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ABSTRACT: Considerable progress has been made in methods for production of transgenic livestock; beginning with pronuclear microinjection over 20 years ago. New methods, including the use of viral vectors, sperm-mediated gene transfer and somatic cell cloning, have overcome many of the limitations of pronuclear microinjection. It is now possible to not only readily make simple insertional genetic modifications, but also to accomplish, more complex, homozygous gene targeting and artificial chromosome transfer in livestock.
Theriogenology 02/2007; 67(1):127-33. · 1.96 Impact Factor
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ABSTRACT: The purpose of this paper is to review the economic and social implications of cloned cattle, their products, and their offspring as related to production agriculture. Cloning technology in cattle has several applications outside of traditional production agriculture. These applications can include bio-medical applications, such as the production of pharmaceuticals in the blood or milk of transgenic cattle. Cloning may also be useful in the production of research models. These models may or may not include genetic modifications. Uses in agriculture include many applications of the technology. These include making genetic copies of elite seed stock and prize winning show cattle. Other purposes may range from "insurance" to making copies of cattle that have sentimental value, similar to cloning of pets. Increased selection opportunities available with cloning may provide for improvement in genetic gain. The ultimate goal of cloning has often been envisioned as a system for producing quantity and uniformity of the perfect dairy cow. However, only if heritability were 100%, would clone mates have complete uniformity. Changes in the environment may have significant impact on the productivity and longevity of the resulting clones. Changes in consumer preferences and economic input costs may all change the definition of the perfect cow. The cost of producing such animals via cloning must be economically feasible to meet the intended applications. Present inefficiencies limit cloning opportunities to highly valued animals. Improvements are necessary to move the applications toward commercial application. Cloning has additional obstacles to conquer. Social and regulatory acceptance of cloning is paramount to its utilization in production agriculture. Regulatory acceptance will need to address the animal, its products, and its offspring. In summary, cloning is another tool in the animal biotechnology toolbox, which includes artificial insemination, sexing of semen, embryo sexing and in vitro fertilization. While it will not replace any of the above mentioned, its degree of utilization will depend on both improvement in efficiency as well as social and regulatory acceptance.
Cloning and Stem Cells 02/2004; 6(2):198-207. · 2.66 Impact Factor
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ABSTRACT: Artificial chromosome vectors are autonomous, replicating DNA sequences containing a centromere, two telomeres and origins of replication. Artificial chromosomes have been proposed as possible vectors for transferring very large sequences of DNA into animals. Our goal has been to insert the entire human heavy- and light-chain immunoglobulin loci into cattle as a step in developing a production system for large quantities of human therapeutic polyclonal antibodies. A mitotically stable fragment of chromosome 14, containing the human heavy-chain locus, was identified. A chromosome cloning system was used to transfer the human lambda locus from an unstable chromosome 22 fragment to the chromosome 14 fragment to create a human artificial chromosome (HAC) carrying both immunoglobulin loci. The HAC vector was introduced into bovine primary fibroblasts. Selected fibroblast clones were rejuvenated and expanded by producing cloned fetuses. Cloned fetal cells were selected and recloned to produce 21 healthy, transchromosomic (Tc) calves. Four were analyzed and shown to functionally rearrange both heavy- and light-chain human immunoglobulin loci and produce human polyclonal antibodies. These results demonstrate the feasibility of using HAC vectors for production of transgenic livestock. More importantly, Tc cattle containing human immunoglobulin genes may be used to produce novel human polyclonal therapeutics.
Theriogenology 02/2003; 59(1):107-13. · 1.96 Impact Factor
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ABSTRACT: Since the landmark study of Wilmut et al. describing the birth of a cloned lamb derived from a somatic cell nucleus, there has been debate about the donor nucleus cell cycle stage required for somatic cell nuclear transfer (NT). Wilmut et al. suggested that induction of quiescence by serum starvation was critical in allowing donor somatic cells to support development of cloned embryos. In a subsequent report, Cibelli et al. proposed that G0 was unnecessary and that calves could be produced from actively dividing fibroblasts. Neither study conclusively documented the importance of donor cell cycle stage for development to term. Other laboratories have had success with NT in several species, and most have used a serum starvation treatment. Here we evaluate methods for producing G0 and G1 cell populations and compare development following NT. High confluence was more effective than serum starvation for arresting cells in G0. Pure G1 cell populations could be obtained using a "shake-off" procedure. No differences in in vitro development were observed between cells derived from the high-confluence treatment and from the "shake-off" treatment. However, when embryos from each treatment were transferred to 50 recipients, five calves were obtained from embryos derived from "shake-off" cells, whereas no embryos from confluent cells survived beyond 180 days of gestation. These results indicate that donor cell cycle stage is important for NT, particularly during late fetal development, and that actively dividing G1 cells support higher development rates than cells in G0.
Nature Biotechnology 01/2002; 19(12):1176-8. · 23.27 Impact Factor
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ABSTRACT: The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6-15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2-3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.
Biology of Reproduction 06/2001; 64(5):1487-93. · 4.01 Impact Factor
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ABSTRACT: Our previous studies have shown that oocytes collected from prepubertal calves lack developmental competence. The overall objective of this study was to assess causes by comparing biochemical and physiologic changes during in vitro maturation of oocytes collected from ovaries of adult cattle at slaughter and from superstimulated calves (<6 mo old) by either laporotomy or ultrasound-guided follicular aspiration. Activity and/or concentrations of maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and inositol 1,4,5-trisphosphate receptor (IP(3)R) were determined by measuring phosphorylation of histone H-1 kinase, phosphorylation of myelin basic protein, or Western blotting, respectively, and were compared between oocytes collected from calves and for those collected from cows. The activities of MPF and MAPK and the relative amount of IP(3)R were significantly lower in calf oocytes. The physiologic significance of these observations was determined by assessing the developmental potential of embryos derived by reciprocal transfer of metaphase II (M-II) chromosomes between cow and calf ooplasts and transfer of adult cumulus cells (G0/G1) into cow and calf ooplasts. Procedural controls consisted of transfer of M-II between adult oocytes and parthenogenic activation of adult and calf oocytes. Adult parthenogenically activated oocytes cleaved and developed to blastocysts at a higher rate than did similarly activated calf oocytes (42.1% vs. 3.4%, P < 0.05). Cleavage was also higher in reciprocal M-II transfer embryos containing adult ooplasm (46.2% vs. 12.0%, P < 0.05). Cleavage (66.7% vs. 21.9%, P < 0.05) and development to blastocyst (20.1% vs. 4.8%, P < 0.05) of nuclear transfer embryos reconstructed from adult cumulus cells was higher after transfer to adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation.
Biology of Reproduction 06/2001; 64(6):1761-8. · 4.01 Impact Factor
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J R Hill,
A J Roussel,
J B Cibelli,
J F Edwards,
N L Hooper,
M W Miller,
J A Thompson,
C R Looney,
M E Westhusin, J M Robl,
S L Stice
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ABSTRACT: The neonatal abnormalities, treatments and outcomes in a group of 13 cloned transgenic calves and fetuses that progressed into the third trimester of pregnancy are described. From these 13 fetuses, 8 calves were born live, 4 stillborn fetuses were recovered from 3 cows that died 7 d to 2 mo before term, and 1 aborted fetus was recovered at 8 mo gestation. All fetuses and calves were derived from the same male fetal Holstein fibroblast cell line transfected with a beta-galactosidase marker gene. Six calves were delivered by Cesarian section and two by vaginal delivery between 278 and 288 d of gestation. Birth weights ranged from 44 to 58.6 kg. Five of the 8 live born calves were judged to be normal within 4 h of birth based on clinical signs and blood gas measurements. One of these 5 calves died at 6 wk of age from a suspected dilated cardiomyopathy. Three of the 8 calves were diagnosed with neonatal respiratory distress immediately following birth, one of which died (at 4 d of age) as a result of pulmonary surfactant deficiency coupled with pulmonary hypertension and elevated systemic venous pressures. Similar findings of chronic pulmonary hypertension were also observed in 2 of 5 fetuses. Placental edema was present in both calves that later died and in the 2 fetuses with cardiopulmonary abnormalities. Hydrallantois occurred with or without placental edema in 6 cows, and only 1 calf from this group survived. The 6 cows without hydrallantois or placental edema produced 5 live calves and 1 aborted fetus. The cardiopulmonary abnormalities observed in the calves and fetuses occurred in utero in conjunction with placental abnormalities, and it is likely that the cloning technique and/or in vitro embryo culture conditions contributed to these abnormalities, although the mechanism remains to be determined.
Theriogenology 07/1999; 51(8):1451-65. · 1.96 Impact Factor
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J M Robl
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ABSTRACT: Mammalian cloning technologies originally developed as methods of testing hypotheses about the mechanisms of cell differentiation. Embryo splitting procedures demonstrated that each of the cells in the early embryo are capable of developing into a complete new individual. Nuclear transplantation technologies have shown that loss of genetic sequences or even irreversible repression of gene function are also not mechanisms of cell differentiation. Therefore, both of these methods can be used for producing genetically identical animals. Nuclear transplantation has the advantage of being able to produce unlimited numbers of identical offspring. Highly efficient procedures have been developed for nuclear transplantation in mammals and several important characteristics of donor cells have been described. Unfortunately, the efficiency of producing cloned offspring is still low and many factors affecting the development of nuclear transfer embryos to term remain to be investigated. The tremendous potential of the technology for use in agriculture and medicine, however, will ensure that these problems are addressed and solved.
Theriogenology 02/1999; 51(2):499-508. · 1.96 Impact Factor
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ABSTRACT: The technology of cloning involves transplanting a diploid nucleus into a mature oocyte cytoplast. The cytoplast is then activated to initiate the first cell cycle of development as a nuclear transplant embryo. Initiation and regulation of events during the first cell cycle are, therefore, critical for proper reprogramming of the donor nucleus and development as a cloned embryo. Activation is normally induced by the sperm and is mediated by a series of intracellular free calcium ([Ca(2+)](i)) oscillations that last for several hours. Although it is not known precisely how the sperm induces activation, current evidence favors the delivery, by the sperm, of a soluble protein factor that causes the production of IP3. IP3 acts to open a Ca(2+) channel in the endoplasmic reticulum and release Ca(2+) into the cytosol. A variety of methods have been used to duplicate or replace the sperm-induced [Ca(2+)](i) increase to cause activation in nuclear transplant embryos. It has been found that treatments that cause a single transient [Ca(2+)](i) activate some oocytes with the level of activation increasing as the oocyte ages. Attempts have been made to extend the period of time over which [Ca(2+)](i) oscillations occur. This has been successful in increasing activation rates of less mature oocytes but the techniques are still cumbersome. An alternative method, that has been very successful, is the combination of a treatment that elevates [Ca(2+)](i) and a treatment that maintains low levels of maturation promoting factor for several hours after the initial [Ca(2+)](i) elevation. The sperm also contributes the centrosome that organizes microtubules during the first cell cycle. One current hypothesis for regulation of sperm centrosomal activity consists of a dephosphorylation of sperm connecting piece proteins following sperm entry into the oocyte and activation of the oocyte. Dephosphorylation of these proteins results in the disassembly of the connecting piece and assembly of a functional centrosome. In nuclear transfer, centrosomal components are contributed by the donor cell. If the cell is fused to the cytoplast before centriole replication then a single aster forms. If the cell is fused after centriole replication then two asters form. In either case and even in parthenogenetic oocytes, which do not have centrioles, the first cell cycle progresses to metaphase. However, progress is slow and some defects are observed in the assembly of chromosomes into a metaphase plate.
Cloning 02/1999; 1(2):89-100.
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ABSTRACT: In this study we evaluated nuclear and ooplasmic maturation of prepuberal calf oocytes to determine a possible cause for their low developmental competency. Calf oocytes resumed meiosis and arrested at the MII stage at rates similar to that of adult animals; however, zygotes derived from calf oocytes cleaved and developed at significantly lower rates. Ooplasmic maturation was assessed during oocyte maturation and fertilization. Transmission electron microscopy revealed that a majority of calf oocytes exhibited some delay in organelle migration and redistribution following maturation. Immunofluorescence microscopy showed that following IVF, a higher percentage of calf oocytes had abnormal chromatin and microtubule configurations than those of adult cattle. These anomalies were characterized by delayed formation of sperm aster and asynchronous pronuclear formation. Microfluorometry was used to characterize the Ca2+ responses of calf oocytes to the addition of agonists or after IVF. The addition of thimerosal demonstrated the presence of Ca2+ stores in calf oocytes. Injection of near threshold concentrations of inositol 1,4,5-trisphosphate (InsP3), used to test the sensitivity of the InsP3R, released significantly less Ca2+ in calf than in cow oocytes, whereas higher concentrations of InsP3 (500 μM) released maximal [Ca2+]i in both oocytes. These results suggested that the Ca2+ content of intracellular stores was similar, but the sensitivity of the InsP3R may be different. Following insemination, calf oocytes exhibiting [Ca2+]i oscillations displayed comparable amplitude and intervals to cow oocytes; however, a significantly higher number of fertilized calf oocytes failed to show oscillations. Our findings suggest that the low developmental competence of calf oocytes can be attributed, at least in part, to incomplete or delayed ooplasmic maturation. © 1996 Wiley-Liss, Inc.
Molecular Reproduction and Development 12/1998; 45(4):521 - 534. · 2.53 Impact Factor
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ABSTRACT: We have developed a method, using nuclear transplantation, to produce transgenic embryonic stem (ES)-like cells from fetal bovine fibroblasts. These cells, when reintroduced into preimplantation embryos, differentiated into derivatives from the three embryonic germ layers, ectoderm, mesoderm, and endoderm, in 5-month-old animals. Six out of seven (86%) calves born were found to be chimeric for at least one tissue. These experiments demonstrate that somatic cells can be genetically modified and then de-differentiated by nuclear transfer into ES-like cells, opening the possibility of using them in differentiation studies and human cell therapy.
Nature Biotechnology 08/1998; 16(7):642-6. · 23.27 Impact Factor
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ABSTRACT: An efficient system for genetic modification and large-scale cloning of cattle is of importance for agriculture, biotechnology, and human medicine. Here, actively dividing fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected, and the cells were fused to enucleated mature oocytes. Out of 28 embryos transferred to 11 recipient cows, three healthy, identical, transgenic calves were generated. Furthermore, the life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone. With the ability to extend the life-span of these primary cultured cells, this system would be useful for inducing complex genetic modifications in cattle.
Science 06/1998; 280(5367):1256-8. · 31.20 Impact Factor
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W M Zawada,
J B Cibelli,
P K Choi,
E D Clarkson,
P J Golueke,
S E Witta,
K P Bell,
J Kane,
F A Ponce de Leon,
D J Jerry, J M Robl,
C R Freed,
S L Stice
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ABSTRACT: Parkinson's disease symptoms can be improved by transplanting fetal dopamine cells into the putamen of parkinsonian patients. Because the supply of human donor tissue is limited and variable, an alternative and genetically modifiable non-human source of tissue would be valuable. We have generated cloned transgenic bovine embryos, 42% of which developed beyond 40 days. Dopamine cells collected from the ventral mesencephalon of the cloned fetuses 42 to 50 days post-conception survived transplantation into immunosuppressed parkinsonian rats and cells from cloned and wild-type embryos improved motor performance. Somatic cell cloning can efficiently produce transgenic animal tissue for treating parkinsonism.
Nature Medicine 06/1998; 4(5):569-74. · 22.46 Impact Factor
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ABSTRACT: Research on cloning animals, again, came to the forefront of public attention in 1997. Most scientists involved in biomedical and agricultural research have emphasized the benefits, of which there are many, of cloning to the public. Basic studies on nuclear transfer have and will continue to contribute to our understanding of how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies, specifically. Also, more basic information on actual mechanisms and specific factors in the oocyte causing nuclear reprogramming is forthcoming. As new molecular approaches in functional genomics are combined with nuclear transfer experiments, new genes involved in nuclear reprogramming will be found. The commercial potentials of products stemming from discoveries in cloning are vast. Cloning will be a more efficient, faster and more useful way of making transgenic fetuses for cell therapies, adult animals for protein production and organs for xenotransplantation. Clearly there are new opportunities in animal cloning technology that will produce many benefits to society.
Theriogenology 02/1998; 49(1):129-38. · 1.96 Impact Factor
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ABSTRACT: Sperm from most mammalian species catalyze the formation of an aster of microtubules in the oocyte after fertilization. One component that may be involved in the regulation of sperm centrosomal activity in the oocyte is a phosphorylated protein complex (MPM-2-reactive sperm protein; MSP) with a molecular mass of 77-85 kDa identified by the MPM-2 antibody. The objective of this study was to compare the MSPs to a previously identified 85-kDa complex (ODF/CP85) that is a component of the outer dense fibers of the sperm midpiece and the segmented columns of the connecting piece. MSPs were isolated from boar sperm using a differential extraction procedure and preparative gel electrophoresis. Three mouse monoclonal and rabbit polyclonal antibodies were made to the isolated complex, and these antibodies labeled similar proteins in rabbit, bull, boar, and mouse sperm. Extraction and solubilization procedures for MSPs and ODF/CP85 required harsh chaotropic and reducing conditions. In addition to migrating at the same molecular mass on gels, proteins from each preparation labeled with MPM-2, an anti-ODF/CP antibody, and the anti-MSP antibody prepared in this study. Amino acid composition was similar to that reported previously for rat and bull ODF/CP85. Furthermore, immunolocalization by both fluorescent and transmission electron microscopy indicated that the MSPs are components of the outer dense fibers and probably the segmented columns of the connecting piece. Taken together, these results indicate that the MSPs are the previously identified 85-kDa complex of the outer dense fibers and connecting piece. Therefore, it is likely that any involvement of these proteins in the regulation of sperm centrosomal activity is through the process of connecting piece disassembly.
Biology of Reproduction 09/1997; 57(2):246-54. · 4.01 Impact Factor
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ABSTRACT: In this study we evaluated nuclear and ooplasmic maturation of prepuberal calf oocytes to determine a possible cause for their low developmental competency. Calf oocytes resumed meiosis and arrested at the MII stage at rates similar to that of adult animals; however, zygotes derived from calf oocytes cleaved and developed at significantly lower rates. Ooplasmic maturation was assessed during oocyte maturation and fertilization. Transmission electron microscopy revealed that a majority of calf oocytes exhibited some delay in organelle migration and redistribution following maturation. Immunofluorescence microscopy showed that following IVF, a higher percentage of calf oocytes had abnormal chromatin and microtubule configurations than those of adult cattle. These anomalies were characterized by delayed formation of sperm aster and asynchronous pronuclear formation. Microfluorometry was used to characterize the Ca2+ responses of calf oocytes to the addition of agonists or after IVF. The addition of thimerosal demonstrated the presence of Ca2+ stores in calf oocytes. Injection of near threshold concentrations of inositol 1,4,5-trisphosphate (InsP3), used to test the sensitivity of the InsP3R, released significantly less Ca2+ in calf than in cow oocytes, whereas higher concentrations of InsP3 (500 microM) released maximal [Ca2+]i in both oocytes. These results suggested that the Ca2+ content of intracellular stores was similar, but the sensitivity of the InsP3R may be different. Following insemination, calf oocytes exhibiting [Ca2+]i oscillations displayed comparable amplitude and intervals to cow oocytes; however, a significantly higher number of fertilized calf oocytes failed to show oscillations. Our findings suggest that the low developmental competence of calf oocytes can be attributed, at least in part, to incomplete or delayed ooplasmic maturation.
Molecular Reproduction and Development 01/1997; 45(4):521-34. · 2.53 Impact Factor
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ABSTRACT: The X chromosome linkage group is conserved in placental mammals. However, X chromosome morphological differences, due to internal chromosome rearrangements, exist among mammalian species. We have developed bovine chromosome painting probes for Xp and Xq to assess segment homologies between the submetacentric bovine X chromosome and the acrocentric sheep and goat X chromosomes. These painting probes and their corresponding DNA libraries were developed by chromosome micromanipulation, DNA micropurification, microcloning, and PCR amplification. The bovine Xp painting probe identified an interstitially located homologous segment in the sheep and goat Xq region, most probably resulting from chromosome inversion. Ten type II (microsatellite) markers obtained from the bovine Xq library and five other X chromosome assigned, but unlinked, markers were used to generate a linkage map for Xq spanning 89.4 centimorgans. The chromosome painting probes and molecular markers generated in this study would be useful for comparative mapping and tracing of internal X chromosome rearrangements in all ruminant species and would contribute to the understanding of mammalian sex chromosome evolution.
Proceedings of the National Academy of Sciences 05/1996; 93(8):3450-4. · 9.68 Impact Factor
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ABSTRACT: Bovine eggs exhibit repetitive rises in intracellular calcium concentration ([Ca2+]i) in response to fertilization. The signaling pathways and Ca2 release mechanisms involved in their generation are not well characterized. This study examined the presence of a GTP-binding protein (G-protein) signaling pathway as well as the role of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R)-mediated Ca2+ release and ryanodine receptor (RyR)-mediated Ca2+ release, the two Ca2+ receptors/channels most often thought to participate in the generation of [Ca2+]i oscillations, by injecting appropriate agonists and antagonists and monitoring their effects on Ca2+ release and pronucleus formation, injection of guanosine 5'-0-(3-thiotriphosphate) (GTP gamma [S]), which promotes G-protein-mediated phosphoinositide turnover, induced, at high concentrations, repetitive [Ca2+]i rises. Low concentrations of GTP gamma [S] were ineffective. Injection of inositol trisphosphorothioate (InsP3S3), a nonmetabolizable analogue of InsP3, evoked an immediate Ca2+ release followed by [Ca2R]i oscillations. The GTP gamma [S]- and InsP3S3-induced oscillations showed a rapid attenuation in amplitude and were terminated in about 30-60 min. Thimerosal, a thiol oxidizing agent, caused repetitive rises in [Ca2+]i by sensitizing Ca2+ injection-induced Ca2+ release. Injection of ryanodine, which stimulates Ca(2+)-induced Ca2+ release via the RyR, did not induce [Ca2+]i oscillations; and eggs into which it was preinjected exhibited normal [Ca2+]i oscillations in response to thimerosal. Preinjection of heparin, a competitive InsP3R antagonist, blocked in a dose-dependent manner the Ca2+ response to InsP3 and thimerosal, and its injection into fertilized oscillating eggs inhibited [Ca2+]i oscillations in all eggs.(ABSTRACT TRUNCATED AT 250 WORDS)
Biology of Reproduction 11/1995; 53(4):766-74. · 4.01 Impact Factor
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ABSTRACT: Mechanisms of nuclear reprogramming and assessment of potential malfunctions that could be deleterious for development were evaluated in rabbit zygotes, parthenotes, and nuclear transfer embryos by analysis of DNA replication, nucleolar fibrillarin label, and localization of nuclear material reactive to the MPM-2 antibody. Nuclear transfer embryos were derived from G1/early S-phase donor nuclei and MII oocytes. In nuclear transfer embryos, DNA rereplication was likely to have occurred because label was incorporated, possibly in the centromeric regions of the chromosomes, prior to premature chromosome condensation and again following pronuclear formation. In parthenotes, DNA replication began very late in the cell cycle, which may be due to deficiencies in the artificial activation stimulus. The presence of fibrillarin label in the nucleolus was used as an indication of nucleolar transcriptional activity. Fibrillarin label was absent in embryos of all types up to the 16-32-cell stage. Although fibrillarin reappeared in nuclear transfer and parthenote embryos at the appropriate stage, not all blastomeres showed label indicating impaired development in these embryos. Labelling of phosphorylated epitopes by MPM-2 antibody showed a change in pattern of labelling during early development. Early cleavage stage embryos did not exhibit labelling over the spindle poles as did blastomeres from 32-cell embryos and tissue culture cells. All cell types exhibited labelling during interphase as dots located primarily over the nucleus in blastomeres from 32-cell embryos and in tissue culture cells, together with cytoplasmic label in embryos at early cleavage stages. Nuclear transplant embryos had a normal pattern of MPM-2 label. In contrast, the appearance of MPM-2 label in parthenotes depended on the type of calcium stimulation. These results demonstrate defects in DNA synthesis, nucleolar activity, and specific phosphorylation events, likely resulting from an improper activation stimulus and chromosome condensation in the transplanted nucleus.
Molecular Reproduction and Development 04/1995; 40(3):292-304. · 2.53 Impact Factor
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ABSTRACT: Changes in intracellular calcium concentrations ([Ca2+]i) occur at regular intervals following fertilization in eggs of all mammalian species studied to date. To investigate the mechanisms of their generation, rabbit eggs were injected with the fluorescent Ca2+ indicator fura-2 dextran. [Ca2+]i oscillations were associated with fertilization (n = 10) and inositol 1,4,5-trisphosphate (InsP3;IICR) appears to participate in their generation because injection of heparin (100 mg/ml in the pipette), a competitive InsP3 receptor antagonist, blocked or considerably delayed the fertilization [Ca2+]i rises in all eggs (8/8). Injection of guanosine 5'-0-(2-thiodiphosphate) (GDP beta[S]), a G-protein antagonist, which possibly reduced the production of InsP3, also resulted in inhibition of [Ca2+]i oscillations (n = 7). Ca2+ injection-induced Ca2+ release in fertilized eggs was observed by injection of CaCl2, which evoked intracellular Ca2+ release in all oscillating eggs (n = 14), but only in a few late fertilization stage nonoscillating eggs (7/19 eggs) and in none of the unfertilized eggs (n = 11). Injection of InsP3 (5 microM) between fertilization [Ca2+]i rises also elicited Ca2+ responses that were similar in peak [Ca2+]i to the fertilization [Ca2+]i rises (n = 5). In unfertilized eggs, injection of guanosine 5'-0-(3-thiotriphosphate) (GTP[S]; 5-20 mM), a stimulator of G-proteins, induced [Ca2+]i oscillations. CaCl2 injections, delivered between GTP[S]-induced [Ca2+]i rises, resulted in increased Ca2+ responses in 5/7 eggs. The results of this study indicate that IICR participates in the generation of fertilization-associated [Ca2+]i rises and that Ca2+ injection-induced Ca2+ release appears to be stimulated by a product of the phosphoinositide pathway. Furthermore, the time to reach threshold levels of InsP3 may dictate the periodicity of fertilization [Ca2+]i rises in rabbit eggs.
Developmental Biology 01/1995; 166(2):634-42. · 4.07 Impact Factor
