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ferT encodes a meiosis-specific nuclear tyrosine kinase.

ferT is a mouse testis-specific mRNA, shown previously to potentially encode a 51 kilodalton tyrosine kinase termed p51ferT. The accumulation of ferT RNA is restricted to primary spermatocytes that are at the prophase stage of the first spermatogenic meiotic division. By using antibodies raised against a synthetic peptide which was designed according to a putative p51ferT unique amino acid sequence, we have shown that testicular cells indeed contain a 51 kilodalton protein that is recognized by the anti-p51ferT antibodies. The protein was not detected in six nontesticular mouse tissues, nor was it detected, like the ferT RNA, in the testes of 14-day-old mice. These findings strongly suggest that the 51 kilodalton protein is p51ferT. Immunohistochemical staining localized p51ferT to meiotically dividing spermatocytes. Transfection experiments in CHO cells confirmed the nuclear localization of p51ferT in eukaryotic cells. p51ferT seems thus to be the first meiosis-specific nuclear tyrosine kinase described to date.

Animals↗

Temporal patterns of A-myb and B-myb gene expression during testis development.

We recently reported the cloning and sequencing of the mouse A-myb proto-oncogene cDNA and the abundant expression of this mRNA primarily in the testis of adult mice. The A-myb mRNA is detectable by in situ hybridization specifically in the spermatogenic cells, and is downregulated during terminal differentiation. A low level of expression is observed in a few other tissues, including ovary, spleen and brain. We have extended those studies by examining A-myb and B-myb expression during testis development in the mouse. The A-myb and B-myb genes are both expressed in a cell- and stage-specific manner during testis development. The B-myb mRNA is expressed most highly in gonocytes of the fetal testis and in spermatogonia and early spermatocytes in the adult. B-myb expression decreases at day 18 post partum, coincident with the initial appearance of late pachytene spermatocytes. B-myb expression was also detectable in some interstitial cells of the fetal and adult testis. The A-myb mRNA was not detectable by in situ hybridization in fetal day 15.5 gonocytes but was detectable at a low abundance by RT-PCR in fetal and newborn mice. A-myb mRNA expression increased at post-natal day 10, when primary spermatocytes first appear. In the adult, the A-myb mRNA was expressed highly in a sub-population of spermatogonia and in primary spermatocytes, but was not detectable in spermatids. This expression of A-myb is consistent with the meiotic arrest that is observed in A-myb-deficient male mice. We conclude that B-myb may play a critical role in controlling the proliferation or differentiation of gonocytes and spermatogonia and possibly the somatic lineages as well, whereas A-myb is required for progression through the first meiotic prophase. These distinct roles for B-myb and A-myb during spermatogenesis may reflect distinct transactivation potentials of the two proteins. Further studies to determine the functions of A-myb and B-myb in the developing testis should improve our understanding of the molecular events associated with spermatogenesis and differentiation of the Sertoli and other somatic cell types of the testis.

Animals↗

Centriole behaviour during meiosis of male germ cells of Dermatobia hominis (Diptera:Cuterebridae).

During the meiotic division of Dermatobia hominis spermatogenesis, the centrioles duplicate only in prophase I, giving rise to short cilia which are exposed on the cellular surface. In metaphase I they are internalized and distributed to the daughter cells. Consequently, the secondary spermatocytes have two centrioles which repeat the cycle of cilia externalization followed by internalization. The spermatids receive only one centriole, which changes into a basal body and originates a flagellum. This centriole behaviour seems to be a general feature in insect male germ cell meiosis.

Animals↗

The synaptonemal complexes of the nematode Caenorhabditis elegans: gametic response to retinol.

Retinol (RO) is essential for normal gene expression and progression of gametogenesis. Excessive amounts of RO in the diet results in the condition termed 'hypervitaminosis A' which severely affects gametogenesis. In the present work, the nematode Caenorhabditis elegans was grown in different concentrations of RO for up to seven generations. Decreased fecundity was positively correlated with increasing concentrations of RO with successive generations. Decreased nuclear volume was positively correlated with increasing RO concentration for each generation. Abnormal chromatin condensation was present along the bivalent and the lengths of the synaptonemal complexes (SC) were consistently longer than in the wild-type. Increasing RO concentration was also negatively correlated with the presence of SCs. The presence of disjunction regulator regions (DRR), which are decondensed regions of chromatin along the bivalent which occur in specific numbers, was negatively correlated with increasing RO concentration. By the third generation, DRRs were no longer observed in any of the organisms. This study shows that high levels of retinol in the diet affect gene expression, via changes in chromosome structure, and interferes with gamete production by inhibiting the pairing of homologous chromosomes during meiotic prophase.

Animals↗

Retinoblastoma and subband deletion of chromosome 13.

Two patients with retinoblastoma and an interstitial deletion of the long arm of chromosome 13 were studied using G-banded metaphase and prophase chromosomes. One patient showed several congenital defects, developmental retardation, and deletion of bands q14 and q21. The second patient showed mild developmental delay, a few minor congenital defects, and a loss of approximately half of band q14. On the basis of this study and nine others from the literature, it is now possible to tentatively assign a predisposition to retinoblastoma to deletion of a specific small region of chromosome 13.

Abnormalities, Multiple↗

Mitotic neuroblasts determine neuritic patterning of progeny.

Neuronal precursor proliferation and axodendritic outgrowth have been regarded as strictly sequential, with process formation presumably beginning after mitotic activity ceases. We now report that sympathetic precursors in vitro often elaborate long neurites before dividing. Of 437 neuroblasts observed in 48 time-lapse recordings, 42 neuroblasts divided. Thirty (71%) of these mitotic neuroblasts had neurites prior to cytokinesis. "Paramitotic" neurites were found to contain microtubules (MTs), indicating that precursors elaborate neuritic cytoskeleton during proliferation. Remarkably, the precise neuritic pattern exhibited by parental neuroblasts was consistently reproduced by daughter cell pairs. Preservation of neuritic morphology occurred through asymmetric division, with individual neurites allocated to specific daughter cells. Paramitotic neurites either remained intact throughout mitosis (12 of 65), or "retracted" into the soma during prophase and then "regrew" within minutes after cytokinesis (53 of 65). "Retraction" and "regrowth" involved resorption of cytoplasm into the soma, then refilling of residual cell membrane, resulting in recapitulation of the parental neurite pattern. Paramitotic neuritogenesis appears to be intrinsically driven, but is responsive to environmental signals. The culture substrate influenced neurite length, but not the response of paramitotic neurites during mitosis or the preservation of neuritic morphology. However, the incidence of neurite-bearing neuroblasts increased from 38 +/- 1.3% to 94 +/- 1.1% with growth factor treatment. The surprisingly high incidence of paramitotic neurites and the fidelity with which patterning was conserved across cell generations raise the possibility that mitotic precursors engage in pathfinding. Our studies suggest a novel link between neurogenesis and cytoarchitectonic patterning.

Animals↗

The localization of p34cdc2 in the cells of normal, hyperplastic, and malignant epithelial and lymphoid tissues of the oral cavity.

The expression of p34cdc2 has been examined in normal, hyperplastic, and malignant oral epithelia and lymphoid tissues. Four monoclonal antibodies were prepared against Xenopus p34cdc2, three of which react specifically with human p34cdc2 and not with p33cdk2. These produced similar patterns of staining in both fixed and frozen sections of human material. Staining occurred mainly in the proliferative compartments of normal and hyperplastic tissues. In normal oral epithelia, parabasal and basal cells were the most strongly stained, with lighter cytoplasmic staining in lower prickle cells. In tonsillar germinal centres, a high proportion of cells was stained, with fewer positive cells in interfollicular zones, a distribution in keeping with the known pattern of cell proliferation. In normal cells, the intracellular location of p34cdc2 was cytoplasmic until early prophase, but in oral squamous cell carcinomas and lymphomas, it was located in both cytoplasm and nucleus during interphase and a larger fraction of cells was positive than in the equivalent normal tissues. Higher-grade neoplasms showed both a higher intensity of staining and a higher proportion of p34cdc2-positive cells.

Antibodies, Monoclonal↗

Partitioning of cytoplasmic organelles during mitosis with special reference to the Golgi complex.

During mitosis, not only the genetic material stored in the nucleus but also the constituents of the cytoplasm should be equally partitioned between the daughter cells. For this sake, the dividing cell goes through an extensive structural reorganization and transport along the endocytic and exocytic pathways is temporarily arrested. Early in prophase, the radiating array of cytoplasmic microtubules disassembles and the membrane systems of the secretory apparatus start to split up. In metaphase, the nuclear envelope fragments and the condensing chromosomes associate with the forming mitotic spindle. The cisternal and tubular elements of the endoplasmic reticulum and the Golgi complex break down into small vesicles, presumably as the result of an imbalance between vesicle budding and fusion. In anaphase, the two sets of chromosomes are pulled apart and a cleavage furrow forms halfway between the spindle poles. Since most organelles occur in multiple and widely dispersed copies at this stage, they will be evenly distributed between the daughter cells. During telophase and cytokinesis, the preceding fragmentation process is reversed. A nuclear envelope reappears around the chromosomes and cytoplasmic microtubules reassemble. The endoplasmic reticulum is rebuilt as a continuous system of flattened cisternae and tubules. Stacks of Golgi cisternae arise from small vesicles and are rearranged in an interconnected network. In parallel, the biosynthetic functions of the cell are normalized and intracellular membrane traffic is resumed.

Cell Compartmentation↗

Cell cycle analysis and synchronization of the Xenopus laevis XL2 cell line: study of the kinesin related protein XlEg5.

Cell free extracts prepared from Xenopus eggs are one of the most powerful in vitro systems to analyze cell cycle-regulated mechanisms such as DNA replication, nuclear assembly, chromosome condensation, or spindle formation. Xenopus embryos can complete several synchronous cell cycles in the absence of transcription, consequently Xenopus extracts are very helpful to study the molecular level of cellular mechanisms. Many key cell cycle regulators like p34cdc2 and cdk2 have been discovered and characterized using those extracts, but their regulation during somatic cell cycles have only been studied in mammalian cultured cells. In this paper, we describe optimized conditions to obtain cell cycle arrested Xenopus XL2 cultured cells. Synchronization of XL2 cells at different stages of the cell cycle was achieved by serum starvation and drug treatments such as aphidicolin, nocodazole, and ALLN. The degree of synchronization was assessed by indirect fluorescence microscopy and FACS analysis. This method was used to study the cell cycle expression of the Xenopus kinesin-related protein, XlEg5, a microtubule-based motor protein involved in movement and cell division in early development. We found that the expression of the protein was maximum in mitosis and minimum in G1, which correlated with the expression of its messenger RNA. XL2 cultured cells were also used to analyze the ultrastructural sub-cellular localization of XlEg5. During mitosis, the protein was found around the centrosome in prophase, on the spindle microtubules in metaphase, and, interestingly, around the minus end of the midbody microtubules in telophase.

Animals↗

Assembly of cytoskeletal proteins into cleavage furrows of tissue culture cells.

We review results obtained after fluorescent actin and myosin II probes were microinjected into interphase and prophase PtK2 and LLC-PK tissue culture cells to follow the changing distribution of these cytoskeletal proteins in the live cells during division. The fluorescent probes first begin to assemble into the future furrow region during mid-anaphase before any sign of initial contractions. The total concentrations of F-actin and myosin in the cleavage furrow begin to decrease a few minutes after the onset of furrow contraction. The cell's shape and the position of its mitotic spindle affect the deposition of cytoskeletal proteins in the forming cleavage furrow. In cells with two spindles, contractile proteins were recruited not only to the cortex bordering the former metaphase plates but also to the cortex midway between each pair of adjacent non-daughter poles or centrosomes. The furrowing between adjacent poles seen in these cultured cells are similar to the furrows observed by Rappaport [(1961) J Exp Zool 148:81-89] when echinoderm eggs were manipulated into a torus shape so that the poles of two mitotic spindles were adjacent to one another. These observations on injected tissue culture cells suggest that vertebrate cells share common mechanisms for the establishment of the cleavage furrow with echinoderm cells.

Animals↗

Chromosome elimination and sex determination in springtails (Insecta, Collembola).

A post-zygotic mechanism of sex determination is described in the two symphypleonans Dicyrtomina ornata (Nicolet) and Ptenothrix italica Dallai. The process consists of the loss of two sex chromosomes from the male embryo. At the end of the first meiotic division of spermatogenesis, a second chromosome elimination occurs, allowing half the secondary spermatocytes, later transformed into spermatids, to receive a complete haploid set of chromosomes. The secondary spermatocytes, which receive an incomplete set of chromosomes, degenerate. Males of the two collembolan species, therefore, produce a reduced number (50%) of spermatozoa. Females of D. ornata have 2n = 12 and males 2n = 10 chromosomes; females of P. italica have 2n = 14 and males 2n = 12 chromosomes. In both species, oogenesis proceeds normally and chromosomes pair and form chiasmata in meiotic prophase. The adaptive significance of this post-zygotic mechanism of sex determination is discussed. The mechanism seems to be a characteristic feature of the suborder Symphypleona. The neanurid Arthropleona Anurida maritima (Guérin), which was studied for comparative analysis, has 2n = 8 chromosomes and normal spermatogenesis producing haploid nuclei with four chromosomes. J. Exp. Zool. (Mol. Dev. Evol.) 285:215-225, 1999.

Animals↗

SF-assemblin in Chlamydomonas: sequence conservation and localization during the cell cycle.

Previously, SF-assemblin has been identified as the filament-forming component of the striated microtubule-associated fibers (SMAFs), which emerge from the basal bodies in several green flagellates. We have sequenced cDNAs coding for SF-assemblin from Chlalmydomonas reinhardtii and C. eugametos. Comparison of the deduced amino acid sequences with the previously described green algal SF-assemblins shows identities between 54 and 71%, indicating a strong drift in sequence. Cells of C. reinhardtii were analyzed by double immunofluorescence using polyclonal anti-SF-assemblin and anti-alpha-tubulin. In interphase cells, SF-assemblin is associated with all four microtubular flagellar roots. During mitosis the SF-assemblin-based cytoskeleton is reorganized; it divides in prophase and is reduced to two dot-like structures at each spindle pole in metaphase. During anaphase, the two dots present at each pole are connected again. In telophase we observed an asymmetrical outgrowth of new fibers. These observations suggest a role for SF-assemblin in reestablishing the microtubular root system characteristic of interphase cells after mitosis.

Amino Acid Sequence↗

Minus end-directed kinesin-like motor protein, Kcbp, localizes to anaphase spindle poles in Haemanthus endosperm.

Microtubule-based motor proteins assemble and reorganize acentrosomal mitotic and meiotic spindles in animal cells. The functions of motor proteins in acentrosomal plant spindles are unknown. The cellulosic cell wall and relative small size of most plant cells precludes accurate detection of the spatial distribution of motors in mitosis. Large cell size and absence of a cellulosic cell wall in Haemanthus endosperm make these cells ideally suited for studies of the spatial distribution of motor proteins during cell division. Immunolocalization of a kinesin-like calmodulin-binding protein (KCBP) in Haemanthus endosperm revealed its mitotic distribution. KCBP appears first in association with the prophase spindle. Highly concentrated within the cores of individual kinetochore fibers, KCBP decorates microtubules of kinetochore-fibers through metaphase. By mid-anaphase (when a barrel-shaped spindle becomes convergent), the protein redistributes and accumulates at the spindle polar regions. In telophase, KCBP relocates toward the phragmoplast and cell plate. These data suggest a role for KCBP in anaphase spindle microtubule convergence, which assures coherence of kinetochore-fibers within each sister chromosome group. Increasing coherence of kinetochore-fibers prevents splitting within each sister chromosome group and formation of multinucleated cells.

Anaphase↗

Dynamic remodeling of nuclear architecture during the cell cycle.

The nuclear matrix is an integral part of nuclear structure which undergoes a profound reorganization during the cell cycle reflecting major changes in functional requirements. This includes the processes of DNA replication and gene expression at interphase and partitioning of the nuclear contents during mitosis. Using a monoclonal antibody (mAb2A) which specifically stains a novel nuclear meshwork which reorganizes during the cell cycle in Drosophila, we have initiated a study to: 1) more closely analyze this structural reorganization; 2) clone and characterize the antigens recognized by this antibody; and 3) isolate other interacting proteins in order to gain insight into the regulation of this process. The mAb2A-labeled structure changes from what appears as a diffuse meshwork at interphase to a distinct spindle-like scaffold at prophase. Since at metaphase the microtubules of the mitotic apparatus co-localize with the mAb2A spindle structure, a model is considered whereby the nuclear mAb2A-labeled scaffolding reorganizes during the cell cycle to provide a guide for the establishment of the mitotic apparatus. The mAb2A has identified two separate antigens, each of which shows similar distribution patterns. One of these antigens has been partially cloned and contains an unusual tandem ser-thr kinase domain. The association of this kinase homologue with a nuclear scaffold which reorganizes during the cell cycle suggests that it may be involved in regulating changes in nuclear architecture during the cell cycle and/or in mediating the downstream consequences of such changes.

Animals↗

Ovarian germinal epithelium and folliculogenesis in the common snook, Centropomus undecimalis (Teleostei: centropomidae).

The ovarian germinal epithelium in the common snook, Centropomus undecimalis, is described. It consists of epithelial and prefollicle cells that surround germ cells, either oogonia or oocytes, respectively. The germinal epithelium borders a body cavity, the ovarian lumen, and is supported by a basement membrane that also separates the epithelial compartment of the ovarian lamellae from the stromal compartment. During folliculogenesis, the epithelial cells, whose cytoplasmic processes encompass meiotic oocytes, transform into prefollicle cells, which become follicle cells at the completion of folliculogenesis. The follicle is a derivative of the germinal epithelium and is composed of the oocyte and surrounding follicle cells. It is separated from the encompassing theca by a basement membrane. The cells that form the theca interna are derived from prethecal cells within the extravascular space of the ovarian stroma. The theca externa differentiates from undifferentiated cells within the stromal compartment of the ovary, from within the extravascular space. The theca interna and the theca externa are not considered to be part of the follicle and are derived from a different ovarian compartment than the follicle. Meiosis commences while oocytes are still within the germinal epithelium and proceeds as far as arrested diplotene of the first meiotic prophase. The primary growth phase of oocyte development also begins while oocytes are still within the germinal epithelium or attached to it in a cell nest. The definitions used herein are consistent between sexes and with the mammalian literature.

Animals↗

Delta-opioid receptor immunoreactivity on astrocytes is upregulated during mitosis.

Endogenous opioid peptides and opioid receptors are expressed by brain cells early during normal development, and exogenous opiate exposure in this period is known to affect brain cell proliferation and maturation. Despite the abundant evidence that opioids affect brain development, little is known about the mechanisms involved. In this study cortical astrocytes in primary culture were examined immunohistochemically by using antibodies against the opioid receptors. The immunoreactivity for delta-opioid receptors was strongly upregulated during mitosis with an increase in immunostaining that started in early prophase and lasted through the M-phase to cytokinesis. Similar effects could not be observed when antibodies against the mu- or kappa-opioid receptor subtypes were used. Cultured neurons and microglia presented a strong and homogenous immunostaining for the delta-opioid receptor and no further upregulation of immunoreactivity could be detected in these cells. The presence of functional delta-opioid receptors on the mitotic astrocytes was verified by using microspectrofluorometry for detection of delta-opioid agonist induced changes in intracellular free calcium concentrations ([Ca2+]i). In these experiments fluo-3/AM incubated cells showed a rapidly induced delta-opioid agonist (DPDPE, 10(-6) M) evoked increase in [Ca2+]i. These results suggest an upregulation of the delta-opioid receptors that could represent a mechanism involved in the response to opioids in the developing brain.

Animals↗

Germ cell mutagenicity of three metabolites of 1,3-butadiene in the rat: induction of spermatid micronuclei by butadiene mono-, di-, and diolepoxides in vivo.

Three metabolites of the industrial chemical 1,3-butadiene (BD), namely butadiene monoepoxide (BMO, 3,4-epoxy-1-butene), diepoxide (DEB, 1,2;3,4-diepoxybutane), and diolepoxide (DE, 3,4- epoxybutane-1,2-diol) were studied for germ cell mutagenicity using the rat spermatid micronucleus (MN) test. All three epoxides increased slightly, but significantly, the frequency of spermatid MN. The most sensitive stage to the action of BMO and DEB was preleptotene (meiotic S phase) harvested at 18-day time intervals after treatment. The dose-response for BMO followed a second order curve at this time interval, with maximum MN induction at the dose of 186 mumol/kg and lower induction of higher doses. Late stages of the meiotic prophase (late pachytene-diplotene-diakinesis) also showed some sensitivity to the three epoxides. Stem cell spermatogonia were affected by DEB as observed by a slight induction of spermatid micronuclei 50 days after treatment. No clear cytotoxic effects were observed by measuring testicular weight or cell numbers of seminiferous epithelial stage 1 18 days after the treatments. DEB at the dose 387 mumol/kg caused a slight inhibition of spermatogonial DNA synthesis in stage I and a delay of meiotic DNA replication observed in stage XII 72 hr after treatment. Since BMO is able to induce spermatid MN in the rat, the present results, together with previous data, indicate that rat bone marrow MN results that are negative for both BD and BMO cannot directly predict mutagenicity in male germ cells. The results also emphasize that tissue; species, and strain-specific differences in metabolism have to be taken into account when the genetic risks of human butadiene exposure are evaluated. The results support the conclusion that 1,3-butadiene is a germ cell mutagen-possibly also in humans.

Animals↗