Homozygous human embryonic stem cells （hESCs） are thought to be better cell sources for hESC banking because their human leukocyte antigen （HLA） haplotype would strongly increase the degree of matching for certain...Homozygous human embryonic stem cells （hESCs） are thought to be better cell sources for hESC banking because their human leukocyte antigen （HLA） haplotype would strongly increase the degree of matching for certain populations with relatively smaller cohorts of cell lines. Homozygous hESCs can be generated from parthenogenetic embryos, but only heterozygous hESCs have been established using the current strategy to artificially activate the oocyte without second polar body extrusion. Here we report the first successful derivation of a human homozygous ESC line （chHES- 32） from a one-pronuclear oocyte following routine in vitro fertilization treatment, chHES-32 cells express common markers and genes with normal hESCs. They have been propagated in an undifferentiated state for more than a year （〉P50） and have maintained a stable karyotype of 46, XX. When differentiated in vivo and in vitro, chHES-32 cells can form derivatives from all three embryonic germ layers. The almost undetectable expression of five paternally expressed imprinted genes and their HLA genotype identical to the oocyte donor indicated their parthenogenetic origin. Using genome-wide single-nucleotide polymorphism analysis and DNA fingerprinting, the homozygosity of chHES-32 cells was further confirmed. The results indicated that ‘ unwanted＇ one-pronuclear oocytes might be a potential source for human homozygous and parthenogenetic ESCs, and suggested an alternative strategyfor obtaining homozygous hESC lines from parthenogenetic haploid oocytes.展开更多
To assess the relationship between pronuclear scoring and day-3 embryo quality and pregnancy outcome and to determine the Clinical value of pronuclear stage scoring system in human in vitro fertilization-embryo transf...To assess the relationship between pronuclear scoring and day-3 embryo quality and pregnancy outcome and to determine the Clinical value of pronuclear stage scoring system in human in vitro fertilization-embryo transfer （IVF-ET） program, a pronuclear scoring system was used to score zygotes 16-20 h after insemination during conventional IVF or intracytoplasmic sperm injection （ICSI）. The embryos were classified into groups Z1, Z2, Z3 and Z4, Comparisons were made of the rates of arrested embryos and excellent embryos on day 3. Comparisons of pregnancy outcome were made only in those patients in whom cohorts of similarly Z-scored embryos were transferred, The results showed that there were less arrested embryos and more excellent embryos on day 3 in groups Z1 and Z2 than those in group Z3 and Z4, More embryos arrested and less excellent embryos developed in group Z4 than group Z3. The clinical pregnancy rates resulting from the transfer of single pronuclear score homologous embryo types were similar among groups Z1, Z2 and Z3. Implantation rates of group Z1 were higher （P〈0.05） than that of group Z3, These findings suggests that pronuclear scoring can predict developmental ability on day 3 and implantation potential. A evaluation that combines the Z-score and day 3 embryo morphology is useful in the determination of the most viable embryos and the number of embryos for transfer.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
Objective To explore the relationship between the patterns of pronucleus and embryo development and pregnancy potential in the pronuclear stageMethods According to the number and distribution of nucleolar precursor bo...Objective To explore the relationship between the patterns of pronucleus and embryo development and pregnancy potential in the pronuclear stageMethods According to the number and distribution of nucleolar precursor bodies, the embryos at pronuclear stage were classified into 6 pronuclear patterns from 0 to 5, 16 - 18 h after in vitro fertilization (IV F) or intracytoplasmic sperm injection (ICSI). For each, pattern, the subsequent embryonic morphology and the pregnancy rate were analyzed.Results Embryos of Pattern 0 developed to significantly more embryos with good quality and higher pregnancy potential than the embryos developing from other patterns (83. 14% and 76. 11% respectively, P<0. 05). The pregnancy rate was decreased as less embryos of Pattern 0 were transferred . The pregnancy rate of the groups of only Pattern 0, with Pattern 0, and without Pattern 0 were 48. 08% , 32. 14% and 21. 28% respectively (P<0. 05).Conclusions The pronuclear patterns are of the predictive value of embryo development and pregnancy potential, which can be used as a new tool for the selection of embryos in IVF and ICSI.展开更多
Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard met...Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.展开更多
Failure of oocyte activation,including polyspermy and defects in pronuclear(PN)formation,triggers early embryonic developmental arrest.Many studies have shown that phospholipase C zeta 1(PLCZ1)mutations cause failure ...Failure of oocyte activation,including polyspermy and defects in pronuclear(PN)formation,triggers early embryonic developmental arrest.Many studies have shown that phospholipase C zeta 1(PLCZ1)mutations cause failure of PN formation following intracytoplasmic sperm injection(ICSI);however,whether PLCZ1 mutation is associated with polyspermy during in vitro fertilization(IVF)remains unknown.Whole-exome sequencing(WES)was performed to identify candidate mutations in couples with primary infertility.Sanger sequencing was used to validate the mutations.Multiple PLCZ1-mutated sperm were injected into human and mouse oocytes to explore whether PN formation was induced.Assisted oocyte activation(AOA)after ICSI was performed to overcome the failure of oocyte activation.We identified three PLCZ1 mutations in three patients who experienced polyspermy during IVF cycles,including a novel missense mutation c.1154C>T,p.R385Q.PN formation failure was observed during the ICSI cycle.However,injection of multiple PLCZ1-mutated sperm induced PN formation,suggesting that the Ca2+oscillations induced by the sperm exceeded the necessary threshold for PN formation.AOA after ICSI enabled normal fertilization,and all patients achieved successful pregnancies.These findings expand the mutational spectrum of PLCZ1 and suggest an important role for PLCZ1 in terms of blocking polyspermy.Furthermore,this study may benefit genetic diagnoses in cases of abnormal fertilization and provide potential appropriate therapeutic measures for these patients with sperm-derived polyspermy.展开更多
Mutations in mitochondrial DNA(mtDNA)are maternally inherited and have the potential to cause severe disorders.Mitochondrial replacement therapies,including spindle,polar body,and pronuclear transfers,are promising st...Mutations in mitochondrial DNA(mtDNA)are maternally inherited and have the potential to cause severe disorders.Mitochondrial replacement therapies,including spindle,polar body,and pronuclear transfers,are promising strategies for preventing the hereditary transmission of mtDNA diseases.While pronuclear transfer has been used to generate mitochondrial replacement mouse models and human embryos,its application in non-human primates has not been previously reported.In this study,we successfully generated four healthy cynomolgus monkeys(Macaca fascicularis)via female pronuclear transfer.These individuals all survived for more than two years and exhibited minimal mtDNA carryover(3.8%–6.7%),as well as relatively stable mtDNA heteroplasmy dynamics during development.The successful establishment of this nonhuman primate model highlights the considerable potential of pronuclear transfer in reducing the risk of inherited mtDNA diseases and provides a valuable preclinical research model for advancing mitochondrial replacement therapies in humans.展开更多
文摘Homozygous human embryonic stem cells （hESCs） are thought to be better cell sources for hESC banking because their human leukocyte antigen （HLA） haplotype would strongly increase the degree of matching for certain populations with relatively smaller cohorts of cell lines. Homozygous hESCs can be generated from parthenogenetic embryos, but only heterozygous hESCs have been established using the current strategy to artificially activate the oocyte without second polar body extrusion. Here we report the first successful derivation of a human homozygous ESC line （chHES- 32） from a one-pronuclear oocyte following routine in vitro fertilization treatment, chHES-32 cells express common markers and genes with normal hESCs. They have been propagated in an undifferentiated state for more than a year （〉P50） and have maintained a stable karyotype of 46, XX. When differentiated in vivo and in vitro, chHES-32 cells can form derivatives from all three embryonic germ layers. The almost undetectable expression of five paternally expressed imprinted genes and their HLA genotype identical to the oocyte donor indicated their parthenogenetic origin. Using genome-wide single-nucleotide polymorphism analysis and DNA fingerprinting, the homozygosity of chHES-32 cells was further confirmed. The results indicated that ‘ unwanted＇ one-pronuclear oocytes might be a potential source for human homozygous and parthenogenetic ESCs, and suggested an alternative strategyfor obtaining homozygous hESC lines from parthenogenetic haploid oocytes.
文摘To assess the relationship between pronuclear scoring and day-3 embryo quality and pregnancy outcome and to determine the Clinical value of pronuclear stage scoring system in human in vitro fertilization-embryo transfer （IVF-ET） program, a pronuclear scoring system was used to score zygotes 16-20 h after insemination during conventional IVF or intracytoplasmic sperm injection （ICSI）. The embryos were classified into groups Z1, Z2, Z3 and Z4, Comparisons were made of the rates of arrested embryos and excellent embryos on day 3. Comparisons of pregnancy outcome were made only in those patients in whom cohorts of similarly Z-scored embryos were transferred, The results showed that there were less arrested embryos and more excellent embryos on day 3 in groups Z1 and Z2 than those in group Z3 and Z4, More embryos arrested and less excellent embryos developed in group Z4 than group Z3. The clinical pregnancy rates resulting from the transfer of single pronuclear score homologous embryo types were similar among groups Z1, Z2 and Z3. Implantation rates of group Z1 were higher （P〈0.05） than that of group Z3, These findings suggests that pronuclear scoring can predict developmental ability on day 3 and implantation potential. A evaluation that combines the Z-score and day 3 embryo morphology is useful in the determination of the most viable embryos and the number of embryos for transfer.
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.
文摘Objective To explore the relationship between the patterns of pronucleus and embryo development and pregnancy potential in the pronuclear stageMethods According to the number and distribution of nucleolar precursor bodies, the embryos at pronuclear stage were classified into 6 pronuclear patterns from 0 to 5, 16 - 18 h after in vitro fertilization (IV F) or intracytoplasmic sperm injection (ICSI). For each, pattern, the subsequent embryonic morphology and the pregnancy rate were analyzed.Results Embryos of Pattern 0 developed to significantly more embryos with good quality and higher pregnancy potential than the embryos developing from other patterns (83. 14% and 76. 11% respectively, P<0. 05). The pregnancy rate was decreased as less embryos of Pattern 0 were transferred . The pregnancy rate of the groups of only Pattern 0, with Pattern 0, and without Pattern 0 were 48. 08% , 32. 14% and 21. 28% respectively (P<0. 05).Conclusions The pronuclear patterns are of the predictive value of embryo development and pregnancy potential, which can be used as a new tool for the selection of embryos in IVF and ICSI.
文摘Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.
基金the General Project of Chongqing Natural Science foundation of China(cstc2021jcyj-msxmX0877)the Chongqing Medical Scientific Research Project(Joint Project of Chongqing Health Commission and Science and Technology Bureau,2023MSXM054)the General Project of Chongqing Health Center for Women and Children(2020YJMS01 and 2021YJMS05).
文摘Failure of oocyte activation,including polyspermy and defects in pronuclear(PN)formation,triggers early embryonic developmental arrest.Many studies have shown that phospholipase C zeta 1(PLCZ1)mutations cause failure of PN formation following intracytoplasmic sperm injection(ICSI);however,whether PLCZ1 mutation is associated with polyspermy during in vitro fertilization(IVF)remains unknown.Whole-exome sequencing(WES)was performed to identify candidate mutations in couples with primary infertility.Sanger sequencing was used to validate the mutations.Multiple PLCZ1-mutated sperm were injected into human and mouse oocytes to explore whether PN formation was induced.Assisted oocyte activation(AOA)after ICSI was performed to overcome the failure of oocyte activation.We identified three PLCZ1 mutations in three patients who experienced polyspermy during IVF cycles,including a novel missense mutation c.1154C>T,p.R385Q.PN formation failure was observed during the ICSI cycle.However,injection of multiple PLCZ1-mutated sperm induced PN formation,suggesting that the Ca2+oscillations induced by the sperm exceeded the necessary threshold for PN formation.AOA after ICSI enabled normal fertilization,and all patients achieved successful pregnancies.These findings expand the mutational spectrum of PLCZ1 and suggest an important role for PLCZ1 in terms of blocking polyspermy.Furthermore,this study may benefit genetic diagnoses in cases of abnormal fertilization and provide potential appropriate therapeutic measures for these patients with sperm-derived polyspermy.
基金supported by the National Natural Science Foundation of China (82021001,31825018)National Key Research and Development Program of China (2022YFF0710901)+3 种基金Shanghai Municipal Science and Technology Major Project (2018SHZDZX05)Strategic Priority Research Program of the Chinese Academy of Sciences (XDB32060100)Biological Resources Program of Chinese Academy of Sciences (KFJ-BRP-005)National Science and Technology Innovation 2030 Major Program 2021ZD0200900。
文摘Mutations in mitochondrial DNA(mtDNA)are maternally inherited and have the potential to cause severe disorders.Mitochondrial replacement therapies,including spindle,polar body,and pronuclear transfers,are promising strategies for preventing the hereditary transmission of mtDNA diseases.While pronuclear transfer has been used to generate mitochondrial replacement mouse models and human embryos,its application in non-human primates has not been previously reported.In this study,we successfully generated four healthy cynomolgus monkeys(Macaca fascicularis)via female pronuclear transfer.These individuals all survived for more than two years and exhibited minimal mtDNA carryover(3.8%–6.7%),as well as relatively stable mtDNA heteroplasmy dynamics during development.The successful establishment of this nonhuman primate model highlights the considerable potential of pronuclear transfer in reducing the risk of inherited mtDNA diseases and provides a valuable preclinical research model for advancing mitochondrial replacement therapies in humans.
