: Spindle disruption and/or chromosome misalignments at both MI and MII are associated with maternal ageing in the SAM mouse. Our findings also suggest that meiotic division lacks a competent checkpoint for spindle integrity and chromosome alignment during reproductive ageing-associated oocyte senescence.
ageing/, meiosis/, oocyte/, Pol-Scope/, senescence-accelerated mouse
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刘林, Lin Liu a, b, *, Eva Czerwiec a, Davi d L. Keefe a
Gene Expression Patterns xx(2004)xxx-xxx,-0001,():
-1年11月30日
The transcription factor Oct-4 is expressed in germ cells and also is considered as a marker for pluripotency of stem cells. We first examined dynamics of Oct-4 protein expression during preimplantation development using both Western blot analysis, and immunofluorescence staining. We show that intact Oct-4 protein is not detected in either ovulated mature oocytes, or in zygotes and 2-4-cell embryos, which are the only known totipotent cell types in mammals. This finding is unexpected, since Oct-4 has been proposed to play a role in the control of totipotency. The results suggest that Oct-4 is not indispensable for fertilization and early cleavage. Rather, expression of Oct-4 protein is first detected in the nuclei of 8-16 cell morula, increases in early blastocysts, and declines in late blastocysts, in which most Oct-4 protein is confined to the inner cell mass (ICM) region, consistent with previous findings. We further compared Oct-4 protein expression in diploid and tetraploid blastocysts derived from normal fertilization or parthenogenesis, as well as expression in diploid androgenetic blastocysts. Expression levels and localization of Oct-4 protein are similar in both diploid and tetraploid early blastocysts, regardless of whether blastocysts are derived from fertilization or parthenogenesis. Androgenetic diploid blastocysts also express similar levels of Oct-4. Late blastocysts generated by both fertilization and parthenogenesis show a similar pattern of Oct-4 expression, suggesting that paternal genome activation is not required for Oct-4 expression. Expression of Oct-4 protein does not differ between diploid and tetraploid embryos, indicating that tetraploidy does not influence Oct-4 expression. Thus, expression of Oct-4 protein is initiated at morula stage in preimplantation embryos and completely controlled by a mechanism activated in oocytes. Downregulation of Oct-4 expression coincides with differentiation of trophectoderm. Similar profiles of Oct-4 expression observed in embryos with different ploidy and genome composition, are suggestive of Oct-4 being necessary but not sufficient for developmental potency.
Oct-4, Mouse, Preimplantation development, Parthenogenesis, Tetraploid
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刘林, Lin Liu, , Maria A. Blasco & David L. Keefe, +
EMBO reports vol. 3 no.3 pp 230-234 2002,-0001,():
-1年11月30日
Telomerase deficiency in the mouse eventually leads to loss of telomeric repeats from chromosome ends and to end-to-end chromosome fusions, which result in defects in highly proliferative tissues. We show that telomere dysfunction resulting from telomerase deficiency leads to disruption of functional meiotic spindles and misalignment of chromosomes during meiotic division of oocytes in late-generation (G4) mice. However, oocytes from first-generation (G1) mice lacking telomerase showed no appreciable telomere dysfunction and exhibited chromosome alignment at the metaphase plates of meiotic spindles, in a manner similar to that of wild-type mouse oocytes. These findings suggest that telomerase does not directly influence chromosome alignment and spindle integrity. Rather, functional telomeres may be involved in mediating metaphase chromosome alignment and maintaining functional spindles during meiotic division.
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刘林, Lin Liu, *, †, Maria A. Blasco, ‡, James R. Trimarchi, † and David L. Keefe*,
,-0001,():
-1年11月30日
Late generations of telomerase-null (TR-/-) mice exhibit progressive defects in highly proliferative tissues and organs and decreased fertility, ultimately leading to sterility. To determine effects of telomerase deficiency on germ cells, weinvestigated the cleavage and preimplantation development of embryos derived from both in vivo and in vitro fertilization of TR-/- or wild-type (TR+/+) sperm with either TR-/- or TR+/+ oocytes. Consistently, fertilization of TR-/- oocytes with either TR+/+ or TR-/- sperm, and TR-/- sperm with TR+/+ oocytes, resulted in aberrant cleavage and development, in contrast to the normal cleavage and development of TR+/+ oocytes fertilized by TR+/+ sperm. Many (>50%) of the fertilized TR-/- eggs developed only one pronucleus, coincident with increased incidence of cytofragmentation, in contrast to the normal formation of two pronuclei and equal cleavage of wild-type embryos. These results suggest that both TR-/- sperm and oocytes contribute to defective fertilization and cleavage. We further found that a subset (7-9%) of telomeres was undetectable at the ends of some metaphase I chromosomes from TR-/- spermatocytes and oocytes, indicating that meiotic germ cells lacking telomerase ultimately resulted in telomere shortening and loss. Dysfunction of meiotic telomeres may contribute to aberrant fertilization of gametes and lead to abnormal cleavage of embryos, implying an important role of functional telomeres for germ cells undergoing fertilization and early cleavage development.
telomerase-null mice, telomere, fertilization, embryo, apoptosis.,
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【期刊论文】Involvement of Mitochondria in Oxidative Stress-Induced Cell Death in Mouse Zygotes1
刘林, Lin Liu, , James R. Trimarchi, and David L. Keefe
BIOLOGY OF REPRODUCTION 62, 1745-1753 (2000),-0001,():
-1年11月30日
Accumulation of reactive oxygen species during aging leads to programmed cell death (PCD) in many cell types but has not been explored in mammalian fertilized eggs, in which mitochondria are "immature," in contrast to "mature" mitochondria in somatic cells. We characterized PCD in mouse zygotes induced by either intensive (1mM for 1.5h) or mild (200mM for 15min) hydrogen peroxide (H2O2) treatment. Shortly after intensive treatment, zygotes displayed PCD, typified by cell shrinkage, cytochrome c release from mitochondria, and caspase activation, then terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in condensed pronuclei. On the other hand, after mild treatment, zygotes arrested developmentally and showed neither cytochrome c release nor caspase activation over 48h; until 72h, 46% zygotes exhibited TUNEL staining, and 88% of zygotes lost plasma membrane integrity. Interestingly, mild oxidative treatment induced a decline in mitochondrial membrane potential and disruption of the mitochondrial matrix. Taken together, these results suggest that oxidative stress caused by H2O2 induces PCD in mouse zygotes and that mitochondria are involved in the early phase of oxidative stress-induced PCD. Furthermore, mitochondrial malfunction also may contribute to cell cycle arrest, followed by cell death, triggered by mild oxidative stress.
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刘林, Lin Liu and Xiangzhong Yang
BIOLOGY OF REPRODUCTION 61, 1-7 (1999),-0001,():
-1年11月30日
The objective of the present study was to examine the activity changes in histone H1 kinase (also known as maturation-promoting factor [MPF]) and mitogen-activated protein kinase (MAPK) and their constituent proteins in in vitro-matured bovine oocytes after in vitro fertilization (IVF) or after parthenogenetic activation induced by calcium ionophore A23187 alone or by the ionophore followed by either 6-dimethylaminopurine (6-DMAP) or cycloheximide (CHX). Inactivation of both H1 kinase and MAPK occurred after both A23187+6-DMAP treatment and IVF; inactivation of H1 kinase preceded inactivation of MAPK. However, MAPK was inactivated much earlier in 6-DMAP-treated oocytes. Further analysis of constituent cell cycle proteins of these kinases by Western blot showed that A23187 alone could not induce changes in cdc2, cdc25, or ERK2 but induced reduction of cyclin B1. IVF and A23187+CHX induced similar changes: cyclin B1 was destroyed shortly after activation followed by accumulation of cyclin B1, phosphorylation of cdc2, and dephosphorylation of ERK2 at pronuclear formation 15 h after activation. No change in cdc25 was observed at this time. In contrast, A23187+6-DMAP treatment resulted in earlier phosphorylation of cdc2 and dephosphorylation of ERK2 at 4 h after treatment when the pronucleus formed. Moreover, accumulation of both cdc25 and cyclin B1 was detected at 15 h. Microinjection of ERK2 antibody into A23187-treated oocytes resulted in pronuclear formation. In conclusion, activation of bovine oocytes with 6-DMAP led to earlier inactivation of MAPK, while CHX induced inactivation of MAPK parallel to that following sperm-induced oocyte activation. Destruction of cyclin B is responsible for inactivation of MPF, while phosphorylation of cdc2 is likely responsible for maintaining its low activity. Inactivation of MAPK is closely associated with pronuclear development regardless of the activation protocol used.
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刘林, Lin Liu, , James R. Trimarchi, Rudolf Oldenbourg, and David L. Keefe
BIOLOGY OF REPRODUCTION 63, 251-258 (2000),-0001,():
-1年11月30日
The newly developed Pol-Scope allows imaging of spindle retardance, which is an optical property of organized macromolecular structures that can be observed in living cells without fixation or staining. Experiments were undertaken to examine changes in meiotic spindles during the initial stages of activation of living mouse oocytes using the Pol-Scope. Parthenogenetic activation of oocytes treated with calcium ionophore evoked a dynamic increase in meiotic spindle retardance, particularly of the midregion, before spindle rotation and second polar body extrusion. The pronounced increase in spindle retardance, which could, for the first time to our knowledge, be quantified in living oocytes, was maintained during polar body extrusion. Spindle retardance of newly in vivo fertilized oocytes was significantly higher than that of ovulated, metaphase Ⅱ oocytes. Pol-Scope imaging of fertilized oocytes did not affect subsequent development. These results establish that increased spindle retardance precedes polar body extrusion and pronuclear formation. The increased birefringence in the spindle provides an early indicator of oocyte activation. Thus, noninvasive, quantitative imaging of the onset of activation in living oocytes might improve the efficiency of assisted fertilization and other embryo technologies.
developmental biology,, fertilization,, IVF/, ART,, oocyte development,, ovum
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刘林, Lin Liu, , James, R. Trimarchi, and David L. Keefe
BIOLOGY OF REPRODUCTION 66, 204-210 (2002),-0001,():
-1年11月30日
Aneuploidy underlies failed development and possibly apoptosis of some preimplantation embryos. We employed a haploid model in the mouse to study the effects of aneuploidy on apoptosis in preimplantation embryos. Mouse metaphase Ⅱ oocytes that were activated with strontium formed haploid parthenogenetic embryos with 1 pronucleus, whereas activation of oocytes with strontium plus cytochalasin D produced diploid parthenogenetic embryo controls with 2 pronuclei. Strontium induced calcium transients that mimic sperm-induced calcium oscillations, and ploidy was confirmed by chromosomal analysis. Rates of development and apoptosis were compared between haploid and diploid parthenogenetic embryos (parthenotes) and control embryos derived from in vitro fertilization (IVF). Haploid mouse parthenotes cleaved at a slower rate, and most arrested before the blastocyst stage, in contrast to diploid parthenotes or IVF embryos. Developmentally retarded haploid parthenotes exhibited apoptosis at a significantly higher frequency than did diploid parthenotes or IVF embryos. However, diploid parthenotes exhibited rates of preimplantation development and apoptosis similar to those of IVF embryos, indicating that parthenogenetic activation itself does not initiate apoptosis during preimplantation development. These results suggest that haploidy can lead to an increased incidence of apoptosis. Moreover, the initiation of apoptosis during preimplantation development does not require the paternal genome.
apoptosis,, developmental biology,, early development,, embryo,, ovum
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刘林, Andras Dinny
BIOLOGY OF REPRODUCTION 64, 257-263 (2001),-0001,():
-1年11月30日
This research was to study the in vitro and in vivo development of cloned embryos derived from adult rabbit fibroblasts following various activation protocols. Effects of serum starvation and passage number of donor cells on the efficiency of cloning were also examined. In experiment I, oocytes were activated either by electric pulses or by electric pulses followed by culture with 6-dimethylaminopurin (DMAP). For experiment Ⅱ, the best activation protocol from experiment I was employed for cloning using adult rabbit fibroblasts that were cultured for 0-15 passages. In experiment Ⅲ, the effect of serum starvation of the donor cells on cloning was examined. Finally, in experiment Ⅳ, embryo transfers were conducted. These experiments showed that combined electrical pulse and DMAP treatment resulted in superior parthenogenetic blastocyst development (up to 29%), and that activation of the cytoplast before versus after fusion was not different in supporting the in vitro development of nuclear transferred embryos (16%–18% blastocysts). Adult fibroblasts derived from nonpassaged cells were less capable of developing into blastocysts than passaged cells (6% vs. 17%). Serum starvation of donor cells improved cleavage (up to 71%) but did not improve blastocyst development (13%), and no progeny was obtained, irrespective of the treatment. Cell-cycle analysis of adult rabbit fibroblast cells showed that passage 6 and 12 cells were more likely to be in G0/G1 than passage 0 cells, which agrees with the improved embryo development in the passaged-cell groups.
developmental biology,, IVF/, ART,, oocyte development,, ovum
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刘林, Lin Liu and David L. Keefe,
BIOLOGY OF REPRODUCTION 62, 1828-1834 (2000),-0001,():
-1年11月30日
Eggs must be the major locus of reproductive aging in women, because donation of eggs from younger to middle-aged women abrogates the effects of age on fertility. Oxidative stress, mitochondrial dysfunction, and apoptosis are associated with senescence. To develop an animal model of egg senescence, we treated mouse zygotes with 175uM H2O2 that induced mitochondrial dysfunction and developmental arrest, followed by delayed cell death, consistent with apoptosis. We reconstructed zygotes with nuclei and cytoplasm from treated or untreated zygotes, then followed development and apoptotic cell death in the reconstituted embryos. Pronuclear exchange between untreated, normal zygotes served as nuclear transfer controls. Rates of cleavage and development to morula and blastocysts were significantly lower (P<0.01) in zygotes reconstituted from untreated pronuclei and H2O2-stressed cytoplasts than those of nuclear transfer controls. Instead, the arrested, reconstituted zygotes displayed TUNEL staining at a similar rate to that of H2O2-treated controls, suggesting that apoptotic potential could be transferred cytoplasmically. On the other hand, rates of cleavage and development to morula and blastocyst of the reconstituted zygotes, derived from stressed pronuclei and untreated cytoplasm, were significantly increased (P<0.05), compared to those of H2O2-treated, control zygotes, indicating that healthy cytoplasm could partly rescue pronuclei from oxidative stress. Although oxidation stressed both nuclei and cytoplasm, cytoplasm was more sensitive than nuclei to oxidative stress. It is suggested that cytoplasm, most likely mitochondria, plays a central role in mediating both development and apoptotic cell death induced by oxidative stress in mouse zygotes.
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