Inositol requiring mutant 80(INO80)is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells.However,the roles and mechanis...Inositol requiring mutant 80(INO80)is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells.However,the roles and mechanisms of INO80 in porcine preimplantation embryonic development remain largely unknown.Here,we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development.The INO80 protein is highly expressed in the nuclei during morula-toblastocyst transition.Functional studies revealed that RNA interference(RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm.Mechanistically,singleembryo RNA sequencing revealed that INO80 regulates multiple genes,which are important for lineage specification,tight junction assembly,and fluid accumulation.Consistent with the altered expression of key genes required for tight junction assembly,a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts.Importantly,aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium.Taken together,these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification,tight junction assembly,and fluid accumulation.展开更多
Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm（TE） lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic l...Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm（TE） lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA（shRNA） and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.展开更多
Zearalenone(ZEA),a mycotoxin produced mainly by fungi belonging to Fusarium species in foods and feeds,causes a serious hazard to humans and animals.Numerous studies have revealed that ingesting ZEA can disrupt the re...Zearalenone(ZEA),a mycotoxin produced mainly by fungi belonging to Fusarium species in foods and feeds,causes a serious hazard to humans and animals.Numerous studies have revealed that ingesting ZEA can disrupt the reproductive function and impair the reproductive process in animals.This experiment was to investigate the toxicological effect and the mechanism of ZEA exposure on reproduction in pigs during early stages of pregnancy.In the present study,we treated with 0 to 80μmol/L ZEA for 12 or 24 h in trophoblast ectoderm(pTr)cells.The results showed that ZEA had significantly decreased cell proliferation(P<0.05),which was accompanied by DNA damage-related cell cycle arrest at G2/M phase,activation of the apoptosis and endoplasmic reticulum(ER)stress,as well as impairment of barrier function(P<0.05).Western blot analysis and transmission electron microscopy(TEM)showed that exposure to ZEA can activation of autophagy in pTr cells.Importantly,pretreatment with chloroquine(CQ)or 3-methyladenine(3-MA)led to increased apoptosis in pTr cells.Interestingly,pTr cells pretreated with 4-phenylbutyric acid(4-PBA),an inhibitor of ER stress,resulted in reduced cell death in pTr cells,indicating a critical role for ER stress in the activation of autophagy.In conclusion,these results reveal that ZEA-triggered ER stress is critical for the cell fate decision of pTr cells during early porcine embryonic development.Application of small molecules with ability of blocking ER stress might be therapeutic option to reduce the deleterious effect of ZEA in pregnant animals.展开更多
Self-organized blastoids from extended pluripotent stem(EPs)cells possess enormous potential for investigating postimplantation embryo development and related diseases.However,the limited ability of postimplantation d...Self-organized blastoids from extended pluripotent stem(EPs)cells possess enormous potential for investigating postimplantation embryo development and related diseases.However,the limited ability of postimplantation development of Eps-blastoids hinders its further application.In this study,single-cell transcriptomic analysis indicated that the“trophectoderm(TE)-like structure”of EPSblastoids was primarily composed of primitive endoderm(PrE)-related cells instead of TE-related cells.We further identified PrE-like cells in EPS cell culture that contribute to the blastoid formation with TE-like structure.Inhibition of PrE cell differentiation by inhibiting MEK signaling or knockout of Gata6 in EPS cells markedly suppressed EPS-blastoid formation.Furthermore,we demonstrated that blastocyst-like structures reconstituted by combining the EPs-derived bilineage embryo-like structure(BLEs)with either tetraploid embryos or tetraploid TE cells could implant normally and develop into live fetuses.In summary,our study reveals that TE improvement is critical for constructing a functional embryo using stem cells in vitro.展开更多
Background: Polyamines stimulate DNA transcription and m RNA translation for protein synthesis in trophectoderm cells, as well as proliferation and migration of cells; therefore, they are essential for development and...Background: Polyamines stimulate DNA transcription and m RNA translation for protein synthesis in trophectoderm cells, as well as proliferation and migration of cells; therefore, they are essential for development and survival of conceptuses(embryo/fetus and placenta). The ovine conceptus produces polyamines via classical and non-classical pathways. In the classical pathway, arginine(Arg) is transformed into ornithine, which is then decarboxylated by ornithine decarboxylase(ODC1) to produce putrescine which is the substrate for the production of spermidine and spermine. In the non-classical pathway, Arg is converted to agmatine(Agm) by arginine decarboxylase(ADC), and Agm is converted to putrescine by agmatinase(AGMAT).Methods: Morpholino antisense oligonucleotides(MAOs) were designed and synthesized to inhibit translational initiation of the m RNAs for ODC1 and ADC, in ovine conceptuses.Results: The morphologies of MAO control, MAO-ODC1, and MAO-ADC conceptuses were normal. Double knockdown of ODC1 and ADC(MAO-ODC1:ADC) resulted in two phenotypes of conceptuses; 33% of conceptuses appeared to be morphological y and functional y normal(phenotype a) and 67% of the conceptuses presented an abnormal morphology and functionality(phenotype b). Furthermore, MAO-ODC1:ADC(a) conceptuses had greater tissue concentrations of Agm,putrescine, and spermidine than MAO control conceptuses, while MAO-ODC1:ADC(b) conceptuses only had greater tissue concentrations of Agm. Uterine flushes from ewes with MAO-ODC1:ADC(a) had greater amounts of arginine, aspartate, tyrosine, citrulline, lysine, phenylalanine, isoleucine, leucine, and glutamine, while uterine flushes of ewes with MAO-ODC1:ADC(b) conceptuses had lower amount of putrescine, spermidine, spermine, alanine, aspartate,glutamine, tyrosine, phenylalanine, isoleucine, leucine, and lysine.Conclusions: The double-knockdown of translation of ODC1 and ADC m RNAs was most detrimental to conceptus development and their production of interferon tau(IFNT). Agm, polyamines, amino acids, and adequate secretion of IFNT are critical for establishment and maintenance of pregnancy during the peri-implantation period of gestation in sheep.展开更多
The first cell fate choice in the mammalian embryo, the segregation of the inner cell mass （ICM） and trophectoderm （TE）, is regulated by the mutually antagonistic effects of the transcription factors, Oct4 and Cdx...The first cell fate choice in the mammalian embryo, the segregation of the inner cell mass （ICM） and trophectoderm （TE）, is regulated by the mutually antagonistic effects of the transcription factors, Oct4 and Cdx2, while the pluripotency factor, Nanog, is essential to specify the epiblast. We have analyzed the promoters of Nanog and Cdx2, and have found that these two transcription factors are likewise regulated reciprocally. Using an embryonic stem cell line with conditional TE differentiation, we show that Nanog overexpression suppresses the upregulation of TE markers, while Nanog knockdown upregulates the expression of TE markers. We further show that Nanog and Cdx2 bind to and repress each other＇s promoters. However, whereas Nanog knockout results in detectable Cdx2 expression in the ICM, we observe no overt disruption of blastocyst development, indicating that Nanog plays a subservient role to Oct4 in segregation of the ICM and TE.展开更多
The influence of inner cell mass （ICM） and trophectoderm （TE） score on pregnancy out- comes in frozen-thawed blastocyst transfer cycles was analyzed. A retrospective analysis of 741 cycles of frozen-thawed blastos...The influence of inner cell mass （ICM） and trophectoderm （TE） score on pregnancy out- comes in frozen-thawed blastocyst transfer cycles was analyzed. A retrospective analysis of 741 cycles of frozen-thawed blastosysts transfer was performed. All cycles were divided into four groups based on the number and morphological score of blastocysts： S-ICM B/TE B group （n=91）, the single blastocyst transfer oflCM B and TE B; D-ICM B/TE B group （n=579）, double blastocysts transfer oflCM B/TE B; D-1CM B/TE C group （n=35）, double blastocysts transfer of ICM B/TE C; and D-ICM C/TE B group （n=36）, double blastocysts transfer ofTE B/ICM C. The pregnancy outcomes were compared among the four groups. As compared with D-ICM B/TE C group, the clinical pregnancy rate, implantation rate and multiple pregnancy rate were increased in D-ICM B/TE B group （74.96% vs. 57.14%, 57.43% vs. 37.14%, and .48.62% vs. 25%, respectively, P〈0.05 for all）. Clinical pregnancy rate and implantation rate in D-ICM B/TE B group were also higher than in D-ICM C/TE B group （74.96% vs. 50%, and 57.43% vs. 33.33%, both P〈0.05）. Multivariable Logistic regression analysis indicated that ICM score was a better predictive parameter for clinical pregnancy （OR=3.05, CI 1.70-5.46, P〈0.001）, while the trophectoderm score was a better one for early abortion （OR=0.074, CI 0.03-0.19, P〈0.001）. Clinical pregnancy rate and multiple pregnancy rate in S-ICM B/TE B group were significantly lower than those in D-ICM B/TE B group （46.15% vs. 74.96%, and 2.38% vs. 48.62%, both P〈0.05）, but there was no si~,,niflcant difference in the implantation rate between the two groups. It was suggested that the higher score of ICM and TE may be indicative of the better pregnancy outcomes. The ICM score is a better predictor of clinical pregnancy than TE, while TE score is a better one in predicting early abortion. Sin- gle ICM B/TE B blastocyst transfer in frozen-thawed cycles can also get satisfactory pregnancy out- comes.展开更多
Through proliferation and differentiation, a single cell, the zygote, can give rise to a complex organism composed of many types of cells. Up to the eight-cell embryo stage, the blastomeres are morphologically identic...Through proliferation and differentiation, a single cell, the zygote, can give rise to a complex organism composed of many types of cells. Up to the eight-cell embryo stage, the blastomeres are morphologically identical and distributed symmetrically in the mammalian embryo. Functionally, in some species, they are all totipotent. However, due to the compaction of blastomeres and the asymmetrical cell division at the late phase of the eight-cell embryo, the blastomeres of the morula are no longer identical. During the transition from morula to blastocyst, blastomeres differentiate, resulting in the first cell fate decision in embryogenesis, namely, the segregation of the inner cell mass and the tropheetoderm. In this review, we will discuss the regulatory mechanisms essential for the cell fate choice during blastocyst development, including transcriptional regulation, epigenetic regulation, mieroRNAs, and signal transduction.展开更多
基金supported by the Anhui Provincial Natural Science Foundation(1908085MC97,2008085MC85)National Natural Science Foundation of China(31802059,31902226)+1 种基金Hefei Innovation and Entrepreneurship Support Plan for Returnee Scholar(03082009)Anhui Provincial Innovation and Entrepreneurship Support Plan for Returnee Scholar(2020LCX015)。
文摘Inositol requiring mutant 80(INO80)is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells.However,the roles and mechanisms of INO80 in porcine preimplantation embryonic development remain largely unknown.Here,we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development.The INO80 protein is highly expressed in the nuclei during morula-toblastocyst transition.Functional studies revealed that RNA interference(RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm.Mechanistically,singleembryo RNA sequencing revealed that INO80 regulates multiple genes,which are important for lineage specification,tight junction assembly,and fluid accumulation.Consistent with the altered expression of key genes required for tight junction assembly,a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts.Importantly,aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium.Taken together,these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification,tight junction assembly,and fluid accumulation.
基金Supported by the Scientifi c Research Fund of Heilongjiang Provincial Education Department(11551039)
文摘Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm（TE） lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA（shRNA） and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.
基金This work was supported by grants from the National Natural Science Foundation of China(No.31625025).
文摘Zearalenone(ZEA),a mycotoxin produced mainly by fungi belonging to Fusarium species in foods and feeds,causes a serious hazard to humans and animals.Numerous studies have revealed that ingesting ZEA can disrupt the reproductive function and impair the reproductive process in animals.This experiment was to investigate the toxicological effect and the mechanism of ZEA exposure on reproduction in pigs during early stages of pregnancy.In the present study,we treated with 0 to 80μmol/L ZEA for 12 or 24 h in trophoblast ectoderm(pTr)cells.The results showed that ZEA had significantly decreased cell proliferation(P<0.05),which was accompanied by DNA damage-related cell cycle arrest at G2/M phase,activation of the apoptosis and endoplasmic reticulum(ER)stress,as well as impairment of barrier function(P<0.05).Western blot analysis and transmission electron microscopy(TEM)showed that exposure to ZEA can activation of autophagy in pTr cells.Importantly,pretreatment with chloroquine(CQ)or 3-methyladenine(3-MA)led to increased apoptosis in pTr cells.Interestingly,pTr cells pretreated with 4-phenylbutyric acid(4-PBA),an inhibitor of ER stress,resulted in reduced cell death in pTr cells,indicating a critical role for ER stress in the activation of autophagy.In conclusion,these results reveal that ZEA-triggered ER stress is critical for the cell fate decision of pTr cells during early porcine embryonic development.Application of small molecules with ability of blocking ER stress might be therapeutic option to reduce the deleterious effect of ZEA in pregnant animals.
基金supported by the National Key R&D Program of China(Nos.2020YFA0112500 and 2021YFA1102900)the National Natural Science Foundation of China(Nos.31721003,81630035,82022027,31871448,32000418 and 31820103009)+2 种基金supported by the key project of the Science and Technology of Shanghai Municipality(Nos.19JC1415300 and 21JC1405500)the Shanghai municipal medical and health discipline construction projects(No.2017ZZ02015)the China Postdoctoral Science Foundation 2021M692437 and the Fundamental Research Funds for the Central Universities.
文摘Self-organized blastoids from extended pluripotent stem(EPs)cells possess enormous potential for investigating postimplantation embryo development and related diseases.However,the limited ability of postimplantation development of Eps-blastoids hinders its further application.In this study,single-cell transcriptomic analysis indicated that the“trophectoderm(TE)-like structure”of EPSblastoids was primarily composed of primitive endoderm(PrE)-related cells instead of TE-related cells.We further identified PrE-like cells in EPS cell culture that contribute to the blastoid formation with TE-like structure.Inhibition of PrE cell differentiation by inhibiting MEK signaling or knockout of Gata6 in EPS cells markedly suppressed EPS-blastoid formation.Furthermore,we demonstrated that blastocyst-like structures reconstituted by combining the EPs-derived bilineage embryo-like structure(BLEs)with either tetraploid embryos or tetraploid TE cells could implant normally and develop into live fetuses.In summary,our study reveals that TE improvement is critical for constructing a functional embryo using stem cells in vitro.
基金supported primarily by the Agriculture and Food Research Initiative Competitive Grants(2016-67,015-24,958 to Fuller W.Bazer and 2015-67,015-23,276 to Guoyao Wu)from the United States Department of Agriculture,National Institute of Food and Agriculturesupported by funding from the Sustainability Strategy2013–2014,from CODI University of Antioquia(Ude A),Medellín,Colombia Scholarship“Becas Doctorado Ude A 2014.”
文摘Background: Polyamines stimulate DNA transcription and m RNA translation for protein synthesis in trophectoderm cells, as well as proliferation and migration of cells; therefore, they are essential for development and survival of conceptuses(embryo/fetus and placenta). The ovine conceptus produces polyamines via classical and non-classical pathways. In the classical pathway, arginine(Arg) is transformed into ornithine, which is then decarboxylated by ornithine decarboxylase(ODC1) to produce putrescine which is the substrate for the production of spermidine and spermine. In the non-classical pathway, Arg is converted to agmatine(Agm) by arginine decarboxylase(ADC), and Agm is converted to putrescine by agmatinase(AGMAT).Methods: Morpholino antisense oligonucleotides(MAOs) were designed and synthesized to inhibit translational initiation of the m RNAs for ODC1 and ADC, in ovine conceptuses.Results: The morphologies of MAO control, MAO-ODC1, and MAO-ADC conceptuses were normal. Double knockdown of ODC1 and ADC(MAO-ODC1:ADC) resulted in two phenotypes of conceptuses; 33% of conceptuses appeared to be morphological y and functional y normal(phenotype a) and 67% of the conceptuses presented an abnormal morphology and functionality(phenotype b). Furthermore, MAO-ODC1:ADC(a) conceptuses had greater tissue concentrations of Agm,putrescine, and spermidine than MAO control conceptuses, while MAO-ODC1:ADC(b) conceptuses only had greater tissue concentrations of Agm. Uterine flushes from ewes with MAO-ODC1:ADC(a) had greater amounts of arginine, aspartate, tyrosine, citrulline, lysine, phenylalanine, isoleucine, leucine, and glutamine, while uterine flushes of ewes with MAO-ODC1:ADC(b) conceptuses had lower amount of putrescine, spermidine, spermine, alanine, aspartate,glutamine, tyrosine, phenylalanine, isoleucine, leucine, and lysine.Conclusions: The double-knockdown of translation of ODC1 and ADC m RNAs was most detrimental to conceptus development and their production of interferon tau(IFNT). Agm, polyamines, amino acids, and adequate secretion of IFNT are critical for establishment and maintenance of pregnancy during the peri-implantation period of gestation in sheep.
文摘The first cell fate choice in the mammalian embryo, the segregation of the inner cell mass （ICM） and trophectoderm （TE）, is regulated by the mutually antagonistic effects of the transcription factors, Oct4 and Cdx2, while the pluripotency factor, Nanog, is essential to specify the epiblast. We have analyzed the promoters of Nanog and Cdx2, and have found that these two transcription factors are likewise regulated reciprocally. Using an embryonic stem cell line with conditional TE differentiation, we show that Nanog overexpression suppresses the upregulation of TE markers, while Nanog knockdown upregulates the expression of TE markers. We further show that Nanog and Cdx2 bind to and repress each other＇s promoters. However, whereas Nanog knockout results in detectable Cdx2 expression in the ICM, we observe no overt disruption of blastocyst development, indicating that Nanog plays a subservient role to Oct4 in segregation of the ICM and TE.
文摘The influence of inner cell mass （ICM） and trophectoderm （TE） score on pregnancy out- comes in frozen-thawed blastocyst transfer cycles was analyzed. A retrospective analysis of 741 cycles of frozen-thawed blastosysts transfer was performed. All cycles were divided into four groups based on the number and morphological score of blastocysts： S-ICM B/TE B group （n=91）, the single blastocyst transfer oflCM B and TE B; D-ICM B/TE B group （n=579）, double blastocysts transfer oflCM B/TE B; D-1CM B/TE C group （n=35）, double blastocysts transfer of ICM B/TE C; and D-ICM C/TE B group （n=36）, double blastocysts transfer ofTE B/ICM C. The pregnancy outcomes were compared among the four groups. As compared with D-ICM B/TE C group, the clinical pregnancy rate, implantation rate and multiple pregnancy rate were increased in D-ICM B/TE B group （74.96% vs. 57.14%, 57.43% vs. 37.14%, and .48.62% vs. 25%, respectively, P〈0.05 for all）. Clinical pregnancy rate and implantation rate in D-ICM B/TE B group were also higher than in D-ICM C/TE B group （74.96% vs. 50%, and 57.43% vs. 33.33%, both P〈0.05）. Multivariable Logistic regression analysis indicated that ICM score was a better predictive parameter for clinical pregnancy （OR=3.05, CI 1.70-5.46, P〈0.001）, while the trophectoderm score was a better one for early abortion （OR=0.074, CI 0.03-0.19, P〈0.001）. Clinical pregnancy rate and multiple pregnancy rate in S-ICM B/TE B group were significantly lower than those in D-ICM B/TE B group （46.15% vs. 74.96%, and 2.38% vs. 48.62%, both P〈0.05）, but there was no si~,,niflcant difference in the implantation rate between the two groups. It was suggested that the higher score of ICM and TE may be indicative of the better pregnancy outcomes. The ICM score is a better predictor of clinical pregnancy than TE, while TE score is a better one in predicting early abortion. Sin- gle ICM B/TE B blastocyst transfer in frozen-thawed cycles can also get satisfactory pregnancy out- comes.
文摘Through proliferation and differentiation, a single cell, the zygote, can give rise to a complex organism composed of many types of cells. Up to the eight-cell embryo stage, the blastomeres are morphologically identical and distributed symmetrically in the mammalian embryo. Functionally, in some species, they are all totipotent. However, due to the compaction of blastomeres and the asymmetrical cell division at the late phase of the eight-cell embryo, the blastomeres of the morula are no longer identical. During the transition from morula to blastocyst, blastomeres differentiate, resulting in the first cell fate decision in embryogenesis, namely, the segregation of the inner cell mass and the tropheetoderm. In this review, we will discuss the regulatory mechanisms essential for the cell fate choice during blastocyst development, including transcriptional regulation, epigenetic regulation, mieroRNAs, and signal transduction.
