TTP, a C3H zinc finger protein gene, is expressed in mouse ovarian oocytes

The gene TTP, encoding a C3H zinc finger protein of the TIS11 family, is expressed in growing mouse oocytes. The gene is downregulated in Graafian follicles shortly before ovulation. This corresponds to a possible function in regulation of maternal mRNA translation, a function attributed to related C3H class genes in Caenorhabditis elegans, zebrafish, and Xenopus.     

一个与小鼠锌指蛋白基因ZF—12相关的假基因的克隆和鉴定

小鼠类Krueppel锌指蛋白基因ZF-12为人的类Krueppel锌指蛋白基因ZNF191的同源基因,它们都编码368个氨基酸残基,N端存在SCAN结构域,C端有4个连续的锌指模体,最近的研究表明ZNF191是肝癌发生的相关基因,我们以人锌指蛋白基因ZNF191的vDNA为探针,筛选小鼠λ噬菌体基因组文库,意外地获得了1个与小鼠锌指蛋白基因ZF-12相类似的基因,多种组织的RT-PCR和启动子序列分析,暗示该基因不表达,且该基因无内含子,与ZF-12高度相似,存在突变,暗示其为与ZF-12相关的假基因序列,经查新证实它为新的序列后,以ZF12p（ZF-12pseudogene)命名在GenBank登录（AY040222）。查录GenBank的人类基因组库以及Southern结果显示人类基因组中无ZNF191假基因序列,ZF12p与ZF-12高度相似,暗示ZF12p在进化过程中产生的时间较晚,这对研究锌指蛋白基因ZF-12的突变与进化具有重要的意义。     

A mouse zinc finger gene which is transiently expressed during spermatogenesis

Zinc finger proteins are polypeptides with sequence-specific, nucleic acid-binding properties. Substantial evidence has established them as a class of trans-acting molecules with regulatory roles in cellular growth and differentiation. We have screened an 11.5 day post coitum urogenital ridge cDNA library with an oligonucleotide encoding a sequence conserved between a variety of zinc finger proteins. By cDNA cloning and sequencing we show that a novel mouse gene, Zfp-35, encodes a protein with a block of 18 zinc finger domains and an N-terminal region rich in acidic residues. The 2.4 kb mRNA encoding this polypeptide is selectively expressed in adult testis, by comparison with other organs. We have analysed Zfp-35 expression in whole testes of sex-reversed mice, whole testes of prepuberal XY animals, germ cell fractions from XY adult testes and by in situ hybridization to sections from adult XY testes. Our studies show that a considerable increase in expression is restricted to spermatocytes at the pachytene stage of meiotic prophase. These experiments suggest that Zfp-35 may act to control gene activity during this particular stage of spermatogenesis.     

Expression of the zinc finger Egr1 gene during zebrafish embryonic development

Egr1 is a highly conserved zinc finger protein which plays important roles in many aspects of vertebrate development and in the adult. The cDNA coding for zebrafish Egr1 was obtained and its expression pattern was examined during zebrafish embryogenesis using whole-mount in situ hybridization. Egr1 mRNA is first detected in adaxial cells in the presomitic mesoderm between 11 and 20 h post-fertilization (hpf), spanning the 4-24 somite stages. Later, Egr1 expression is observed only in specific brain areas, starting at 21 hpf and subsequently increasing in distinct domains of the central nervous system, e.g. in the telencephalon, diencephalon and hypothalamus. Between 24 and 48 hpf, Egr1 is expressed in specific domains of the hypothalamus, mesencephalon, tegmentum, pharynx, retina, otic vesicle and heart.     

CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.     

Expression analysis of an Arabidopsis C2H2 zinc finger protein gene

C₂H₂ zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity. AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C₂H₂ zinc finger-encoding sequences previously characterized from Arabidopsis. The genomic sequence corresponding to the AtZFP1 cDNA has been determined. Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems. A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the -glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots. Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promotor:-glucuronidase fusion shows good correlation with RNA blot hybridization analysis. This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function.     

Regulation of asymmetrical cytokinesis by cAMP during meiosis I in mouse oocytes

Mammalian oocytes undergo an asymmetrical first meiotic division, extruding half of their chromosomes in a small polar body to preserve maternal resources for embryonic development. To divide asymmetrically, mammalian oocytes relocate chromosomes from the center of the cell to the cortex, but little is known about the underlying mechanisms. Here, we show that upon the elevation of intracellular cAMP level, mouse oocytes produced two daughter cells with similar sizes. This symmetrical cell division could be rescued by the inhibition of PKA, a cAMP-dependent protein kinase. Live cell imaging revealed that a symmetrically localized cleavage furrow resulted in symmetrical cell division. Detailed analyses demonstrated that symmetrically localized cleavage furrows were caused by the inappropriate central positioning of chromosome clusters at anaphase onset, indicating that chromosome cluster migration was impaired. Notably, high intracellular cAMP reduced myosin II activity, and the microinjection of phospho-myosin II antibody into the oocytes impeded chromosome migration and promoted symmetrical cell division. Our results support the hypothesis that cAMP plays a role in regulating asymmetrical cell division by modulating myosin II activity during mouse oocyte meiosis I, providing a novel insight into the regulation of female gamete formation in mammals.     

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 pronuclei 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.     

Chromosomal localization of zinc finger protein genes in man and mouse

We have determined the mouse and human chromosomal location of a gene (Zfp-3) that codes for a protein that contains potential DNA zinc-binding fingers. An analysis of the segregation of restriction fragment length polymorphisms in recombinant inbred strains and in an interspecific backcross demonstrated that Zfp-3 is located on mouse chromosome 11. Zfp-3 is very closely linked to the Trp53-1 locus but unlinked to another finger protein gene Zfp-4 located on mouse chromosome 8. In humans ZFP3 has been localized to chromosome 17p12-17pter and thus is part of the conserved linkage group between this chromosome and the distal half of mouse chromosome 11.     

Rad51 immunocytology in rat and mouse spermatocytes and oocytes

On the assumption that Rad51 protein plays a role in early meiotic chromosomal events, we examine the location and time of appearance of immuno-reactive Rad51 protein in meiotic prophase chromosomes. The Rad51 foci in mouse spermatocytes appear after the emergence of, and attached to, short chromosomal core segments that we visualize with Cor1-specific antibody. These foci increase in number to about 250 per nucleus at the time when core formation is extensive. The numbers are higher in mouse oocytes and lower in rat spermatocytes, possibly correlating with recombination rates in those cases. In the male mouse, foci decrease in number to approximately 100 while chromosome synapsis is in progress. When synapsis is completed, the numbers of autosomal foci decline to near 0 while the X chromosome retains about 15 foci throughout this time. This stage coincides with the appearance of testis-specific histone H1t at mid- to late pachytene. Electron microscopy reveals that at first Rad51 immunogold-labeled 100 nm nodules are associated with single cores, and that they come to lie between the chromosome cores during synapsis. It appears that these nodules may be the homologs of the Rad51-positive early nodules that are well documented in plants. The reciprocal recombination-correlated late nodules appear after the Rad51 foci are no longer detectable. The absence of Rad51 foci in the chromatin loops suggests that in wild-type mice Rad51/DNA filaments are restricted to DNA at the cores/synaptonemal complexes. The expected association of Rad51 protein with Rad52 could not be verified immunocytologically. Received: 12 December 1996; in revised form: 3 April 1997 / Accepted: 4 April 1997     

A gene that encodes a protein consisting solely of zinc finger domains is preferentially expressed in transformed mouse cells.

We describe the cloning and characterization of the mouse MOK-2 gene, a new member of the Krüppel family of zinc finger proteins. Sequencing of both cDNA and genomic clones showed that the predicted MOK-2 protein consists of seven zinc finger domains with only five additional amino acids. The finger domains of MOK-2 are highly homologous to one another but not to those of other zinc finger proteins. MOK-2 is preferentially expressed in transformed cell lines, brain tissue, and testis tissue. Its possible role in cellular transformation is discussed.     

Spindle formation and dynamics of gamma-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.