There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be alt...There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be altered in case of high body mass index （BMI）. A few studies assessing the impact of BMI on sperm DNA integrity have been published, but they did not lead to a strong consensus. Our objective was to explore further the relationship between sperm DNA integrity and BMI, through a 3-year multicentre study. Three hundred and thirty male partners in subfertile couples were included. Using the terminal uridine nick-end labelling （TUNEL） assay, we observed an increased rate of sDerm DNA damage in obese men （odds ratio （95% confidence interval）： 2.5 （1.2-5.1））.展开更多
In vertebrates, the skeletal muscles of the body and their associated stem cells originate from muscle progenitor cells,during development. The specification of the muscles of the trunk, head and limbs, relies on the ...In vertebrates, the skeletal muscles of the body and their associated stem cells originate from muscle progenitor cells,during development. The specification of the muscles of the trunk, head and limbs, relies on the activity of distinctgenetic hierarchies. The major regulators of trunk and limb muscle specification are the paired-homeobox transcriptionfactors PAX3 and PAX7. Distinct gene regulatory networks drive the formation of the different muscles of thehead. Despite the redeployment of diverse upstream regulators of muscle progenitor differentiation, the commitmenttowards the myogenic fate requires the expression of the early myogenic regulatory factors MYF5, MRF4, MYOD andthe late differentiation marker MYOG. The expression of these genes is activated by muscle progenitors throughoutdevelopment, in several waves of myogenic differentiation, constituting the embryonic, fetal and postnatal phases ofmuscle growth. In order to achieve myogenic cell commitment while maintaining an undifferentiated pool of muscleprogenitors, several signaling pathways regulate the switch between proliferation and differentiation of myoblasts.The identification of the gene regulatory networks operating during myogenesis is crucial for the development ofin vitro protocols to differentiate pluripotent stem cells into myoblasts required for regenerative medicine.展开更多
Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer (SCNT), holds great promise for treating many human diseases using regene...Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer (SCNT), holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells (hESCs) have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmission, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi-or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC (ntESC) lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results indicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.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
The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodel-ing of cloned embryos.In this study we investigated the effect of DNA methylation state of donor cells on the d...The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodel-ing of cloned embryos.In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem(ES)cell nuclei.Our results confirmed that deletion of the DNA methyltransferase 3a(Dnmt3a)and DNA methyltransferase 3b(Dnmt3b)distinctly decreases the level of DNA methylation in ES cells.In contrast to wild type ES cells(J1),Dnmt3a–/–3b–/–(DKO)and Dnmt3b–/–(3bKO)donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer(ECNT)morula,blastocysts and postimplantation embryos(P<0.05).However,the efficiency of establish-ment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved,and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postim-plantation embryos was not altered either.Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.展开更多
Background:There has been a growing interest in camel anaplasmosis due to its recent emergence in this reservoir species and concerns for its zoonotic potential.The epidemiology of anaplasmosis in camels therefore rem...Background:There has been a growing interest in camel anaplasmosis due to its recent emergence in this reservoir species and concerns for its zoonotic potential.The epidemiology of anaplasmosis in camels therefore remains poorly understood mostly because camels belong to marginalised poor and often transhumant populations whose interests are largely neglected.Most studies of anaplasmosis in camels have relied on microscopy and serology for diagnosis and only three studies,undertaken in Tunisia,Saudia Arabia and China,have used molecular diagnostics.The present work characterises Anaplasmataceae strains circulating in the Camelus dromedarius reservoir in Morocco using PCR.Methods:Camels(n=106)were randomly sampled from 6 regions representing different agro-ecological areas in southern Morocco.Whole blood was collected and screened using PCR methods targeting the gene groEL.Anaplasmataceae strains were characterised by sequence analysis of the gene groEL.Results:A total of 39.62%(42/106)camels screened were positive for Anaplasmataceae spp.GenBank BLAST analysis of five positive sequenced samples revealed that all strains were 100%identical to“Candidatus Anaplasma camelii”.Phylogenetic investigation and genetic characterisation of the aligned segment(650 bp)of the gene groEL confirmed high similarity with A.platys.Conclusion:This study demonstrates the circulation of a previously unidentified species of the genus Anaplasma in Morocco which is genetically close to the agent causing canine anaplasmosis but whose main reservoir is thought to be Camelus dromedarius.Trial registration number:This study is not a clinical trial and therefore a trial registration number does not apply.展开更多
Exome sequencing(ES)generates secondary findings(SFs)in 2%of tested individuals if one follows the American College of Medical Genetics and Genomics(ACMG)guide-lines.i,z2 However,the rate of incidental and secondary f...Exome sequencing(ES)generates secondary findings(SFs)in 2%of tested individuals if one follows the American College of Medical Genetics and Genomics(ACMG)guide-lines.i,z2 However,the rate of incidental and secondary findings(ISFs)is higher in routine clinical practice because of(i)the use of trio ES instead of solo sequencing and(i)the exclusion of the incidental findings(IFs)of medical value concerning genes in the ACMG list.Hence,it is not clear how sufficient is a restricted list of genes to detect every ISF of major clinical value;and what is the amount of additional workload for the laboratory.展开更多
The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies,yet the underlying cellular and molecular mechanisms remain elusive.In this study,we investigate the role of HACD1/PTPLA,which ...The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies,yet the underlying cellular and molecular mechanisms remain elusive.In this study,we investigate the role of HACD1/PTPLA,which is involved in the elongation of the very long chain fatty acids,in muscle fibre formation.In humans and dogs,HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscleweakness.Throughanalysis of HACD1-deficient Labradors,Hacd1-knockout mice,and Hacd1-deficient myoblasts,we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration.We further demonstrate that in normal differentiating myoblasts,expression of the catalytically active HACD1 isoform,which is encoded by a muscle-enriched splice variant,yields decreased lysophosphatidylcholine content,a potent inhibitor of myoblast fusion,and increased concentrations of≥C18 and monounsaturated fatty acids of phospholipids.These lipid modifications correlate with a reduction in plasma membrane rigidity.In conclusion,we propose that fusion impairment constitutes a novel,non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism.展开更多
文摘There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be altered in case of high body mass index （BMI）. A few studies assessing the impact of BMI on sperm DNA integrity have been published, but they did not lead to a strong consensus. Our objective was to explore further the relationship between sperm DNA integrity and BMI, through a 3-year multicentre study. Three hundred and thirty male partners in subfertile couples were included. Using the terminal uridine nick-end labelling （TUNEL） assay, we observed an increased rate of sDerm DNA damage in obese men （odds ratio （95% confidence interval）： 2.5 （1.2-5.1））.
基金FR laboratory is supported by funding from Association Française contre les Myopathies(AFM)via TRANSLAMUSCLE(PROJECT 19507 and 22946)Agence Nationale pour la Recherche(ANR)grant Epimuscle(ANR 11 BSV201702)RHU CARMMA(ANR-15-RHUS-0003).
文摘In vertebrates, the skeletal muscles of the body and their associated stem cells originate from muscle progenitor cells,during development. The specification of the muscles of the trunk, head and limbs, relies on the activity of distinctgenetic hierarchies. The major regulators of trunk and limb muscle specification are the paired-homeobox transcriptionfactors PAX3 and PAX7. Distinct gene regulatory networks drive the formation of the different muscles of thehead. Despite the redeployment of diverse upstream regulators of muscle progenitor differentiation, the commitmenttowards the myogenic fate requires the expression of the early myogenic regulatory factors MYF5, MRF4, MYOD andthe late differentiation marker MYOG. The expression of these genes is activated by muscle progenitors throughoutdevelopment, in several waves of myogenic differentiation, constituting the embryonic, fetal and postnatal phases ofmuscle growth. In order to achieve myogenic cell commitment while maintaining an undifferentiated pool of muscleprogenitors, several signaling pathways regulate the switch between proliferation and differentiation of myoblasts.The identification of the gene regulatory networks operating during myogenesis is crucial for the development ofin vitro protocols to differentiate pluripotent stem cells into myoblasts required for regenerative medicine.
基金the National Nature Science Foundation of China (Grant Nos. 30525040 and 30670229)
文摘Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer (SCNT), holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells (hESCs) have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmission, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi-or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC (ntESC) lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results indicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.
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.
基金supported by grants from the National Natural Science Foundation of China(Grant No.90919060)from the National Basic Research Program of China(No.2006CB944003).
文摘The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodel-ing of cloned embryos.In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem(ES)cell nuclei.Our results confirmed that deletion of the DNA methyltransferase 3a(Dnmt3a)and DNA methyltransferase 3b(Dnmt3b)distinctly decreases the level of DNA methylation in ES cells.In contrast to wild type ES cells(J1),Dnmt3a–/–3b–/–(DKO)and Dnmt3b–/–(3bKO)donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer(ECNT)morula,blastocysts and postimplantation embryos(P<0.05).However,the efficiency of establish-ment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved,and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postim-plantation embryos was not altered either.Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.
基金This work was supported by the PRAD project under grant agreement number 28027YMthe Institut Agronomique et Veterinaire Hassan II,BIPAR and Ecole Nationale Veterinaire de Toulouse。
文摘Background:There has been a growing interest in camel anaplasmosis due to its recent emergence in this reservoir species and concerns for its zoonotic potential.The epidemiology of anaplasmosis in camels therefore remains poorly understood mostly because camels belong to marginalised poor and often transhumant populations whose interests are largely neglected.Most studies of anaplasmosis in camels have relied on microscopy and serology for diagnosis and only three studies,undertaken in Tunisia,Saudia Arabia and China,have used molecular diagnostics.The present work characterises Anaplasmataceae strains circulating in the Camelus dromedarius reservoir in Morocco using PCR.Methods:Camels(n=106)were randomly sampled from 6 regions representing different agro-ecological areas in southern Morocco.Whole blood was collected and screened using PCR methods targeting the gene groEL.Anaplasmataceae strains were characterised by sequence analysis of the gene groEL.Results:A total of 39.62%(42/106)camels screened were positive for Anaplasmataceae spp.GenBank BLAST analysis of five positive sequenced samples revealed that all strains were 100%identical to“Candidatus Anaplasma camelii”.Phylogenetic investigation and genetic characterisation of the aligned segment(650 bp)of the gene groEL confirmed high similarity with A.platys.Conclusion:This study demonstrates the circulation of a previously unidentified species of the genus Anaplasma in Morocco which is genetically close to the agent causing canine anaplasmosis but whose main reservoir is thought to be Camelus dromedarius.Trial registration number:This study is not a clinical trial and therefore a trial registration number does not apply.
文摘Exome sequencing(ES)generates secondary findings(SFs)in 2%of tested individuals if one follows the American College of Medical Genetics and Genomics(ACMG)guide-lines.i,z2 However,the rate of incidental and secondary findings(ISFs)is higher in routine clinical practice because of(i)the use of trio ES instead of solo sequencing and(i)the exclusion of the incidental findings(IFs)of medical value concerning genes in the ACMG list.Hence,it is not clear how sufficient is a restricted list of genes to detect every ISF of major clinical value;and what is the amount of additional workload for the laboratory.
基金This work was supported by the Agence Nationale de la Recherche(ANR-12-JSV1-0005)the Association Franc¸aise contre les Myopathies(14577,15882,and 16143)+4 种基金the CNM Project(www.labradorcnm.com)the Alliance program(22866ZM)the Myotubular Trust and Grants-in-Aid for Scientific Research(B)to A.K.from Japan Society for the Promotion of Science(23370057)J.B.was supported by the French Ministry of Research and Technologies and the Universite´Paris 6(Paris)V.G.,A.P.,and A.R.were supported by the ANR,N.B-G.and I.B.were supported by the AFM,and G.W.was supported by the BBSRC CASE and the Myotubular Trust.
文摘The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies,yet the underlying cellular and molecular mechanisms remain elusive.In this study,we investigate the role of HACD1/PTPLA,which is involved in the elongation of the very long chain fatty acids,in muscle fibre formation.In humans and dogs,HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscleweakness.Throughanalysis of HACD1-deficient Labradors,Hacd1-knockout mice,and Hacd1-deficient myoblasts,we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration.We further demonstrate that in normal differentiating myoblasts,expression of the catalytically active HACD1 isoform,which is encoded by a muscle-enriched splice variant,yields decreased lysophosphatidylcholine content,a potent inhibitor of myoblast fusion,and increased concentrations of≥C18 and monounsaturated fatty acids of phospholipids.These lipid modifications correlate with a reduction in plasma membrane rigidity.In conclusion,we propose that fusion impairment constitutes a novel,non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism.
