Cytogenetic analysis of the 1-st cleavage division of rat embryos

The chromosome number in 174 zygotes on the stage of the 1st cleavage division was counted. In 113 zygotes (64.94%) euploidy (metaphase with 42 chromosomes) and in 57 zygotes (32.77%) aneuploidy was found. Frequencies of spontaneous chromosomal aberration were 14.93%. 33 zygotes were hypoploid, 6 (3.44%)--hyperploid (with 43 and 46 chromosomes), and 3 (5.76%)--polyploid, 4 zygotes (2.29%) had structural anomalies of chromosomes. Hypoploidy may be often due to artifacts. Superovulation induced by gonadotrophin results in an increased polyspermia.     

Gene conversion in transgenic maize plants expressing FLP/FRT and Cre/loxP site-specific recombination systems

DNA recombination reactions (site-specific and homologous) were monitored in the progeny of transgenic maize plants by bringing together two recombination substrates (docking sites and shuttle vectors) in the zygotes. In one combination of transgenic events, the recombination marker gene (yellow fluorescent protein gene, YFP) was activated in 1%-2% of the zygotes receiving both substrates. In other crosses, chimeric embryos and plants were identified, indicative of late recombination events taking place after the first mitotic division of the zygotes. The docking site structure remained unchanged; therefore, all recovered recombination events were classified as gene conversions. The recombinant YFP-r gene segregated as a single locus in subsequent generations. The recombination products showed evidence of homologous recombination at the 5' end of the YFP marker gene and recombinational rearrangements at the other end, consistent with the conclusion that DNA replication was involved in generation of the recombination products. Here, we demonstrate that maize zygotes are efficient at generating homologous recombination products and that the homologous recombination pathways may successfully compete with other possible DNA repair/recombination mechanisms such as site-specific recombination. These results indicate that maize zygotes provide a permissive environment for homologous recombination, offering a new strategy for gene targeting in maize.     

Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1

Transgenic Research - CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using...     

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.     

Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). After 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.     

从烟草EST微卫星标记中筛选合子胚中优势表达的基因

为获得烟草合子胚中的优势表达基因,利用CAP3程序对来自烟草公共数据库的EST序列进行组装,利用MISA程序从组装后的EST中筛选SSR位点,将多态性的SSR位点在烟草合子文库中进行扩增并对等位基因进行分析。结果表明:具有多态性的16个SSR标记中,有9个基因能从烟草的合子库中成功扩增得到。该研究为筛选烟草合子胚中优势表达基因提供新的途径。     

Effect of culture-medium supplementation with alpha-mannosidase and/or beta-N-acetyloglucosaminidase on in vitro bovine embryonic development

Glycosidases are enzymes with a potential role in embryonic development. The objectives of this study were to assess: (a) whether in vitro bovine embryonic development is affected by the addition of beta-N-acetyloglucosaminidase (beta-NAGASE) and/or alpha-mannosidase to the culture medium and (b) whether these enzymes are utilized by bovine embryos during their development in vitro. Bovine embryos were produced using standard methods of IVM, IVF and IVC. Presumptive zygotes were cultured in groups of 20 in 50 microl drops of SOF medium (plus 5% FBS after 24 h culture) incubated in 5% CO2, 5% O2 and 90% N2 at 38.5 degrees C. The groups of zygotes were allocated to four treatments in which the culture medium was supplemented with: (1) beta-NAGASE, (2) alpha-mannosidase, (3) beta-NAGASE plus alpha-mannosidase, and (4) control (no supplement). Embryos were evaluated and samples of culture medium collected and frozen prior to assay for glycosidases at day 7 of culture. The experimental design was a randomised block arrangement of 4 treatments x 7 replicates with 20 zygotes per plot (culture droplet). Data were analysed by ANOVA and presented as mean +/- S.E.M. The osmolarity of the control culture medium was 272 mOsm. This was increased to 279 mOsm by the addition of alpha-mannosidase, 424 mOsm by beta-NAGASE and 337 mOsm with a combination of the two enzymes. The beta-NAGASE supplemented medium and the combined supplement reduced (0%) the development of zygotes to morula or blastocyst stages (P < 0.002) relative to control medium (35.7 +/- 8.4%). Embryo development was also reduced to 21.9 +/- 3.2 (P< 0.002), relative to control, by alpha-mannosidase supplementation. The reduced embryo development in the beta-NAGASE-supplemented medium was attributed to increased osmolarity of the culture medium. Embryos appeared to utilize alpha-mannosidase because its concentration decreased from 600.95 +/- 174.03 IU/l in drops without zygotes/embryos to 211.01 +/- 71.59 IU/l in drops with zygotes/embryos. Other culture media supplementation showed no significant differences between droplets, with or without zygotes/embryos. It was concluded that beta-NAGASE increased medium osmolarity, embryos utilized alpha-mannosidase and both glycosidases (singly or in combination) inhibited the development of bovine zygotes to morulae/blastocysts.     

Oxidative stress-induced DNA damage of mouse zygotes triggers G2/M checkpoint and phosphorylates Cdc25 and Cdc2

In vitro fertilized (IVF) embryos show both cell cycle and developmental arrest. We previously showed oxidative damage activates the ATM?→?Chk1?→?Cdc25B/Cdc25C cascade to mediate G2/M cell cycle arrest for repair of hydrogen peroxide (H₂O₂)-induced oxidative damage in sperm. However, the mechanisms underlying the developmental delay of zygotes are unknown. To develop a model of oxidative-damaged zygotes, we treated mouse zygotes with different concentrations of H₂O₂ (0, 0.01, 0.02, 0.03, 0.04, 0.05 mM), and evaluated in vitro zygote development, BrdU incorporation to detect the duration of S phase. We also examined reactive oxygen species level and used immunofluorescence to detect activation of γH2AX, Cdc2, and Cdc25. Oxidatively damaged zygotes showed a delay in G2/M phase and produced a higher level of ROS. At the same time, γH2AX was detected in oxidatively damaged zygotes as well as phospho-Cdc25B (Ser323), phospho-Cdc25C (Ser216), and phospho-Cdc2 (Tyr15). Our study indicates that oxidative stress-induced DNA damage of mouse zygotes triggers the cell cycle checkpoint, which results in G2/M cell cycle arrest, and that phospho-Cdc25B (Ser323), phospho-Cdc25C (Ser216), and phospho-Cdc2 (Tyr15) participate in activating the G2/M checkpoint.     

A possible aid in targeted insertion of large DNA elements by CRISPR/Cas in mouse zygotes

The CRISPR/Cas system has rapidly emerged recently as a new tool for genome engineering, and is expected to allow for controlled manipulation of specific genomic elements in a variety of species. A number of recent studies have reported the use of CRISPR/Cas for gene disruption (knockout) or targeted insertion of foreign DNA elements (knock‐in). Despite the ease of simple gene knockout and small insertions or nucleotide substitutions in mouse zygotes by the CRISPR/Cas system, targeted insertion of large DNA elements remains an apparent challenge. Here the generation of knock‐in mice with successful targeted insertion of large donor DNA elements ranged from 3.0 to 7.1 kb at the ROSA26 locus using the CRISPR/Cas system was achieved. Multiple independent knock‐in founder mice were obtained by injection of hCas9 mRNA/sgRNA/donor vector mixtures into the cytoplasm of C57BL/6N zygotes when the injected zygotes were treated with an inhibitor of actin polymerization, cytochalasin. Successful germ line transmission of three of these knock‐in alleles was also confirmed. The results suggested that treatment of zygotes with actin polymerization inhibitors following microinjection could be a viable method to facilitate targeted insertion of large DNA elements by the CRISPR/Cas system, enabling targeted knock‐in readily attainable in zygotes. genesis 54:65–77, 2016. © 2016 Wiley Periodicals, Inc.     

Mutagenic DNA repair in Escherichia coli

Summary Escherichia coli K12 Hfr H Tsx^s Str^s and F^- Pro^- Tsx^r His^- Arg^- Str^r bacteria were conjugated in the absence of arginine with or without glucose. The efficiency of conjugation, measured by the frequency of Pro⁺ and His⁺ recombinants was not affected. Arginine starvation alone did not affect the tsx ^s gene expression which occurred in all the zygotes which had received the gene. In contrast, argine and glucose starvation allows tsx ^s expression only in those zygotes in which the donor gene had been integrated in the genome. As the glucose starvation brings on a destabilization of the messenger RNA synthesized by the F^- cells in absence of arginine, the results can be interpreted as follows: the transferred tsx ^s genes are transitorily expressed in all the zygotes at the unintegrated state. After this transient period, only those genes integrated in the chromosomes of the zygotes continue to be expressed.     

血红素加氧酶-1显性负性突变体转基因小鼠模型的建立与鉴定

目的建立血红素加氧酶-1(heme oxygenase-1,HO-1)显性负性突变体G143H转基因小鼠模型。方法通过SalI/DraI酶切pCAGGHO-1G143H转基因表达载体,纯化回收HO-1G143H表达盒片段;通过显微注射把表达目的基因的DNA片段导入FVB小鼠受精卵原核,并移植给同期发情的假孕受体母鼠,获得子代小鼠;用PCR对子代鼠尾DNA进行鉴定,并用Southern blot对结果做进一步验证;通过RT-PCR、免疫组化和Western blot方法检测HO-1基因的表达。结果表达盒回收片段正确;假孕鼠出生的17只子代小鼠共有3只阳性,均为雄性;RT-PCR、免疫组化和Western blot结果表明,阳性小鼠体内的HO-1 mRNA与蛋白表达水平增高。结论成功建立HO-1显性负性突变体G143H表达的转基因小鼠,该模型为研究HO-1在体内的作用机制奠定了基础。     

PYRENOID FORMATION ASSOCIATED WITH THE CELL CYCLE IN THE BROWN ALGA,SCYTOSIPHON LOMENTARIA (SCYTOSIPHONALES,PHAEOPHYCEAE)1

Vegetative cells of the brown alga Scytosiphon lomentaria (Lyngbye) Link characteristically have only one chloroplast with a prominent protruding pyrenoid, whereas zygotes have both paternal and maternal chloroplasts. In zygotes, before cell and chloroplast division, each chloroplast has an old and a new pyrenoid. In this study, we raised a polyclonal antibody to RUBISCO and examined the distribution of RUBISCO by immunofluorescence microscopy, focusing on new pyrenoid formation in vegetative cells of gametophytes and zygotes in Scytosiphon. In interphase, only one old pyrenoid was positively indicated by anti‐RUBISCO antibody in vegetative cells of gametophytes. From mid‐S phase, small fluorescence aggregates reflecting RUBISCO localization started to appear at stroma positions other than adjacent to the old protruding pyrenoid. The fluorescent spots eventually coalesced into a protrusion into the adjacent cytoplasm. We also used inhibitors to clarify the relationship between the cell cycle and new pyrenoid formation, using zygotes after fertilization. When DNA replication was blocked by aphidicolin, new pyrenoid formation was also inhibited. Washing out aphidicolin permitted new pyrenoid formation with the progression of the cell cycle. When mitosis was prolonged by nocodazole, which disrupted the spindle microtubules, the fluorescent masses indicating RUBISCO localization continued to increase when compared with pyrenoid formation in untreated zygotes. During treatment with chloramphenicol, mitosis and cytokinesis were completed. However, there was no occurrence of new RUBISCO localization within the chloroplast stroma beyond the old pyrenoid. From these observations, it seems clear that new pyrenoid formation in the brown alga Scytosiphon depends on the cell cycle.     

Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein

The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9–FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.     

MORPHOLOGY AND PAEDOGAMOUS SEXUAL REPRODUCTION IN CHLOROGONIUM CAPILLATUM SP. NOV. (VOLVOCALES, CHLOROPHYTA)1

Morphology and sexual reproduction in Chlorogonium capillatum Nozaki, Watanabe & Aizawa sp. nov. (Volvocales, Chlorophyta) originating from Miyatoko Mire, Japan, were studied under controlled laboratory conditions. Vegetative cells of this new species were fusiform with blunt anterior and posterior ends, and they had a massive parietal chloroplast and numerous contractile vacuoles distributed throughout the protoplast. Several to many pyrenoids were randomly distributed in the chloroplast, but they disappeared under the light microscope when grown photoheterotrophically. During asexual reproduction, the first division took place transversely without a preceding rotation of the parental protoplast. In sexual reproduction, the parental protoplast divided successively to form 32 or 64 small, biflagellate isogametes. After gametogenesis, the gametes did not escape from the parental cell (gametangial) wall, within which pairs of the adjoining gametes fused to form quadriflagellate zygotes. Such zygotes were then released from the parental cell wall and developed into hypnozygotes, which at maturity developed numerous thin spines or hairs on the zygote wall. On zygote germination, four biflagellate germ cells were released from the zygote wall separately. This type of gametic union, "paedogamy," has not previously been described in the green algae except for Chlorococcum echinozygotum Starr . Chlorogonium capillatum can be clearly distinguished from other described species of Chlorogonium by its numerous contractile vacuoles and blunt anterior and posterior ends in vegetative cells as well as by its unique sexual reproduction, in which paedogamous conjugation occurs, and numerous thin spines or hairs that develop on the hypnozygote walls .     

人二倍体化合子的DNA 甲基化变化模式

目的:探讨人类三原核合子及二倍体化合子中DNA甲基化模式的变化情况。方法:我们采用显微操作技术去除三原核合子中两个雄原核中的一个,观察恢复了二倍体状态的胚胎的发育情况,并检测了三原核和二倍体化的合子及早期胚胎中DNA甲基化模式的动态变化。结果:二倍体化的合子的囊胚形成率与三原核合子的囊胚形成率无显著性差异;在人三原核合子中两个雄原核发生主动地DNA去甲基化而雌原核在受精后的20h后仍保持甲基化。三原核与二倍体化合子中,DNA甲基化模式没有差别。结论:去除一个雄原核不会影响合子和胚胎的DNA甲基化模式。去除多余雄原核并不能改善胚胎的发育。     

MATURATION OF ALGAL ZYGOTES: ALTERNATIVE EXPERIMENTAL APPROACHES FOR CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Young zygotes from crosses of Chlamydomonas reinhardtii Dang. mutant and wild-type strains were incubated, in the presence or absence of light and/or nitrogen to determine whether continuation of conditions inducing gamete formation permits zygospore formation without loss of viability. Different culture media, continuous illumination vs. dark incubation and various durations of the maturation period were tested, for effect on zygospore germination efficiency, zygospore “burst size” and zoospore viability. Following either the routine maturation procedure of dark incubation on standard minimal medium, or following a new procedure of incubation under continuous illumination on N-free medium, zygospore formation can be ensured and high germination efficiencies obtained within 3 days after mating. Tetrad analysis indicates meiosis occurs normally whether zygotes have been matured in the presence or absence of light or nitrogen. Preliminary data suggest an effect of increased maturation time on the transmission of cytoplasmic genes, if a N-free continuous illumination maturation protocol is followed. Two experimental approaches for the maturation of C. reinhardtii zygotes are suggested and advantages of each are discussed.     

Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.     

Developmental capacity of rat embryos produced by in vivo or in vitro fertilization

This work compares the ability of rat zygotes fertilized in vitro or in vivo to develop into viable embryos. All oocytes were from adult cyclic females. After the first cleavage, the zygotes were transferred to oviducts of pseudopregnant recipients. Their fate was examined on day 13 at laparotomy and again on day 20. Ninety-five of 146 in vivo fertilized zygotes developed into normal sized 13-day fetuses and 72 (55%) to apparently normal near-term fetuses. Forty-six of 135 in vitro fertilized zygotes developed up to day 13, and 30 (24%) developed to term. It appears that the probability that in vitro fertilized rat zygotes will develop into viable embryos is about half the chance of in vivo fertilized zygotes. Since the two types of zygotes were morphologically identical, the morphological appearance of the two-cell stage is not an adequate criterion for judging developmental potential.     

Chromosome engineering in zygotes with CRISPR/Cas9

Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection. genesis 54:78–85, 2016. © 2016 The Authors. genesis Published by Wiley Periodicals, Inc.