Methods in molecular biology (Clifton, N.J.), 2012
Nonhuman primates are the closest relatives to humans and therefore our most evolutionary close c... more Nonhuman primates are the closest relatives to humans and therefore our most evolutionary close cousins. While marvelous insights are gleaned from studying rodents and other systems, it is impossible to envision how those mechanistic findings can be responsibly translated to the clinic without the appropriate use of nonhuman primates. Thankfully, noninvasive technologies now permit nonhuman primate studies without endangering the model itself. Work with primates is predicted to continue to lead the fields of reproductive and regenerative medicine for the rest of the twenty-first century.
In this study, we used a large non-human primate model, the baboon, to establish a step-wise prot... more In this study, we used a large non-human primate model, the baboon, to establish a step-wise protocol to generate CD34+ endothelial progenitor cells (EPCs) from embryonic stem cells (ESCs) and to demonstrate their reparative effects. Baboon ESCs were sequentially differentiated from embryoid body cultures for 9 days and then were specified into EPCs by culturing them in monolayer for 12 days. The resulting EPCs expressed CD34, CXCR4 and UEA-1, but neither CD31 nor CD117. The EPCs were able to form intact lumen structures when seeded on Matrigel, took up Dil-LDL, and responded to TNF-α. Angioblasts specified in EGM-2 medium and ECGS medium had 6.41 ± 1.16% (n = 3) and 9.32 ± 3.73% CD34+ cells (n = 3). The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM-2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ± 1.32%, n = 3) than those specified in EGM-2 medium (0.49 ± 0.52%, n = 3). To o...
We have studied the accumulation and localization of U1 RNA during mouse embryo development by in... more We have studied the accumulation and localization of U1 RNA during mouse embryo development by in situ hybridization with a U1 RNA probe and immunofluorescence microscopy using a mouse monoclonal antibody to U1 snRNP. There is a substantial amount of U1 RNA present in the oocyte that is present in both the germinal vesicle and the cytoplasm although the concentration is higher in the nuclear compartment. Following the germinal vesicle breakdown that accompanies ovulation and meiotic maturation, the U1 RNA is uniformly distributed throughout the unfertilized oocyte. In the fertilized egg, the silver grain density from in situ hybridization is higher over pronuclei and this enrichment is maintained at the two-cell and later stages. Similar results were obtained for the distribution of the U1 snRNP as assayed by immunofluorescence microscopy: U1 RNA is predominantly localized in all nuclei except polar body nuclei. The U1 RNA in the oocyte and two-cell embryo is predominantly (greater than 85%) U1a RNA. By the eight-cell stage there is a two to three-fold increase in the amount of total U1 RNA and the proportion of U1b RNA has increased to about 40%. The amount of U1 RNA continues to increase through the blastocyst stage and the proportion of the U1b RNA increases to 60%.
Microtubule assembly is required for the formation of the male and female pronuclei during mouse,... more Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 microM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the Pl nuclear peripheral antigen, appears on both. DNA synthesis does not occur in these fertilized, colcemid-arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4-6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 microM taxol nor microfilament inhibition with 10 microM cytochalasin D or 2.2 micrograms/ml latrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment does not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.
Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and seve... more Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the 'Weismann barrier', which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis.
Exogenous DNA transfer, mediated by intracytoplasmic sperm injection (ICSI) with plasmid-bound sp... more Exogenous DNA transfer, mediated by intracytoplasmic sperm injection (ICSI) with plasmid-bound spermatozoa, results in the production of transgene expressing embryos in rhesus macaques (Macaca mulatta, mean 34.6%; n 81). Rhodamine-tagged DNA encoding the green fluorescent protein (GFP) gene binds avidly to spermatozoa. The rhodamine signal, while lost at the egg surface during in-vitro fertilization (IVF), is traced by dynamic imaging
Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome ar... more Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents g-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm micro- tubule-nucleating capability on reconstituted centrosomes. g-Tubulin is biparentally inherited in humans (maternal . . than paternal): Western blots detect the presence of paternal g-tubulin. Recruitment of maternal g-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm "priming" induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosom...
Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspr... more Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor.
Successful fertilization in humans, achieved when parental chromosomes intermix at first mitosis,... more Successful fertilization in humans, achieved when parental chromosomes intermix at first mitosis, requires centrosome restoration and microtubule-mediated motility. Imaging of inseminated human oocytes reveals that the sperm introduces the centrosome. The centrosome then nucleates the new microtubule assembly to form the sperm aster--a step essential for successful fertilization. Oocytes from some infertile patients failed to complete fertilization because of defects in uniting the sperm and egg nuclei, indicating that failure to properly effect the cytoplasmic motions uniting the nuclei results in human infertility. These discoveries have important implications for infertility diagnosis and managing reproduction.
Methods in molecular biology (Clifton, N.J.), 2012
Nonhuman primates are the closest relatives to humans and therefore our most evolutionary close c... more Nonhuman primates are the closest relatives to humans and therefore our most evolutionary close cousins. While marvelous insights are gleaned from studying rodents and other systems, it is impossible to envision how those mechanistic findings can be responsibly translated to the clinic without the appropriate use of nonhuman primates. Thankfully, noninvasive technologies now permit nonhuman primate studies without endangering the model itself. Work with primates is predicted to continue to lead the fields of reproductive and regenerative medicine for the rest of the twenty-first century.
In this study, we used a large non-human primate model, the baboon, to establish a step-wise prot... more In this study, we used a large non-human primate model, the baboon, to establish a step-wise protocol to generate CD34+ endothelial progenitor cells (EPCs) from embryonic stem cells (ESCs) and to demonstrate their reparative effects. Baboon ESCs were sequentially differentiated from embryoid body cultures for 9 days and then were specified into EPCs by culturing them in monolayer for 12 days. The resulting EPCs expressed CD34, CXCR4 and UEA-1, but neither CD31 nor CD117. The EPCs were able to form intact lumen structures when seeded on Matrigel, took up Dil-LDL, and responded to TNF-α. Angioblasts specified in EGM-2 medium and ECGS medium had 6.41 ± 1.16% (n = 3) and 9.32 ± 3.73% CD34+ cells (n = 3). The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM-2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ± 1.32%, n = 3) than those specified in EGM-2 medium (0.49 ± 0.52%, n = 3). To o...
We have studied the accumulation and localization of U1 RNA during mouse embryo development by in... more We have studied the accumulation and localization of U1 RNA during mouse embryo development by in situ hybridization with a U1 RNA probe and immunofluorescence microscopy using a mouse monoclonal antibody to U1 snRNP. There is a substantial amount of U1 RNA present in the oocyte that is present in both the germinal vesicle and the cytoplasm although the concentration is higher in the nuclear compartment. Following the germinal vesicle breakdown that accompanies ovulation and meiotic maturation, the U1 RNA is uniformly distributed throughout the unfertilized oocyte. In the fertilized egg, the silver grain density from in situ hybridization is higher over pronuclei and this enrichment is maintained at the two-cell and later stages. Similar results were obtained for the distribution of the U1 snRNP as assayed by immunofluorescence microscopy: U1 RNA is predominantly localized in all nuclei except polar body nuclei. The U1 RNA in the oocyte and two-cell embryo is predominantly (greater than 85%) U1a RNA. By the eight-cell stage there is a two to three-fold increase in the amount of total U1 RNA and the proportion of U1b RNA has increased to about 40%. The amount of U1 RNA continues to increase through the blastocyst stage and the proportion of the U1b RNA increases to 60%.
Microtubule assembly is required for the formation of the male and female pronuclei during mouse,... more Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 microM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the Pl nuclear peripheral antigen, appears on both. DNA synthesis does not occur in these fertilized, colcemid-arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4-6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 microM taxol nor microfilament inhibition with 10 microM cytochalasin D or 2.2 micrograms/ml latrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment does not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.
Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and seve... more Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the 'Weismann barrier', which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis.
Exogenous DNA transfer, mediated by intracytoplasmic sperm injection (ICSI) with plasmid-bound sp... more Exogenous DNA transfer, mediated by intracytoplasmic sperm injection (ICSI) with plasmid-bound spermatozoa, results in the production of transgene expressing embryos in rhesus macaques (Macaca mulatta, mean 34.6%; n 81). Rhodamine-tagged DNA encoding the green fluorescent protein (GFP) gene binds avidly to spermatozoa. The rhodamine signal, while lost at the egg surface during in-vitro fertilization (IVF), is traced by dynamic imaging
Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome ar... more Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents g-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm micro- tubule-nucleating capability on reconstituted centrosomes. g-Tubulin is biparentally inherited in humans (maternal . . than paternal): Western blots detect the presence of paternal g-tubulin. Recruitment of maternal g-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm "priming" induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosom...
Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspr... more Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor.
Successful fertilization in humans, achieved when parental chromosomes intermix at first mitosis,... more Successful fertilization in humans, achieved when parental chromosomes intermix at first mitosis, requires centrosome restoration and microtubule-mediated motility. Imaging of inseminated human oocytes reveals that the sperm introduces the centrosome. The centrosome then nucleates the new microtubule assembly to form the sperm aster--a step essential for successful fertilization. Oocytes from some infertile patients failed to complete fertilization because of defects in uniting the sperm and egg nuclei, indicating that failure to properly effect the cytoplasmic motions uniting the nuclei results in human infertility. These discoveries have important implications for infertility diagnosis and managing reproduction.
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Papers by Calvin Simerly