Techniques for localization of specific molecules in oocytes and embryos.
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Animals↗
Biomedical subjects
C Simerly
Publications and source records attributed to C Simerly.
Gamma-tubulin reorganization during mouse fertilization and early development.
gamma-Tubulin, a component of spindle pole bodies in fungal cells and pericentriolar material in vertebrate cells, is thought to play a role in the nucleation of microtubule growth and to define their polarity. In contrast to the adult somatic cells, microtubules are nucleated in the absence of centrioles in mammalian oocytes and early embryos. By studying acentriolar mouse oocytes and their early development following fertilization, we show that gamma-tubulin antibody crossreacts with a 50,000 M(r) protein in unfertilized mouse oocytes and demonstrate that gamma-tubulin distribution is rearranged dramatically during fertilization. In unfertilized mouse oocytes, gamma-tubulin is concentrated in the broad spindle poles of meiotic spindle (MII) and as the distinct foci which form the centers of the cytoplasmic microtubule asters (cytasters). The integrity of these gamma-tubulin foci and their cytoplasmic location is maintained during the drug- or cold-induced depolymerization of microtubules. gamma-Tubulin is also found in the basal body of the mouse sperm. During fertilization, the gamma-tubulin is found at the cytastral centers as well as in the incorporated sperm basal body complex, and the gamma-tubulin foci coalesce at the perinuclear microtubule organizing regions of the two pronuclei at the first mitotic prophase. During mitosis, gamma-tubulin is found associated with broad bands that form the poles of the first mitotic spindle. By the late preimplantation stage, when newly generated centrioles have been reported to arise, gamma-tubulin remains localized at the centrosome of mitotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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Microgravity effects on sea urchin fertilization and development.
Gravity has been a pervasive influence on all living systems and there is convincing evidence to suggest that it alters fertilization and embryogenesis in several developmental systems. Notwithstanding the global importance of gravity on development, it has only been recently possible to begin to design experiments which might directly investigate the specific effects of this vector. The goal of this research program is to explore and understand the effects of gravity on fertilization and early development using sea urchins as a model system. Sea urchin development has several advantages for this project including the feasibility of maintaining and manipulating these cells during spaceflight, the high percentage of normal fertilization and early development, and the abundant knowledge about molecular, biochemical, and cellular events during embryogenesis which permits detailed insights into the mechanism by which gravity might interfere with development. Furthermore, skeletal calcium is deposited into the embryonic spicules within a day of fertilization permitting studies of the effects of gravity on bone calcium deposition.
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Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte.
During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.
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Maternal inheritance of centrosomes in mammals? Studies on parthenogenesis and polyspermy in mice.
The centrosome, the microtubule-organizing center of the cell, is introduced typically by the sperm at fertilization. In some mammals, however, this paternal pattern of inheritance appears to be violated. The hypothesis that the centrosome is maternally inherited was tested during parthenogenesis, polyspermy, and polygyny as well as after recovery from microtubule inhibition at first mitosis. During parthenogenesis the paternal contribution was absent, and in polyspermy the paternal contribution was multiplied. Haploid and diploid parthenogenotes as well as polyspermic and digynic fertilized eggs each segregated their centrosomes to organize a bipolar mitotic apparatus. Oocytes recovering from a nocodazole block formed two normal bipolar mitotic apparatus; the paternal chromosomes aligned at one spindle equator, while the maternal chromosomes were found at the other. These results show that the centrosome is maternally inherited from cytoplasmic sites in the mouse. The evolutionary switch from paternal to maternal inheritance in mammals might be related to the additional dangers that parthenogenesis represents: a threat to the life of the mother as well as to the life of the fetus.
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Three-dimensional imaging of fertilization and early development.
The field of biological microscopy has recently enjoyed major technical advances, exemplified by the development of field-emission low-voltage scanning electron microscopes and laser scanning confocal light microscopes. In addition, computer processing of microscopical data is revolutionizing the way morphological information is imaged. In this paper, we illustrate methods by which this new technology can be used to examine events in fertilization and early development in three dimensions. Different types of specimen preparation protocols, using both echinoderm and mammalian gametes and embryos, are evaluated for their ability to preserve accurately the three-dimensional organization of these specimens for imaging by both low-voltage scanning electron microscopy and laser scanning confocal light microscopy.
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U3 snRNPs and nucleolar development during oocyte maturation, fertilization and early embryogenesis in the mouse: U3 snRNA and snRNPs are not regulated coordinate with other snRNAs and snRNPs.
U3 small nuclear ribonucleic acids (snRNA) and U3 small nuclear ribonucleoprotein (snRNP), which are thought to be responsible for ribosomal RNA processing, are quantitated and localized during oocyte maturation, fertilization, and early embryogenesis in the mouse. On the basis of Northern blot and nuclease protection experiments, it is estimated that there are about 5 x 10(4) U3 snRNA molecules in an ovulated oocyte and in a two-cell embryo. This number then increases roughly 50-fold to 2.7 x 10(6) molecules per embryo by the blastocyst stage. At all stages of development U3 snRNP antigens colocalize with nucleoli, as defined by differential interference contrast microscopy and an antibody to a nucleolar epitope. The synthesis and distribution of U3 snRNA and U3 snRNP follow a pattern independent from other major U snRNPs and snRNAs.
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Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.
After ovulation mammalian oocytes arrest in second meiotic metaphase. We asked whether the microtubules that comprise the meiotic spindle of mouse oocytes were stable or were undergoing rapid cycles of assembly and disassembly. Porcine brain tubulin, derivatized with biotin or x-rhodamine [5- (and -6)-carboxy-x-rhodamine], was microinjected into living oocytes. Biotinylated tubulin incorporated into the meiotic spindle to apparent equilibrium within 15 min. To assess quantitatively the rates of disassembly and assembly of the microtubules, small domains within the spindles of oocytes injected with x-rhodamine-tubulin were photobleached and their recovery was analyzed by digital imaging microscopy. Fluorescence recovery in the spindles was rapid and extensive, plateauing to an average of 83% at 4 min. The calculated half-time for turnover of the spindle microtubules was 77 sec. In contrast, fluorescence recovery of the spindle midbodies in telophase oocytes was much more limited, averaging approximately 22% at 4 min. These data indicate that most microtubules within the arrested metaphase spindle of the mouse oocyte undergo rapid cycles of assembly and disassembly. Microtubules of the telophase midbody are more stable.
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Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes.
Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.
Anaphase↗
Microtubule assembly is required for the formation of the pronuclei, nuclear lamin acquisition, and DNA synthesis during mouse, but not sea urchin, fertilization.
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.
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The small nuclear RNAs for pre-mRNA splicing are coordinately regulated during oocyte maturation and early embryogenesis in the mouse.
The abundance and localization of snRNAs and snRNPs involved in processing and splicing of pre-mRNA has been studied during early mouse embryogenesis. The amount of U1, U2, U4, U5 and U6 RNA remains relatively constant between the postovulatory oocyte and 2-cell stage but increases three- to ten-fold in quantity between the 2-cell and blastocyst stages. Localization was examined by in situ hybridization with U1, U2 and U6 riboprobes and immunofluorescence microscopy using a monoclonal antibody to snRNP antigens. The snRNAs and snRNPs are primarily localized to the germinal vesicle in the preovulatory oocyte but are released and diluted within the cytoplasm of the oocyte during germinal vesicle breakdown and meiotic maturation. They subsequently relocalize to both pronuclei following fertilization and the nuclei of the 2-cell embryo following the first cleavage division. Since the amount of snRNA is constant during the first cleavage, the small amount of pre-mRNA that is synthesized at the time of transcriptional activation in the 2-cell embryo may be spliced and processed by snRNPs of maternal origin.
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Confocal fluorescence microscopy with the tandem scanning light microscope.
Applications of the tandem scanning confocal microscope (TSM) to fluorescence microscopy and its ability to resolve fluorescent biological structures are described. The TSM, in conjunction with a cooled charge-coupled device (cooled CCD) and conventional epifluorescence light source and filter sets, provided high-resolution, confocal data, so that different fluorescent cellular components were distinguished in three dimensions within the same cell. One of the unique features of the TSM is the ability to image fluorochromes excited by ultraviolet light (e.g. Hoechst, DAPI) in addition to fluorescein and rhodamine. Since the illumination is dim, photobleaching is insignificant and prolonged viewing of living specimens is possible. Series of optical sections taken in the Z-axis with the TSM were reproduced as stereo images and three-dimensional reconstructions. These data show that the TSM is potentially a powerful tool in fluorescence microscopy for determining three-dimensional relationships of complex structures within cells labeled with multiple fluorochromes.
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Kinetochore appearance during meiosis, fertilization and mitosis in mouse oocytes and zygotes.
The events of mammalian fertilization overlap with the completion of meiosis and first mitosis; the pro-nuclei never fuse, instead the parental genomes first intermix at the mitotic spindle equator at metaphase. Since kinetochores are essential for the attachment of chromosomes to spindle microtubules, this study explores their appearance and behavior in mouse oocytes, zygotes and embryos undergoing the completion of meiosis, fertilization and mitoses. Kinetochores are traced with immunofluorescence microscopy using autoimmune sera from patients with CREST (CREST = calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) scleroderma. These sera cross-react with the 17 kDa centromere protein (CENP-A) and the 80 kDa centromere protein (CENP-B) found at the kinetochores in human cell cultures. The unfertilized oocyte is ovulated arrested at second meiotic metaphase and kinetochores are detectable as paired structures aligned at the spindle equator. At meiotic anaphase, the kinetochores separate and remain aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis is lost. The female pronucleus and the second polar body nucleus each receive a detectable complement of kinetochores. Mature sperm have neither detectable centrosomes nor detectable kinetochores, and shortly after sperm incorporation kinetochores become detectable in the decondensing male pronucleus. In pronuclei, the kinetochores are initially distributed randomly and later found in apposition with nucleoli. At mitosis, the kinetochores behave in a pattern similar to that observed at meiosis or mitosis in somatic cells: irregular distribution at prophase, alignment at metaphase, separation at anaphase and redistribution at telophase. They are also detectable in later stage embryos. Colcemid treatment disrupts the meiotic spindle and results in the dispersion of the meiotic chromosomes along the oocyte cortex; the chromosomes remain condensed with detectable kinetochores. Fertilization of Colcemid-treated oocytes results in the incorporation of a sperm which is unable to decondense into a male pronucleus. Remarkably kinetochores become detectable at 5 h post-insemination, suggesting that the emergence of the paternal kinetochores is not strictly dependent on male pronuclear decondensation.
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Localization and expression of U1 RNA in early mouse embryo development.
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%.
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Acetylated alpha-tubulin in microtubules during mouse fertilization and early development.
alpha-Tubulin in the microtubules of mouse oocytes and embryos is acetylated in a specific spatial and temporal sequence. In the unfertilized oocyte, a monoclonal antibody to the acetylated form of alpha-tubulin is bound predominantly at the poles of the arrested metaphase meiotic spindle. The labeling intensity of the spindle microtubules is weaker as observed by immunofluorescence using oocytes double-labeled for total tubulin and acetylated alpha-tubulin, and as measured by immuno high-voltage electron microscopy (immunoHVEM) with colloidal gold; cytasters are not acetylated. At meiotic anaphase, the spindle becomes labeled, and by telophase and during second polar body formation only the meiotic midbody is acetylated. The sperm axoneme retains its acetylation after incorporation though the interphase microtubules are not detected. First mitosis follows a pattern similar to that observed at the second meiosis and during interphase only the mitotic midbodies are acetylated. After treatment with cold, colcemid, or griseofulvin, the remaining stable microtubules are acetylated, but immunoHVEM observations suggest that these fibers might not have been acetylated prior to microtubule disruption. Taxol stabilization does not alter acetylation patterns. Acetylated microtubules are not necessarily old microtubules since acetylated fibers are observed at 30 sec after cold recovery. These results show the presence of acetylated microtubules during meiosis and mitosis and demonstrate a cell-cycle-specific pattern of acetylation, with acetylated microtubules found at the centrosomes at metaphase, an increase in spindle labeling at anaphase, and the selective deacetylation of all but midbody microtubules at telophase.
Acetylation↗
Localization of fodrin during fertilization and early development of sea urchins and mice.
Fodrin, a spectrin-like protein, is localized in gametes, zygotes, and embryos from sea urchins and mice. Mammalian fodrin comprises two polypeptides with molecular weights of approximately 240 kDa (alpha) and 235 kDa (beta). An antibody specific for mammalian alpha-fodrin cross-reacted with a 240-kDa polypeptide from sea urchin egg extracts. This indicates that sea urchins contain a protein of similar electrophoretic mobility and immunological properties to mammalian alpha-fodrin. When this antibody was used to stain the sea urchin gametes with indirect immunofluorescence, fodrin-specific fluorescence was localized to the acrosome of the sperm and was distributed over the entire egg near the surface in a punctate pattern similar to the distribution of polymeric actin. During sperm incorporation, the fodrin-specific fluorescence is found at the site of sperm incorporation, in the fertilization cone. After fertilization, the intensity of fodrin fluorescence increases. During mitosis and cytokinesis in sea urchins, the entire surface of the egg remains stained; the cleavage furrow also was stained but no more intensely than was the rest of the egg surface. Antibody labeling with colloidal gold followed by electron microscopy showed that fodrin was loated in the cytoplasm immediately beneath the plasma membrane. In unfertilized mouse oocytes, both actin and fodrin were stained most intensely beneath the membrane adjacent to the meiotic spindle. After insemination, the cell surfaces of the pronucleate egg and the second polar body were stained; however, the actin matrix surrounding the apposed pronuclei did not bind the fodrin antibody. During cytokinesis in the mouse, the cleavage furrow stained more intensely than did the rest of the egg cortex, and in embryos the cell borders were delineated. These results indicate that organisms as unrelated to mammals as sea urchins have fodrin-like proteins; the rearrangements of such proteins suggest that they participate in the actin-mediated events at the cell surface during fertilization and early development in both mice and sea urchins.
Actins↗
Latrunculin inhibits the microfilament-mediated processes during fertilization, cleavage and early development in sea urchins and mice.
Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.
Acrosome↗
Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs.
The forms and locations of centrosomes in mouse oocytes and in sea urchin eggs were followed through the whole course of fertilization and first cleavage by immunofluorescence microscopy. Centrosomes were identified with an autoimmune antiserum to centrosomal material. Staining of the same preparations with tubulin antibody and with the DNA dye Hoechst 33258 allowed the correlation of the forms of the centrosomes with the microtubule structures that they generate and with the stages of meiosis, syngamy, and mitosis. The results with sea urchin eggs conform to Boveri's view on the paternal origin of the functional centrosomes. Centrosomes are seen in spermatozoa and enter the egg at fertilization. Initially, the centrosomes are compact, but as the eggs enter the mitotic cycle the forms of the centrosomes go through a cycle in which they spread during interphase, apparently divide, and condense into two compact poles by metaphase. In anaphase, they spread to form flat poles. In telophase and during reconstitution of the daughter nuclei, the centrosomal material is disposed as hemispherical caps around the poleward surfaces of the nuclei. Mouse sperm lack centrosomal antigen. In the unfertilized mouse oocyte, the meiotic spindle poles are displayed as broad-beaded centrosomes. In addition, centrosomal material is detected in the cytoplasm as particles, about 16 in number, which are foci of small aster-like arrays of microtubules. The length and number of astral microtubules correlate with the size of the centrosomal foci. After sperm incorporation, as the pronuclei develop and more cytoplasmic microtubules assemble, a few of the foci associate with the peripheries of the nuclei. The number of foci multiplies during the first cell cycle. At the end of interphase, all of the centrosomal foci have concentrated on the nuclear peripheries and the cytoplasmic microtubules have disappeared. At prophase, the centrosomes are seen as two irregular clusters, marking the poles which, at metaphase and anaphase, appear as rough bands with foci, and the spindle is typically barrel-shaped. At telophase, the centrosomes are seen as arcs that lie on the nuclear peripheries after cleavage. The ordering of microtubules in all the stages reflects the shapes of the centrosomes. The findings on the sea urchin confirm the classical theory of the paternal origin of centrosomes and contrast with observations tracing the mitotic poles of the mouse egg to maternal centrosomal material. This evidence strengthens the conclusion that mouse centrosomes derive from the oocyte.
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