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Biomedical subjects

C Simerly

Publications and source records attributed to C Simerly.

At least 19 recordsLinked to original sources
PubMed2002

Arrest of cell cycle progression during first interphase in murine zygotes microinjected with anti-PCM-1 antibodies.

To investigate the function of the centrosome protein PCM-1, antibodies against PCM-1 were microinjected into either germinal vesicle stage meiotic oocytes or fertilized mouse eggs, and cell cycle progression events (i.e., microtubule assembly, chromosome and centrosome organization, meiotic maturation) were assayed. These studies determined that microinjected PCM-1 antibodies arrested cell cycle progression, with anti-PCM-1 arresting fertilized eggs at the pronucleate stage when injected during G1. Analysis of the injected eggs determined that centrosome disruption and microtubule cytaster disorganization accompanied the cell cycle arrest. Anti-PCM-1 blocked neither pronuclear centration, completion of mitosis when microinjected into zygotes at G2, nor meiotic maturation when microinjected into immature oocytes. These results identify a novel role for PCM- 1 in cell cycle regulation, and indicate that PCM-1 must fulfill an essential function for cells to complete interphase.

Animals↗
PubMed2001

Transgenic monkeys produced by retroviral gene transfer into mature oocytes.

Transgenic rhesus monkeys carrying the green fluorescent protein (GFP) gene were produced by injecting pseudotyped replication-defective retroviral vector into the perivitelline space of 224 mature rhesus oocytes, later fertilized by intracytoplasmic sperm injection. Of the three males born from 20 embryo transfers, one was transgenic when accessible tissues were assayed for transgene DNA and messenger RNA. All tissues that were studied from a fraternal set of twins, miscarried at 73 days, carried the transgene, as confirmed by Southern analyses, and the GFP transgene reporter was detected by both direct and indirect fluorescence imaging.

Animals↗
PubMed2001

Fertilization imaged in 2-, 3- and four dimensions: molecular insights for treating infertility.

Fertilization in humans follows a complex series of events including binding of the sperm to the oocyte plasma membrane, oocyte activation, the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei and the restoration of the sperm centrosome. This duplicates and separates, forming two mitotic spindle poles upon which the parental genomes can intermix to complete fertilization. The use of intracytoplasmic sperm injection (ICSI) has been highly effective as a treatment for severe male infertility and thousands of ICSI babies have been born world-wide. Working with rhesus monkey gametes, we have developed a preclinical animal model for understanding the cell biological basis of ICSI. Typically, ICSI results in abnormal nuclear remodeling during sperm decondensation due to the presence of the sperm acrosome and perinuclear structures normally removed at the oolemma during in vitro fertilization. These unusual modifications raise concerns that the ICSI procedure itself might lead to the observed increase in chromosome anomalies reported for

Animals↗
PubMed2000

Cellular and molecular events after in vitro fertilization and intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has heralded an era of tremendous improvements in treating male infertility leading to the births of thousands of babies. However, recent concerns over possible long-term effects of ICSI on offspring has prompted the development of a preclinical, nonhuman primate model to assess the safety of ICSI. Fluorescent imaging of rhesus macaque IVF zygotes revealed that this species shares many similarities with humans in terms of cytoskeletal and chromatin dynamics during fertilization. However, rhesus monkey zygotes fertilized by ICSI resulted in abnormal nuclear remodeling leading to asynchronous chromatin decondensation in the apical region of the sperm head, delaying the onset of DNA synthesis. The persistence of the acrosome and perinuclear theca on the apex of sperm introduced into the oocyte by ICSI may constrict the DNA in this region. Despite these differences, normal rhesus monkey ICSI embryos have been produced and have lead to several births after transfer. The irregularities described in this paper raise concerns that the ICSI procedure may result in chromatin damage during DNA decondensation and further highlight the need for devising improved pre-clinical assessment prior to global acceptance of this, and other, novel methods of assisted reproduction.

Animals↗
PubMed2000

Microfilament stabilization by jasplakinolide arrests oocyte maturation, cortical granule exocytosis, sperm incorporation cone resorption, and cell-cycle progression, but not DNA replication, during fertilization in mice.

Jasplakinolide (JAS), which induces microfilament polymerization and stabilization, inhibits microfilament-mediated events in murine oocyte maturation and fertilization in a fashion unlike the effects of cytochalasin B (CCB) and latranculin A (LAT A). JAS prevents egg polar body emission at a much lower concentration than either CCB or LAT A. Microfilament bundles were detected on the entire egg cortex after JAS exposure. Conversely, microfilament patterns did not change after exposure to CCB, and few microfilaments were observed after exposure to LAT A. Eggs that were allowed to recover from JAS were unable to recover normal microfilament organization. During oocyte maturation, JAS prevented both spindle migration to the oocyte cortex and first polar body emission. During in vitro fertilization, sperm head entered the eggs and formed pronuclei, but sperm tail entry, pronuclear centration, and second polar body emission were not detected. DNA synthesis occurs in these JAS-treated zygotes. JAS inhibited not only the formation, but also the disassembly, of incorporation cones. JAS was also found to prevent cortical granule exocytosis following artificial activation, and cortical granules were still beneath the plasma membrane even after activation. Finally, incorporation of microinjected nonmuscle actin into the microfilament network of mice eggs was delayed by JAS. We conclude that JAS acts as a microfilament inhibitor during maturation and fertilization and is more powerful than other inhibitors. Its mechanism differs in that it promotes assembly and stabilization of microfilaments. JAS is a novel cell permeable tool for the investigation of microfilament-dependent events in early mammalian development.

Actin Cytoskeleton↗
PubMed2000

Cytoskeletal aspects of assisted fertilization.

During mammalian fertilization, the zygotic centrosome organizes a large sperm aster, critical for uniting the male and female pronuclei prior to first mitosis. Fluorescent imaging of inseminated human oocytes has shown that centrosomal defects may result in abnormal microtubule nucleation preventing genomic union, suggesting a novel cause of fertilization failure. Working with rhesus monkey gametes, we have developed a preclinical model for understanding the cell biological basis of intracytoplasmic sperm injection (ICSI). Typically, ICSI results in abnormal nuclear remodeling during sperm decondensation due to the presence of the sperm acrosome and perinuclear theca, structures normally removed at the oolemma during IVF; this is turn causes a delay in the onset of DNA synthesis. These unusual modifications raise concerns that the ICSI procedure itself may result in chromatin damage during DNA decondensation and further highlight the need for a more rigorous assessment of methods of assisted reproduction prior to their global application.

Acrosome↗
PubMed2000

ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.

We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle.

Acrosome↗
PubMed2000

Highly degenerated distal centrioles in rhesus and human spermatozoa.

In humans and other mammals except rodents, the spermatozoa contribute the proximal centriole during fertilization. The inheritance of the distal centriole is not yet fully clear. In the present work, the distal centrioles of rhesus and human spermatozoa have been studied by transmission electron microscopy. The round and elongating rhesus spermatids possess both proximal and distal centrioles. The distal centriole extends posteriorly as an axoneme while the proximal centriole produces a microtubular adjunct. Ejaculated rhesus and human spermatozoa have intact proximal centrioles, but the distal centrioles have degenerated. The central pair of microtubules of the axoneme extends continuously into the distal centriolar region up to the sperm head. Serial transverse and longitudinal sections of the sperm neck region reveal few scattered microtubule duplexes or triplets in the distal centriolar region. The loss of the centriolar microtubules is more extensive on the ventral side of the neck region, the side where the proximal centriole resides. The distal centriole degenerates caudally from the rostral area. Immunogold electron microscopy with anti-beta-tubulin antibody showed that the distal centriolar regions possess 50% fewer gold particles than the proximal centrioles, indicating a significant loss of centriolar microtubules in the distal centriolar region. The A-tubules of the remaining triplets are filled with a dense material, as observed in the axoneme. Thus, rhesus and human spermatozoa introduce only proximal centrioles intact, whereas the distal centrioles are mostly disorganized in the mature spermatozoa.

Animals↗
PubMed2000

Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidanceof parthenogenesis, and successful embryogenesis.

Manipulations of DNA and cellular structures are essential for the propagation of genetically identical animals by nuclear transfer. However, none of the steps have been optimized yet. This study reports a protocol that improves live dynamic imaging of the unfertilized bovine oocyte's meiotic spindle microtubules with microinjected polymerization-competent X-rhodamine-tubulin and/or with vital long-wavelength excited DNA fluorochrome Sybr14 so that the maternal chromosomes can be verifiably removed to make enucleated eggs the starting point for cloning. Suitability of the new fluorochromes was compared to the conventional UV excitable Hoechst 33342 fluorochrome. Enucleation removed the smallest amount of cytoplasm (4-7%) and was 100% efficient only when performed under continuous fluorescence, i.e., longer fluorescence exposure. This was in part due to the finding that the second metaphase spindle is frequently displaced (60.7 +/- 10%) from its previously assumed location subjacent to the first polar body. Removal of as much as 24 +/- 3% of the oocyte cytoplasm underneath the polar body, in the absence of fluorochromes, often resulted in enucleation failure (36 +/- 6%). When labeled oocytes were exposed to fluorescence and later activated, development to the blastocyst stage was lowest in the group labeled with Hoechst 33342 (3%), when compared to Sybr14 (19%), rhodamine-tubulin (23%), or unlabeled oocytes (37%). This suggests that longer wavelength fluorochromes can be employed for live visualization of metaphase spindle components, verification of their complete removal during enucleation, and avoidance of the confusion between artifactual parthenogenesis versus "cloning" success, without compromising the oocyte's developmental potential after activation.

Animals↗
PubMed2000

Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos.

The strictly maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) in mammals is a developmental paradox promoted by an unknown mechanism responsible for the destruction of the sperm mitochondria shortly after fertilization. We have recently reported that the sperm mitochondria are ubiquitinated inside the oocyte cytoplasm and later subjected to proteolysis during preimplantation development (P. Sutovsky et al., Nature 1999; 402:371-372). Here, we provide further evidence for this process by showing that the proteolytic destruction of bull sperm mitochondria inside cow egg cytoplasm depends upon the activity of the universal proteolytic marker, ubiquitin, and the lysosomal apparatus of the egg. Binding of ubiquitin to sperm mitochondria was visualized by monospecific antibodies throughout pronuclear development and during the first embryonic divisions. The recognition and disposal of the ubiquitinated sperm mitochondria was prevented by the microinjection of anti-ubiquitin antibodies and by the treatment of the fertilized zygotes with lysosomotropic agent ammonium chloride. The postfecundal ubiquitination of sperm mitochondria and their destruction was not seen in the hybrid embryos created using cow eggs and sperm of wild cattle, gaur, thus supporting the hypothesis that sperm mitochondrion destruction is species specific. The initial ligation of ubiquitin molecules to sperm mitochondrial membrane proteins, one of which could be prohibitin, occurs during spermatogenesis. Even though the ubiquitin cross-reactivity was transiently lost from the sperm mitochondria during epididymal passage, likely as a result of disulfide bond cross-linking, it was restored and amplified after fertilization. Ubiquitination therefore may represent a mechanism for the elimination of paternal mitochondria during fertilization. Our data have important implications for anthropology, treatment of mitochondrial disorders, and for the new methods of assisted procreation, such as cloning, oocyte cytoplasm donation, and intracytoplasmic sperm injection.

Animals↗
PubMed2000

Contractile apparatus of the normal and abortive cytokinetic cells during mouse male meiosis.

Mouse male meiotic cytokinesis was studied using immunofluorescent probes against various elements of cytokinetic apparatus and electron microscopy. In normal mice, some spermatocytes fail to undergo cytokinesis after meiotic I or II nuclear divisions, forming syncytial secondary spermatocytes and spermatids. Abnormal cytokinetic cells develop sparse and dispersed midzone spindles during the early stage. However, during late stages, single and compact midzone spindles are formed as in normal cells, but localize asymmetrically and attach to the cortex. Myosin and f-actin were observed in the midzone spindle and midbody regions of normally cleaving cells as well as in those cells that failed to develop a cytokinetic furrow, implying that cytokinetic failure is unlikely to be due to defect in myosin or actin assembly. Depolymerization of microtubules by nocodazole resulted in the loss of the midbody-associated f-actin and myosin. These observations suggest that actin-myosin localization in the midbody could be a microtubule-dependent process that may not play a direct role in cytokinetic furrowing. Anti-centrin antibody labels the putative centrioles while anti-(gamma)-tubulin antibody labels the minus-ends of the midzone spindles of late-stage normal and abnormal cytokinetic cells, suggesting that the centrosome and midzone spindle nucleation in abnormal cytokinetic cells is not different from those of normally cleaving cells. Possible use of mouse male meiotic cells as a model system to study cytokinesis has been discussed.

Actins↗
PubMed1999

The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle.

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.

Fungal Proteins↗
PubMed1999

Centriole and centrin degeneration during mouse spermiogenesis.

Centrosome reduction during mouse spermiogenesis has been studied by immunofluorescent microscopy using anticentrin antibody (20H5) and TEM. Centrin is detected as two spots in round spermatids, corresponding to a pair of centrioles. In elongating spermatids, centrin spots colocalize with the centrioles in the neck region, while the perinuclear ring from which manchette microtubules arise, does not label with the antibody 20H5. The proximal centriole of the elongating spermatids develops a prominent adjunct, which assembles an aster of microtubules. TEM studies after immunogold labeling revealed that centrin is associated with the distal and the proximal centrioles, but not with the adjunct. Centrin labeling in the neck region diminishes after spermiation stage, although it is not completely lost from all testicular sperm. Mature epididymal sperm do not display centrin labeling. Mouse sperm lose both distal and proximal centrioles at maturity. Loss of centrin staining appears to correlate with the degeneration of centrioles during mouse spermiogenesis.

Animals↗
PubMed1999

Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys.

Intracytoplasmic sperm injection has begun an era of considerable improvements in treating male infertility. Despite its success, questions remain about the dangers of transmitting traits responsible for male infertility, sex and autosomal chromosome aberrations and possible mental, physical and reproductive abnormalities. We report here the first births of rhesus monkeys produced by intracytoplasmic sperm injection at rates greater or equal to those reported by clinics. Essential assumptions about this process are flawed, as shown by results with the preclinical, nonhuman primate model and with clinically discarded specimens. Dynamic imaging demonstrated the variable position of the second meiotic spindle in relation to the first polar body; consequently, microinjection targeting is imprecise and potentially lethal. Intracytoplasmic sperm injection resulted in abnormal sperm decondensation, with the unusual retention of vesicle-associated membrane protein and the perinuclear theca, and the exclusion of the nuclear mitotic apparatus from the decondensing sperm nuclear apex. Male pronuclear remodeling in the injected oocytes was required before replication of either parental genome, indicating a unique G1-to-S transition checkpoint during zygotic interphase (the first cell cycle). These irregularities indicate that the intracytoplasmic sperm injection itself might lead to the observed increased chromosome anomalies.

Animals↗
PubMed1999

Optimization strategies for production of mammalian embryos by nuclear transfer.

In order to optimize each of the individual steps in the nuclear transfer procedure, we report alternative protocols useful for producing recipient cytoplasts and for improving the success rate of nuclear transfer embryos in cattle, rhesus monkey, and hamster. Vital labeling of maternal chromatin/spindle is accomplished by long wavelength fluorochromes Sybr14 and rhodamine labeled tubulin allowing constant monitoring and verification during enucleation. The use of Chinese hamster ovary (CHO) donor cells expressing the viral influenza hemagglutinin fusion protein (HA-300a+), to adhere and induce fusion between the donor cells and enucleated cow, rhesus and hamster oocytes was examined. Cell surface hemagglutinin was activated with trypsin prior to nuclear transfer and fusion was induced by a short incubation of a newly created nuclear transfer couplet at pH 5.2 at room temperature. Donor cell cytoplasm was dynamically labeled with CMFDA, or further transfected with the green fluorescence protein (GFP) gene, so that fusion could be directly monitored using live imaging. High rates of fusion were observed between CHO donor cells and hamster (100%), rhesus (100%), and cow recipient cytoplasts (81.6%). Live imaging during fusion revealed rapid intermixing of cytoplasmic components between a recipient and a donor cell. Prelabeled donor cytoplasmic components were uniformly distributed throughout the recipient cytoplast, within minutes of fusion, while the newly introduced nucleus remained at the periphery. The fusion process did not induce activation as evidenced by unchanged distribution and density of cortical granules in the recipient cytoplasts. After artificial activation, the nuclear transfer embryos created in this manner were capable of completing several embryonic cell divisions. These procedures hold promise for enhancing the efficiency of nuclear transfer in mammals of importance for biomedical research, agriculture, biotechnology, and preserving unique, rare, and endangered species.

Animals↗