Aim： To examine the possible effect of heat treatment on expression of heat shock proteins （Hsps） 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. Methods： Western blot...Aim： To examine the possible effect of heat treatment on expression of heat shock proteins （Hsps） 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. Methods： Western blot analysis, realtime polymerase chain reaction （PCR）, and confocal immunohistochemistry were used to analyze mRNA and protein levels of the Hsps in response to 43~C treatment of Sertoli cells isolated from pubertal monkey testes. Results： Staining with Hoechst 33342 indicated Sertoli cells did not undergo apoptosis after heat treatment. Hspl05 was expressed in cytoplasm of untreated Sertoli cells. Both Hspl05 mRNA and protein levels were increased approximately 20-fold compared to those of the untreated controls at 12 h after heat treatment. Untreated Sertoli cells did not express Hsp70, but heat stress induced its expression in the cell cytoplasm. The time-course of changes in Hsp70 was similar to that of Hsp105. In contrast to Hsp105 and Hsp70, the change in Hsp60 expression was much less obvious. The protein level between 12 h and 48 h after heat treatment was only approximately 1.5-fold that of the untreated control. Extracellular regulated kinase （ERK） 1/2 inhibitor （U0126） or phosphoinositide kinase-3 （PI3K） inhibitor （LY294002） could partially block the response of Hspl05 and Hsp70 induced by heat treatment. Conclusion： These results indicate that the heat-induced expression of the three types of Hsp in monkey Sertoli cells might be regulated by ERK and/or PI3K signal pathways, but the profile of their expression is different, suggesting that they might have different regulatory functions in Sertoli cells.展开更多
Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in respon...Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.展开更多
Dear Editor, It is now well known that somatic cells can be efficiently reprogrammed into induced pluripotent stem cells （iPSCs） by forced expression of defined factors [1- 3]. These cells, like embryonic stem cel...Dear Editor, It is now well known that somatic cells can be efficiently reprogrammed into induced pluripotent stem cells （iPSCs） by forced expression of defined factors [1- 3]. These cells, like embryonic stem cells （ESCs）, have true pluripotency as shown by the live, fertile mice that can be generated through the tetraploid complementation assay using these iPSCs [4, 5]. So far, iPSCs have been generated from many species including mice, primate,展开更多
High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the importa...High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.展开更多
Mouse and human somatic cells can be induced to become pluripotent stem （iPS） cells by retroviral transduction with defined transcription factors . Reprogrammed pluripotent stem cells have great potential for regene...Mouse and human somatic cells can be induced to become pluripotent stem （iPS） cells by retroviral transduction with defined transcription factors . Reprogrammed pluripotent stem cells have great potential for regenerative medicine and also provide a good experi- mental system for studying epigenetic reprogramming and differentiation. However, at present, the reprogram- ming process is still somewhat slow and ineffective.展开更多
The enrichment and identification of human epidermal stem cells （EpSCs） are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been...The enrichment and identification of human epidermal stem cells （EpSCs） are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rap- idly adherent keratinocytes into three populations that range in size from 5-7 μm （population A）, to 7-9 μm （population B）, to ≥9μm （population C） in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of plintegrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency （CFE）. When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to pl-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.展开更多
Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chro...Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.展开更多
Background： Heat stress is known to alter follicular dynamics and granulosa cell function and may contribute to the diminished reproductive efficiency commonly observed in mammals during the summer. Although several ...Background： Heat stress is known to alter follicular dynamics and granulosa cell function and may contribute to the diminished reproductive efficiency commonly observed in mammals during the summer. Although several investigators have studied heat-induced ovarian injury, few reports have focused on the effects of chronic heat stress on ovarian function and the molecular mechanisms through which it induces ovarian injury.Methods： In Exp. 1, 48 female mice were assigned to a control or heat-stressed treatment. After exposure to a constant temperature of 25 ℃ for 7, 14, 21 or 28 d（n = 6） or to 42 ℃ for 3 h per d for 7, 14, 21 or 28 d（n = 6）, the mice were euthanized and their ovaries were analyzed for follicular atresia, granulosa cell apoptosis, changes in the abundance of HSP70 protein and serum concentrations of estradiol. In Exp. 2, the expression of HSP70 and aromatase was quantified in antral follicles cultured in vitro at 37 or 42 ℃ for 24 h. In Exp. 3, granulosa cells from ovaries maintained at 37 or 41 ℃ for 2 h were analyzed for their expression of HSP70, Bim, caspase-3 and cleaved caspase-3.Results： In Exp. 1, body weight and food intake of heat-stressed mice decreased（P 〈 0.05） compared with control mice while the concentration of estradiol in serum was lower（P 〈 0.05） in heat-stressed mice than in control mice. Compared with control mice, the percentage of atretic follicles and the number of antral follicles with severe apoptotic signals were increased（P 〈 0.05） after 21 d of heat-stressed treatment. HSP70 protein was more abundant（P 〈 0.05） in heat-stressed mice than control mice. In Exp. 2, heat stress increased HSP70 and decreased aromatase proteins（P 〈 0.05） in antral follicles. In Exp. 3, TUNEL-positive granulosa cells from heat-stressed ovaries were observed concomitant with a significant increase in HSP70, Bim and cleaved caspase-3 protein.Conclusion： Heat-stress in mice decrease estradiol in serum and aromatase in antral follicles but increased number of atretic follicles and granulosa cell undergoing apoptosis which may explain the decreased fertility commonly observed in heat-stressed animals.展开更多
Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell a...Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons（breeding and non-breeding season） on superovulation and the imported exogenous gene on growth.Results： In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep.Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons（P 〉 0.05）. The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group（P 〈 0.05）. The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.Conclusions： Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.展开更多
The Chinese Grouse(Tetrastes sewerzowi) and Hazel Grouse(T. bonasia) are sibling species that are well-adapted to harsh high-altitude and latitude habitats. In the current study, we sampled and sequenced 29 Chinese Gr...The Chinese Grouse(Tetrastes sewerzowi) and Hazel Grouse(T. bonasia) are sibling species that are well-adapted to harsh high-altitude and latitude habitats. In the current study, we sampled and sequenced 29 Chinese Grouse(n=16) and Hazel Grouse(n=13) from eight locations in China, Sweden,Germany, and northeast Poland to analyze population genetic diversity and structure, introgression, and local adaptation.展开更多
Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear tr...Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC （NTESC） lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage RI donor cells were able to form full term developed pups, whereas those from late passage RI ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-RI-ESC lines that were developed from NT blastocyst of late passage R 1 ESC donors. However, these NT-R I-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.展开更多
Embryonic stem（ES） cells are pluripotent cells that can give rise to derivatives of all three embryonic germ layers. Due to its characteristics, the patient-specific ES cells are of great potential for transplantati...Embryonic stem（ES） cells are pluripotent cells that can give rise to derivatives of all three embryonic germ layers. Due to its characteristics, the patient-specific ES cells are of great potential for transplantation therapies. Several strategies can reprogramme somatic cells back to pluripotent stem cells： nuclear transfer, fusion with ES cells, treatment with cell extract and induction by specific factors. Considering the future clinical use, the differentiation from ES to neurons, cardiomyocytes and many other types of cells currently provide basic cognition and experience to regenerative medicine. This article will review two courses, the reprogramming of differentiated cells and the differentiation of ES cells to specific cell types.展开更多
It is here reported for the first time that luteal cells are capable of secreting plasminogen activators(PA),(both tissue-type,tPA,and urokinase-type,uPA),and plasminogen activator inhibitor type-1(PAl-1).Using organ ...It is here reported for the first time that luteal cells are capable of secreting plasminogen activators(PA),(both tissue-type,tPA,and urokinase-type,uPA),and plasminogen activator inhibitor type-1(PAl-1).Using organ culture model,we have demonstrated that tPA,but not uPA,showed markedchange during luteolytic period in rat corpus luteum.A great amount oftPA was secreted in corpusluteum on D 14 and D 17 while very low level of tPA activity was detected before D 12.Correspondingly,the progesterone production in the corpus luteum increased gradually in a time-dependent manner from D 1 to D 12 but dropped abruptly to a very low level on D 14.Additionof exogenous tPA to the CL culture caused considerable decrease in progesterone secretion whileinclusion of purified monoclone tPA antibodies in the culture augmented progesterone productionof CL.It is therefore suggested that tPA may play an important role in luteolytic process.展开更多
We have demonstrated that prolactin inhibits gonadotropin-induced ovulation in PMSG-primed mice.The number of ova in oviducts considerably decreased in the group of hCG plus prolactin (PRL)(19.7 + 4.9) as compared wit...We have demonstrated that prolactin inhibits gonadotropin-induced ovulation in PMSG-primed mice.The number of ova in oviducts considerably decreased in the group of hCG plus prolactin (PRL)(19.7 + 4.9) as compared with that of hCG alone (31.7 + 6.7). PRL inhibition of hCG-inducedovulation in mice may be through decreasing the ovarian plasminogen activator (PA) activity on onehand, and inhibiting the preovulatory increase in estrogen (E) secretion on the other.展开更多
Effects of forskolin on progesterone and plasminogen activator production in pseudopregnant ratcorpora lutea was investigated using isolated in vitro perfused ovaries.Progesterone andplasminogen activator production w...Effects of forskolin on progesterone and plasminogen activator production in pseudopregnant ratcorpora lutea was investigated using isolated in vitro perfused ovaries.Progesterone andplasminogen activator production were measured on day 1,8 and 18 of hCG-inducedpseudopregnancy.The results indicated:different concentrations of forskolin(100,200,400 and800 μg)administered to ovaries on the 8th day of pseudopregnancy caused elevation of progesteronesecretion in a dose-dependent manner.After 8 hours of perfusion,PA contents increasedsignificantly in ovaries treated with forskolin.With exogenous PA-urokinase(800 U)added to theperfusion solution,progesterone secretion increased significantly as compared to control group andremained on high level throughout the perfusion period.Though exerting no apparent effects in lowdosage(5 mM),AMCHA,a PA inhibitor,administered in higher dosage(10 and 15 mM)led tomarked reduction in PA activity and progesterone secretion as compared to control group.Thusforskolin causes significant elevation of level of progesterone secretion and PA activity inpseudopregnant rat ovary perfused in vitro.And PA seems to regulate progesterone secretion in theperfused rat corpora luteum.展开更多
Since a GnRH was isolated from mammalian hypothalamus and purified in 1971 and 1972, severalvariants have been identified in various forms of lower vertebrates. However, the presence of GnRHin amphioxus is still an op...Since a GnRH was isolated from mammalian hypothalamus and purified in 1971 and 1972, severalvariants have been identified in various forms of lower vertebrates. However, the presence of GnRHin amphioxus is still an open question. Chang et al. (1984) observed the presence of immunopositivegranules for GnRH in Hatschek's pit of amphioxus. In this paper, HPLC was used for the isolationand purification of GnRH-like peptide from amphioxus tissues, while radioimmunoassay was appliedto determine the immunoreactivity of the peptide. Based on the immunological and chromatographiccharacteristics, two kinds of GnRH (mGnRH and sGchH) were identified in amphioxus and theseGnRH-like peptide were found to be present in the 'head', 'middle' and 'tail' regions ofamphioxus.展开更多
The presont study was carried out to investigate the effect of different amounts of gossypol on bovineblastocyst attachment and trophoblastic outgrowth in vitro. Bovine oocyte were collected from theovaries of slaught...The presont study was carried out to investigate the effect of different amounts of gossypol on bovineblastocyst attachment and trophoblastic outgrowth in vitro. Bovine oocyte were collected from theovaries of slaughtered cows and were matured and fertilized in vitro. Cleaved oocyte were culturedin CRlaa + BOEC and TC-199 + 10% FCS combined in an 1:1 ratio. After 8 days of co-culture,the hatched blastocysts were randomly allotted to different treatment groups. All were cultured ona fetal fibroblast monolaycr (prepared from bovine fetuses) in TC-199 culture medium supplementedwith 10% fetal calf serum (TCFCS). But the groups differed from one another in the dose ofgossypol given: 0.01 μg, 0.1 μg, 1 μg, 10 μs/ml, and no gossypol as control. All cultures wereperformed in 24-well culture plates at 39℃ with 5% CO_2 in air. The results indicate that the ratesof attached and outgrowing blastocysts in the medium containing 1 μg/ml gossypol were significantlylowcr than the control group (p<0.01) and outgrowth were inhibited by gossypol in a dose-dependent manner.展开更多
Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by using in situ hybridization and in situ 3’-end l...Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by using in situ hybridization and in situ 3’-end labeling of DNA fragments (T躈EL). The results show that: (i) TR2 mRNA is specifically expressed in the germ cells, mainly in the spermatocytes, round and elongated spermatids. The expression level of TR2 mRNA varies with the seminiferous cycle. (ii) in the rat cryptorchid testes on days 3 and 5 after the surgery, the germ cells began to undergo apoptosis with no evident decrease in TR2 mRNA level. On day 7.5, however, most germ cells underwent apoptosis, while the expression level of TR2 mRNA declined markedly, and TR2 mRNA was rarely expressed on day 10 thereafter. (iii) On days 15 and 20 of the cryptorchid testes of rhesus monkey, TR2 mRNA was only expressed in a few of primary spermatocytes and the mRNA was almost undetectable on days 30, 45, 60. These results suggest that TR2 mRNA展开更多
The pluripotent state between human and mouse embryonic stem cells is different.Pluripotent state of human embryonic stem cells(ESCs)is believed to be primed and is similar with that of mouse epiblast stem cells(EpiSC...The pluripotent state between human and mouse embryonic stem cells is different.Pluripotent state of human embryonic stem cells(ESCs)is believed to be primed and is similar with that of mouse epiblast stem cells(EpiSCs),which is different from the naïve state of mouse ESCs.Human ESCs could be converted into a naïve state through exogenous expression of defined transcription factors(Hanna et al.,2010).Here we report a rapid conversion of human ESCs to mouse ESC-like naïve states only by modifying the culture conditions.These converted human ESCs,which we called mhESCs(mouse ESC-like human ESCs),have normal karyotype,allow single cell passage,exhibit domed morphology like mouse ESCs and express some pluripotent markers similar with mouse ESCs.Thus the rapid conversion established a naïve pluripotency in human ESCs like mouse ESCs,and provided a new model to study the regulation of pluripotency.展开更多
The corpus luteum(CL)is a transient endocrine organ that secretes progesterone to support early pregnancy.If implantation is unsuccessful,luteolysis is initiated.Extensive tissue re-modeling occurs during CL formation...The corpus luteum(CL)is a transient endocrine organ that secretes progesterone to support early pregnancy.If implantation is unsuccessful,luteolysis is initiated.Extensive tissue re-modeling occurs during CL formation and luteolysis.In this study,we have studied the possible in-volvement of MMP-2,-9,-14,and their inhibitors,TIMP-1,-2,-3 in the CL of cycling rhesus monkey at various stages by in situ hybridization,immunohistochemistry and microscopic assessment.The re-sults showed that the MMP-2 mRNA and protein were mainly expressed in the endothelial cells at the early and middle stages of the CL development,while their expressions were observed in the luteal cells at the late stage during luteal regression.MMP-9 protein was detected in the CL at the early and middle stages,and obviously increased at the late stage.The expressions of MMP-14 and TIMP-1 mRNA were high at the early and late stages,and low at the middle stage.TIMP-2 mRNA was high throughout all the stages,the highest level could be observed at the late stage.The TIMP-3 produc-tion was detected throughout all the stages,but obviously declined during CL regression.MMP-9,-14 and TIMP-1,-2,-3 were mainly localized in the cytoplasm of the steroidogenic cells.The results suggest that the MMP/TIMP system is involved in regulation of CL development in the primate,and the coordinated expression of MMP-2,-14 and TIMP-1,-3 may have a potential role in the CL forma-tion and the functional maintaining,while the interaction of MMP-2,-9,-14 and TIMP-1,-2,-3 might also play a role in CL regression at the late stage of CL development in the primate.展开更多
基金Acknowledgment This study was supported by the ＂973＂ project （No. 2006CB504001）, the Major Research Plan （No. 2006CB944001）, the CAS Innovation Project （KSCA2- YW-R-55）, the National Natural Science Foundation of China （No. 3061800530230190 30600311）, and the Beijing Natural Science Foundation （No. 5073032）.
文摘Aim： To examine the possible effect of heat treatment on expression of heat shock proteins （Hsps） 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. Methods： Western blot analysis, realtime polymerase chain reaction （PCR）, and confocal immunohistochemistry were used to analyze mRNA and protein levels of the Hsps in response to 43~C treatment of Sertoli cells isolated from pubertal monkey testes. Results： Staining with Hoechst 33342 indicated Sertoli cells did not undergo apoptosis after heat treatment. Hspl05 was expressed in cytoplasm of untreated Sertoli cells. Both Hspl05 mRNA and protein levels were increased approximately 20-fold compared to those of the untreated controls at 12 h after heat treatment. Untreated Sertoli cells did not express Hsp70, but heat stress induced its expression in the cell cytoplasm. The time-course of changes in Hsp70 was similar to that of Hsp105. In contrast to Hsp105 and Hsp70, the change in Hsp60 expression was much less obvious. The protein level between 12 h and 48 h after heat treatment was only approximately 1.5-fold that of the untreated control. Extracellular regulated kinase （ERK） 1/2 inhibitor （U0126） or phosphoinositide kinase-3 （PI3K） inhibitor （LY294002） could partially block the response of Hspl05 and Hsp70 induced by heat treatment. Conclusion： These results indicate that the heat-induced expression of the three types of Hsp in monkey Sertoli cells might be regulated by ERK and/or PI3K signal pathways, but the profile of their expression is different, suggesting that they might have different regulatory functions in Sertoli cells.
基金Acknowledgment This study was supported by the National Natural Science Foundation of China （30230190）, the National Basic Science Research and Development Project （973） （G1999055901） and the Chinese Academy of Sciences （CAS） Knowledge Innovation Program （KSCX-2-SW-201）.
文摘Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.
文摘Dear Editor, It is now well known that somatic cells can be efficiently reprogrammed into induced pluripotent stem cells （iPSCs） by forced expression of defined factors [1- 3]. These cells, like embryonic stem cells （ESCs）, have true pluripotency as shown by the live, fertile mice that can be generated through the tetraploid complementation assay using these iPSCs [4, 5]. So far, iPSCs have been generated from many species including mice, primate,
基金the National Basic Re-search Program of China (973 Program) (No. 2006CB504004 and 2006CB944004)the National Natural Science Foundation of China (No. 30430530)the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KSCX2-YW-N-017).
文摘High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.
基金These three authors contributed equally to this work. This study was supported in part by grants from the Hi-Tech Research and Development Program of China （2006AA02A101 to QZ）, the National Natural Science Foundation of China （30670229 to QZ）, China National Basic Research Program （2006CB701500, 2007CB947700 and 2007CB947800）, the Shanghai Leading Academic Discipline Project （$30201）, and STCSM Project （08dj 1400502）.
文摘Mouse and human somatic cells can be induced to become pluripotent stem （iPS） cells by retroviral transduction with defined transcription factors . Reprogrammed pluripotent stem cells have great potential for regenerative medicine and also provide a good experi- mental system for studying epigenetic reprogramming and differentiation. However, at present, the reprogram- ming process is still somewhat slow and ineffective.
文摘The enrichment and identification of human epidermal stem cells （EpSCs） are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rap- idly adherent keratinocytes into three populations that range in size from 5-7 μm （population A）, to 7-9 μm （population B）, to ≥9μm （population C） in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of plintegrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency （CFE）. When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to pl-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.
基金supported by the National Natural Science Foundation of China(No.30930065 and No.31271605)
文摘Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.
基金The design of the study and collection,analysis,and interpretation of data and in writing the manuscript were supported by the Specialized Research Fund for the Doctoral Program of Higher Education(20130008130001)
文摘Background： Heat stress is known to alter follicular dynamics and granulosa cell function and may contribute to the diminished reproductive efficiency commonly observed in mammals during the summer. Although several investigators have studied heat-induced ovarian injury, few reports have focused on the effects of chronic heat stress on ovarian function and the molecular mechanisms through which it induces ovarian injury.Methods： In Exp. 1, 48 female mice were assigned to a control or heat-stressed treatment. After exposure to a constant temperature of 25 ℃ for 7, 14, 21 or 28 d（n = 6） or to 42 ℃ for 3 h per d for 7, 14, 21 or 28 d（n = 6）, the mice were euthanized and their ovaries were analyzed for follicular atresia, granulosa cell apoptosis, changes in the abundance of HSP70 protein and serum concentrations of estradiol. In Exp. 2, the expression of HSP70 and aromatase was quantified in antral follicles cultured in vitro at 37 or 42 ℃ for 24 h. In Exp. 3, granulosa cells from ovaries maintained at 37 or 41 ℃ for 2 h were analyzed for their expression of HSP70, Bim, caspase-3 and cleaved caspase-3.Results： In Exp. 1, body weight and food intake of heat-stressed mice decreased（P 〈 0.05） compared with control mice while the concentration of estradiol in serum was lower（P 〈 0.05） in heat-stressed mice than in control mice. Compared with control mice, the percentage of atretic follicles and the number of antral follicles with severe apoptotic signals were increased（P 〈 0.05） after 21 d of heat-stressed treatment. HSP70 protein was more abundant（P 〈 0.05） in heat-stressed mice than control mice. In Exp. 2, heat stress increased HSP70 and decreased aromatase proteins（P 〈 0.05） in antral follicles. In Exp. 3, TUNEL-positive granulosa cells from heat-stressed ovaries were observed concomitant with a significant increase in HSP70, Bim and cleaved caspase-3 protein.Conclusion： Heat-stress in mice decrease estradiol in serum and aromatase in antral follicles but increased number of atretic follicles and granulosa cell undergoing apoptosis which may explain the decreased fertility commonly observed in heat-stressed animals.
基金supported by grants from National Transgenic Creature Breeding Grand Project (2014ZX08008-005)
文摘Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons（breeding and non-breeding season） on superovulation and the imported exogenous gene on growth.Results： In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep.Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons（P 〉 0.05）. The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group（P 〈 0.05）. The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.Conclusions： Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.
基金supported by the National Natural Science Foundation of China(31520103903)。
文摘The Chinese Grouse(Tetrastes sewerzowi) and Hazel Grouse(T. bonasia) are sibling species that are well-adapted to harsh high-altitude and latitude habitats. In the current study, we sampled and sequenced 29 Chinese Grouse(n=16) and Hazel Grouse(n=13) from eight locations in China, Sweden,Germany, and northeast Poland to analyze population genetic diversity and structure, introgression, and local adaptation.
文摘Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC （NTESC） lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage RI donor cells were able to form full term developed pups, whereas those from late passage RI ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-RI-ESC lines that were developed from NT blastocyst of late passage R 1 ESC donors. However, these NT-R I-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.
文摘Embryonic stem（ES） cells are pluripotent cells that can give rise to derivatives of all three embryonic germ layers. Due to its characteristics, the patient-specific ES cells are of great potential for transplantation therapies. Several strategies can reprogramme somatic cells back to pluripotent stem cells： nuclear transfer, fusion with ES cells, treatment with cell extract and induction by specific factors. Considering the future clinical use, the differentiation from ES to neurons, cardiomyocytes and many other types of cells currently provide basic cognition and experience to regenerative medicine. This article will review two courses, the reprogramming of differentiated cells and the differentiation of ES cells to specific cell types.
基金Project supported by HRP,WHO,The State Family Planning commission State Key Laboratory of Reproductive Biology.
文摘It is here reported for the first time that luteal cells are capable of secreting plasminogen activators(PA),(both tissue-type,tPA,and urokinase-type,uPA),and plasminogen activator inhibitor type-1(PAl-1).Using organ culture model,we have demonstrated that tPA,but not uPA,showed markedchange during luteolytic period in rat corpus luteum.A great amount oftPA was secreted in corpusluteum on D 14 and D 17 while very low level of tPA activity was detected before D 12.Correspondingly,the progesterone production in the corpus luteum increased gradually in a time-dependent manner from D 1 to D 12 but dropped abruptly to a very low level on D 14.Additionof exogenous tPA to the CL culture caused considerable decrease in progesterone secretion whileinclusion of purified monoclone tPA antibodies in the culture augmented progesterone productionof CL.It is therefore suggested that tPA may play an important role in luteolytic process.
基金The project is supported by Chinese Academy of SciencesThe State Family Planning Commission+1 种基金The Natural Science Foundation of chinaRockefeller Foundation,New York.
文摘We have demonstrated that prolactin inhibits gonadotropin-induced ovulation in PMSG-primed mice.The number of ova in oviducts considerably decreased in the group of hCG plus prolactin (PRL)(19.7 + 4.9) as compared with that of hCG alone (31.7 + 6.7). PRL inhibition of hCG-inducedovulation in mice may be through decreasing the ovarian plasminogen activator (PA) activity on onehand, and inhibiting the preovulatory increase in estrogen (E) secretion on the other.
文摘Effects of forskolin on progesterone and plasminogen activator production in pseudopregnant ratcorpora lutea was investigated using isolated in vitro perfused ovaries.Progesterone andplasminogen activator production were measured on day 1,8 and 18 of hCG-inducedpseudopregnancy.The results indicated:different concentrations of forskolin(100,200,400 and800 μg)administered to ovaries on the 8th day of pseudopregnancy caused elevation of progesteronesecretion in a dose-dependent manner.After 8 hours of perfusion,PA contents increasedsignificantly in ovaries treated with forskolin.With exogenous PA-urokinase(800 U)added to theperfusion solution,progesterone secretion increased significantly as compared to control group andremained on high level throughout the perfusion period.Though exerting no apparent effects in lowdosage(5 mM),AMCHA,a PA inhibitor,administered in higher dosage(10 and 15 mM)led tomarked reduction in PA activity and progesterone secretion as compared to control group.Thusforskolin causes significant elevation of level of progesterone secretion and PA activity inpseudopregnant rat ovary perfused in vitro.And PA seems to regulate progesterone secretion in theperfused rat corpora luteum.
文摘Since a GnRH was isolated from mammalian hypothalamus and purified in 1971 and 1972, severalvariants have been identified in various forms of lower vertebrates. However, the presence of GnRHin amphioxus is still an open question. Chang et al. (1984) observed the presence of immunopositivegranules for GnRH in Hatschek's pit of amphioxus. In this paper, HPLC was used for the isolationand purification of GnRH-like peptide from amphioxus tissues, while radioimmunoassay was appliedto determine the immunoreactivity of the peptide. Based on the immunological and chromatographiccharacteristics, two kinds of GnRH (mGnRH and sGchH) were identified in amphioxus and theseGnRH-like peptide were found to be present in the 'head', 'middle' and 'tail' regions ofamphioxus.
基金Research was supported by the Rockefeller FoundationCollege of Agricultural and Life Sciences,University of Wisconsin-Madion.
文摘The presont study was carried out to investigate the effect of different amounts of gossypol on bovineblastocyst attachment and trophoblastic outgrowth in vitro. Bovine oocyte were collected from theovaries of slaughtered cows and were matured and fertilized in vitro. Cleaved oocyte were culturedin CRlaa + BOEC and TC-199 + 10% FCS combined in an 1:1 ratio. After 8 days of co-culture,the hatched blastocysts were randomly allotted to different treatment groups. All were cultured ona fetal fibroblast monolaycr (prepared from bovine fetuses) in TC-199 culture medium supplementedwith 10% fetal calf serum (TCFCS). But the groups differed from one another in the dose ofgossypol given: 0.01 μg, 0.1 μg, 1 μg, 10 μs/ml, and no gossypol as control. All cultures wereperformed in 24-well culture plates at 39℃ with 5% CO_2 in air. The results indicate that the ratesof attached and outgrowing blastocysts in the medium containing 1 μg/ml gossypol were significantlylowcr than the control group (p<0.01) and outgrowth were inhibited by gossypol in a dose-dependent manner.
文摘Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by using in situ hybridization and in situ 3’-end labeling of DNA fragments (T躈EL). The results show that: (i) TR2 mRNA is specifically expressed in the germ cells, mainly in the spermatocytes, round and elongated spermatids. The expression level of TR2 mRNA varies with the seminiferous cycle. (ii) in the rat cryptorchid testes on days 3 and 5 after the surgery, the germ cells began to undergo apoptosis with no evident decrease in TR2 mRNA level. On day 7.5, however, most germ cells underwent apoptosis, while the expression level of TR2 mRNA declined markedly, and TR2 mRNA was rarely expressed on day 10 thereafter. (iii) On days 15 and 20 of the cryptorchid testes of rhesus monkey, TR2 mRNA was only expressed in a few of primary spermatocytes and the mRNA was almost undetectable on days 30, 45, 60. These results suggest that TR2 mRNA
基金by grants from the China National Basic Research Program(Grant No.2011CB965300)to L.W.grants from the National Natural Science Foundation of China(Grant No.90919060)to Q.Z.grants from the"Strategic Priority Research Program"of the Chinese Academy of Sciences(No.XDA01020101)to Q.Z.
文摘The pluripotent state between human and mouse embryonic stem cells is different.Pluripotent state of human embryonic stem cells(ESCs)is believed to be primed and is similar with that of mouse epiblast stem cells(EpiSCs),which is different from the naïve state of mouse ESCs.Human ESCs could be converted into a naïve state through exogenous expression of defined transcription factors(Hanna et al.,2010).Here we report a rapid conversion of human ESCs to mouse ESC-like naïve states only by modifying the culture conditions.These converted human ESCs,which we called mhESCs(mouse ESC-like human ESCs),have normal karyotype,allow single cell passage,exhibit domed morphology like mouse ESCs and express some pluripotent markers similar with mouse ESCs.Thus the rapid conversion established a naïve pluripotency in human ESCs like mouse ESCs,and provided a new model to study the regulation of pluripotency.
基金supported by WHO/Rockefeller Foundation Project,National Natural Science Foundation of China(Grant Nos.90208025,30370200,30270196&30170452)Knowledge Innovation Program of CAS(Grant No.KSCX2-SW-201).
文摘The corpus luteum(CL)is a transient endocrine organ that secretes progesterone to support early pregnancy.If implantation is unsuccessful,luteolysis is initiated.Extensive tissue re-modeling occurs during CL formation and luteolysis.In this study,we have studied the possible in-volvement of MMP-2,-9,-14,and their inhibitors,TIMP-1,-2,-3 in the CL of cycling rhesus monkey at various stages by in situ hybridization,immunohistochemistry and microscopic assessment.The re-sults showed that the MMP-2 mRNA and protein were mainly expressed in the endothelial cells at the early and middle stages of the CL development,while their expressions were observed in the luteal cells at the late stage during luteal regression.MMP-9 protein was detected in the CL at the early and middle stages,and obviously increased at the late stage.The expressions of MMP-14 and TIMP-1 mRNA were high at the early and late stages,and low at the middle stage.TIMP-2 mRNA was high throughout all the stages,the highest level could be observed at the late stage.The TIMP-3 produc-tion was detected throughout all the stages,but obviously declined during CL regression.MMP-9,-14 and TIMP-1,-2,-3 were mainly localized in the cytoplasm of the steroidogenic cells.The results suggest that the MMP/TIMP system is involved in regulation of CL development in the primate,and the coordinated expression of MMP-2,-14 and TIMP-1,-3 may have a potential role in the CL forma-tion and the functional maintaining,while the interaction of MMP-2,-9,-14 and TIMP-1,-2,-3 might also play a role in CL regression at the late stage of CL development in the primate.
