fertilization

施肥
  • 文章类型: Journal Article
    在过去的几十年中,欧洲农业实践的强化极大地改变了农业生态系统。景观的简化,半自然栖息地的丧失和化学品在作物上的应用导致农业景观的生物多样性下降,引起人们对基本生态系统服务损失的重大担忧,如授粉或害虫防治。根据位置,规模和区域背景,定期调查不同的指示物种组(ISG),以评估农业生态系统中生物多样性变化的状态和趋势。尽管这些ISG的高度多样性允许评估不同的生物多样性方面(例如,营养水平,生物物理隔间,指示刻度),它使结果的解释变得复杂,从而使它们的实际应用变得复杂。此外,物种多样性指标多种多样,从简单的物种计数到更复杂的多样性指数测量,有时会有对抗反应。这里,为了满足这个复杂主题中对综合的迫切需要,我们遵循标准化的系统地图协议,以收集和总结报告主要农业管理实践(AMPs)在耕地作物中的影响的实地证据的文献,欧洲低地农业区一组ISG上的草原和生态基础设施。
    使用在线出版物数据库进行文献搜索,英文搜索引擎和专业网站。根据先前方案中公布的纳入/排除标准,筛选收集的出版物的相关性。我们提取并绘制了有关实验设计的信息,监测方法,研究了ISG和AMP,并在每个出版物中介绍了多样性措施。这些参数在可用的数据编码表中被结构化。
    搜索收集了20,162个参考文献,在全文资格筛选后,仍有1208个参考文献。研究的主要地区是西欧,从1984年到2022年,研究数量呈指数增长。大多数出版物都是实验和农场研究,这些研究在田间规模上评估了AMP的影响。主要研究的AMP是施肥,放牧,有机农业,耕作,割草和除草剂的应用。大多数用来研究其影响的ISG是植物区系,海盗,蜘蛛,鸟,蜜蜂和环节动物,经常与其他ISG结合使用。研究了AMP和ISG之间的组合以及监测方法。最常用的多样性指标是丰度,物种丰富度,香农指数,均匀度,和社区组成。
    我们确定了几个知识集群:(1)有机农业,受精,耕作,放牧和割草对各种ISG的影响,(2)植物区系对农业实践的反应,(3)环节动物对影响土壤结构的农艺干预措施的反应(例如,耕作,受精,作物轮作,作物残留物管理),(4)蝴蝶和直翅目动物对草原割草和放牧效应的反应,(5)使用鸟类监测对评估AES在景观尺度上实施的效率的影响。我们强调,应该对迄今为止关于农业实践研究不足的ISG进行进一步研究,比如两栖动物,爬行动物,腹足类,千足虫和cent。更多的实地证据表明,间作等多样化做法的影响,undersowing,中间种植,和农林业需要就它们对生物多样性的好处得出结论。
    在线版本包含补充材料,可在10.1186/s13750-024-00347-0获得。
    UNASSIGNED: The intensification of the agricultural practices in Europe over the last decades has drastically transformed the agroecosystems. The simplification of the landscape, the loss of semi-natural habitats and the application of chemicals on crops led to biodiversity decline in agricultural landscapes, raising substantial concerns about the loss of essential ecosystem services, such as pollination or pest control. Depending on the location, the scale and the regional context, different indicator species groups (ISGs) are regularly surveyed to assess the state and trend of biodiversity changes in agroecosystems. Although the high diversity of these ISGs allows assessing different biodiversity aspects (e.g., trophic levels, bio-physical compartments, scale of indication), it complicates the interpretation of the results and thus their practical application. In addition, species diversity metrics are various, from simple species counts to more complex measurements of diversity indices, sometimes with antagonistic responses. Here, to meet the pressing need for synthesis in this complex topic, we follow a standardized systematic map protocol to collect and summarize the literature reporting field evidence of the effects of the main agricultural management practices (AMPs) in arable crops, grasslands and ecological infrastructures on a set of ISGs in European lowland farming areas.
    UNASSIGNED: Searches of literature were made using online publication databases, search engine and specialist websites in English. Gathered publications were screened for relevance following inclusion/exclusion criteria published in a prior protocol. We extracted and mapped information about experimental design, monitoring methods, ISGs and AMPs studied and the diversity measures presented in each included publication. These parameters are structured in available data coding sheets.
    UNASSIGNED: The search gathered 20,162 references from which 1208 remained after full text eligibility screening. Main areas studied are in Western Europe, and the number of studies increased exponentially from 1984 to 2022. Most publications are experimental and on-farm studies which assess AMPs effects at the field scale. Main studied AMPs are fertilization, grazing, organic farming, tillage, mowing and herbicide application. Most ISGs used to study their impacts are flora, carabids, spiders, birds, bees and annelids, often combined with other ISGs. The combinations between AMPs and ISGs studied are detailed as well as monitoring methods. The most used diversity measures are abundance, species richness, Shannon index, evenness, and community composition.
    UNASSIGNED: We identified several knowledge clusters: (1) organic farming, fertilization, tillage, grazing and mowing impact on a wide range of ISGs, (2) flora response to agricultural practices, (3) annelids response to agronomic interventions that impact soil structure (e.g., tillage, fertilization, crop rotation, crop residue management), (4) butterflies and orthopterans response to mowing and grazing effects in grasslands, (5) the use of bird monitoring for the impact for assessing the efficiency of AES implementation at the landscape scale. We highlight that further research should be conducted on ISGs that are until now poorly studied regarding agricultural practices, such as amphibians, reptiles, gastropods, millipedes and centipedes. More field evidence of the effects of diversification practices such as intercropping, undersowing, intermediate cropping, and agroforestry are needed to draw conclusions on their benefits on biodiversity.
    UNASSIGNED: The online version contains supplementary material available at 10.1186/s13750-024-00347-0.
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  • 文章类型: Journal Article
    在经典的体外受精程序(IVF)或胞质内单精子注射(ICSI)后的第18小时进行受精检查是辅助生殖的关键阶段。治疗的成功很大程度上取决于显示两个原核的受精卵的数量。因此,低受精率或完全受精失败对患者和生殖专家都是非常不希望的结果.ICSI后应用额外的钙离子载体进行卵母细胞激活可能会带来益处,并可能增强治疗效果。特别是对于在以前的治疗周期中经历过低受精率或无受精率(FR)的患者。该研究的目的是评估Ca2离子载体应用于卵母细胞活化的功效。
    回顾性分析从120例患者中获得的924个卵母细胞,这些患者因先前的治疗失败而经历了ICSI周期,具有低受精或无受精史。在下一个ART周期之后,在57例病例中应用了额外的卵母细胞Ca2离子载体激活,以优化治疗过程(第1组)。纳入63例患者,其结局作为对照组(第2组).我们对两组的结果进行了比较分析。该研究的主要结果包括受精,卵裂胚胎质量,囊胚率,并确定了临床妊娠。
    在第1天受精检查时,我们在第1组中有274/386个受精卵(71%FR),在第2组中有132/410个受精卵(32.2%FR),(P<0.0001)。第2组的22个(34.9%)周期导致总受精失败(TFF)。在卵裂阶段,与第2组相比,第1组的优质胚胎明显更高(P=0.0021)。第1组进行了48个胚胎移植(ET),导致41.67%的临床妊娠,而第2组只有33个ET,仅4个妊娠(12.12%)(P=0.0044)。
    结果证实了在先前受精失败周期的情况下,辅助卵母细胞激活作为附加方法的适当性。
    UNASSIGNED: Fertilization check performed at the 18th hour following classic in vitro fertilization procedure (IVF) or intracytoplasmic sperm injection (ICSI) is a critical stage in assisted reproduction. The success of the treatment is significantly reliant on the quantity of zygotes exhibiting two pronuclei. Consequently, low fertilization rates or complete fertilization failure are highly undesirable outcomes for both patients and reproductive specialists. Applying additional calcium ionophore for oocyte activation subsequent to ICSI may offer benefits and potentially enhance treatment outcomes, particularly for patients who have experienced low or absent fertilization rates (FR) in previous treatment cycles. The aim of the study is to evaluate the efficacy of Ca2+ ionophore application for oocyte activation.
    UNASSIGNED: A retrospective analysis of 924 oocytes obtained from 120 patients who underwent ICSI cycles with a history of low or no fertilization as a result of previous unsuccessful treatment rounds. The next ART cycle followed with additional oocyte Ca2+ ionophore activation applied in 57 of the cases in order to optimize the treatment process (Group 1), and 63 patients were included and their outcomes followed as a control group (Group 2).We conducted a comparative analysis of results in both groups. The study\'s primary outcomes encompassed fertilization, cleavage embryo quality, blastocyst rate, and established clinical pregnancies.
    UNASSIGNED: At day 1 fertilization check we had 274/386 zygotes (71%FR) in group 1 and 132/410 in group 2 (32.2%FR), (P < 0.0001). Twenty-two (34.9%) cycles in group 2 resulted in total fertilization failure (TFF). At the cleavage stage top-quality embryos from group 1 were significantly higher (P = 0.0021) in comparison to group 2. Forty-eight embryo transfers (ET) were performed in group 1 resulting in 41.67% clinical pregnancies versus 33 ET and only 4 pregnancies (12.12%) for group 2 (P = 0.0044).
    UNASSIGNED: The results confirm the appropriateness of assisted oocyte activation as an additional method in cases of previous fertilization failure cycles.
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  • 文章类型: Journal Article
    哺乳动物胚胎发育是由父系和母系配子的结合启动的。受精后,他们的表观基因组景观是通过一系列精心策划的机制转化的,这些机制对生存和成功的胚胎发生至关重要。具体来说,母体或卵母细胞特异性重编程因子将生殖细胞特异性表观遗传标记调节到胚胎状态.在早期胚胎发育期间观察到表观遗传标记如DNA甲基化和组蛋白修饰的快速和动态变化。这些变化在合子基因组激活之前控制胚胎基因组的结构。在父系和母系基因组之间观察到表观遗传标记的差异变化,因为亲本基因组的结构允许与特定卵母细胞重编程因子相互作用。例如,父系基因组由TET家族的酶靶向,该酶将5-甲基胞嘧啶(5mC)表观遗传标记氧化为5-羟甲基胞嘧啶(5hmC)以降低DNA甲基化水平。母体基因组主要受母体因子的TET3介导的氧化保护,斯特拉.TET3介导的DNA去甲基化发生在全球水平上,并且在许多哺乳动物物种中清楚地观察到。其他表观遗传调节酶,如DNA甲基转移酶,通过启动从头甲基化来提供DNA甲基化水平的微调。启动配子表观遗传重编程的机制对于胚胎基因组的适当激活以及随后建立多能性和正常发育至关重要。存在与重编程调节剂突变相关的临床病例或疾病,强调需要了解这些调制器的机械行为。此外,通过体外胚胎生产系统产生的胚胎通常存在表观遗传异常。了解表观遗传调节剂的机制作用将潜在地改善患有这些表观遗传疾病的个体的健康并纠正体外产生的胚胎中的表观遗传异常。这篇综述将总结当前对早期胚胎发育过程中TET酶的表观遗传重编程的理解,并强调其临床相关性和辅助生殖技术的潜在意义。
    Mammalian embryo development is initiated by the union of paternal and maternal gametes. Upon fertilization, their epigenome landscape is transformed through a series of finely orchestrated mechanisms that are crucial for survival and successful embryogenesis. Specifically, maternal or oocyte-specific reprogramming factors modulate germ cell specific epigenetic marks into their embryonic states. Rapid and dynamic changes in epigenetic marks such as DNA methylation and histone modifications are observed during early embryo development. These changes govern the structure of embryonic genome prior to zygotic genome activation. Differential changes in epigenetic marks are observed between paternal and maternal genomes because the structure of the parental genomes allows interaction with specific oocyte reprogramming factors. For instance, the paternal genome is targeted by the TET family of enzymes which oxidize the 5-methylcytosine (5mC) epigenetic mark into 5-hydroxymethylcytosine (5hmC) to lower the level of DNA methylation. The maternal genome is mainly protected from TET3-mediated oxidation by the maternal factor, STELLA. The TET3-mediated DNA demethylation occurs at the global level and is clearly observed in many mammalian species. Other epigenetic modulating enzymes, such as DNA methyltransferases, provide fine tuning of the DNA methylation level by initiating de novo methylation. The mechanisms which initiate the epigenetic reprogramming of gametes are critical for proper activation of embryonic genome and subsequent establishment of pluripotency and normal development. Clinical cases or diseases linked to mutations in reprogramming modulators exist, emphasizing the need to understand mechanistic actions of these modulators. In addition, embryos generated via in vitro embryo production system often present epigenetic abnormalities. Understanding mechanistic actions of the epigenetic modulators will potentially improve the well-being of individuals suffering from these epigenetic disorders and correct epigenetic abnormalities in embryos produced in vitro. This review will summarize the current understanding of epigenetic reprogramming by TET enzymes during early embryogenesis and highlight its clinical relevance and potential implication for assisted reproductive technologies.
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  • 文章类型: Journal Article
    植物界提供了丰富的分子,这些分子对各种人类具有潜在的功效,动物,和植物作物感染和疾病。大麻由于其广泛的生物活性,在近几十年来在科学界引起了极大的关注。从这种植物中分离出关键的生物活性化合物,如大麻素和酚类化合物,驱动它的生物活性。许多研究强调了不同农艺做法的影响,特别是施肥,关于植物化学成分,特别是改变各种化学基团的百分比。这篇综述旨在提出更新的施肥建议,作物需求,以及它们对苜蓿植物化学成分的影响,以及过去五年文献中记录的主要生物学特性。利用各种数据库来总结有关施肥和作物需求的信息,化学成分,使用的生物测定,天然产物(提取物或分离的化合物),和生物活性结果。通过这次审查,很明显,C.sativa有望成为治疗多种人类疾病的新型分子的来源。尽管如此,仔细考虑农艺实践对于优化化学成分和最大化治疗潜力至关重要。
    The plant kingdom offers a wealth of molecules with potential efficacy against various human, animal, and plant crop infections and illnesses. Cannabis sativa L. has garnered significant attention in recent decades within the scientific community due to its broad biological activity. Key bioactive compounds such as cannabinoids and phenolic compounds have been isolated from this plant, driving its bioactivity. Numerous studies have highlighted the impact of different agronomic practices, particularly fertilization, on the phytochemical composition, notably altering the percentage of various chemical groups. This review aims to present updated fertilization recommendations, crop requirements, and their implications for the chemical composition of C. sativa plants, along with major biological properties documented in the literature over the past five years. Various databases were utilized to summarize information on fertilization and crop requirements, chemical composition, bioassays employed, natural products (extracts or isolated compounds), and bioactivity results. Through this review, it is evident that C. sativa holds promise as a source of novel molecules for treating diverse human diseases. Nonetheless, careful consideration of agronomic practices is essential to optimize chemical composition and maximize therapeutic potential.
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  • 文章类型: Editorial
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  • 文章类型: Journal Article
    背景:在被子植物中,植物物种的延续错综复杂地依赖于营养运输中的真菌多方面的作用,机械支撑,和种子的开裂。SEEDSTICK(STK)是一种参与种子大小和脱落的MADS-box转录因子,和少数被确定为影响真菌生长的基因之一。鉴于真菌对正确种子发育的重要性,与以前的stk突变体的表型观察结果有关,我们从花期17进行了STK真菌的转录组学分析,使用RNA测序,来推断基因放松的网络。
    结果:生成的差异表达基因的数据集富含细胞壁生物发生,细胞周期,糖代谢和运输术语,所有这些都符合从花期17开始在真菌中观察到的stk表型。我们选择了八个差异表达的基因用于使用qPCR和/或启动子报告系的转录组验证。这些基因与脱落有关,种子发育或stk真菌中的新功能,如激素/次级代谢产物转运。
    结论:总体而言,在这项研究中进行的分析允许深入研究在拟南芥真菌中建立的STK网络,填补了文学空白。同时,我们的发现加强了转录组的可靠性,使其成为真菌功能遗传研究候选基因选择的宝贵资源。这将加深我们对STK控制的监管网络的理解,关于真菌的作用以及它们如何影响种子发育。
    BACKGROUND: In Angiosperms, the continuation of plant species is intricately dependent on the funiculus multifaceted role in nutrient transport, mechanical support, and dehiscence of seeds. SEEDSTICK (STK) is a MADS-box transcription factor involved in seed size and abscission, and one of the few genes identified as affecting funiculus growth. Given the importance of the funiculus to a correct seed development, allied with previous phenotypic observations of stk mutants, we performed a transcriptomic analysis of stk funiculi from floral stage 17, using RNA-sequencing, to infer on the deregulated networks of genes.
    RESULTS: The generated dataset of differentially expressed genes was enriched with cell wall biogenesis, cell cycle, sugar metabolism and transport terms, all in accordance with stk phenotype observed in funiculi from floral stage 17. We selected eight differentially expressed genes for transcriptome validation using qPCR and/or promoter reporter lines. Those genes were involved with abscission, seed development or novel functions in stk funiculus, such as hormones/secondary metabolites transport.
    CONCLUSIONS: Overall, the analysis performed in this study allowed delving into the STK-network established in Arabidopsis funiculus, fulfilling a literature gap. Simultaneously, our findings reinforced the reliability of the transcriptome, making it a valuable resource for candidate genes selection for functional genetic studies in the funiculus. This will enhance our understanding on the regulatory network controlled by STK, on the role of the funiculus and how seed development may be affected by them.
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  • 文章类型: Journal Article
    当卵在雌性卵巢中成熟时,鱼交配中的成功受精发生,排卵,男性睾丸中的精子成熟,两性的生殖行为是同步触发的。鱼的男性性行为是由激素和信息素诱导的。在之前的研究中,我们证明,在水中添加外用激素可以诱导雌性斑马鱼的卵母细胞成熟和排卵。这里,我们试图建立一种类似的方法来诱导雄性斑马鱼的性行为。当直接施用到周围的水中时,男性类固醇睾丸激素(Tes)在体内数小时内引发性行为。膜孕激素受体(mPR)的选择性激动剂,OrgOD-02(Org),也诱发了性行为。通过各种化合物组合的试验,我们发现,最有效的条件是通过用睾酮(Tes)和Org的混合物治疗。通过与体内诱导排卵的雌性配对获得的受精卵数量来评估治疗效果。通过时程实验评估了诱导男性性行为所需的时间。在处理3-4小时时,交配成功率和受精卵数量达到最高水平。激素治疗的持续时间通过计数钩住事件的数量来确认,这是诱导雌性产卵的最终线索。总之,我们建立了一种在斑马鱼体内诱导男性性行为的方法。该方法可以通过简单地将试剂添加到水中来获得斑马鱼的受精卵。
    Successful fertilization in fish mating occurs when egg maturation in the ovary of the female, ovulation, sperm maturation in the testis of the male, and reproductive behaviors in both sexes are triggered in synchrony. The male sexual behavior of fish is induced by hormones and pheromones. In a previous study, we demonstrated that externally applied hormones added to the water can induce oocyte maturation and ovulation in female zebrafish. Here, we attempted to establish a similar method to induce the sexual behavior of male zebrafish. The male sex steroid testosterone (Tes) triggered sexual behavior within several hours in vivo when administered directly into the surrounding water. A selective agonist for membrane progesterone receptor (mPR), Org OD-02 (Org), also induced sexual behavior. Through trials of various combinations of compounds, we found that the most effective conditions were achieved by treatment with a mixture of testosterone (Tes) and Org. The effect of treatment was evaluated by the number of fertilized eggs obtained by pairing with females with induced ovulation in vivo. The period necessary for the induction of male sexual behavior was evaluated by time course experiments. The success rate of mating and the number of fertilized eggs reached the maximum level at 3-4 hours of treatment. The duration of hormonal treatment was confirmed by counting the number of hooking occurrences, which is the final cue to induce spawning by females. In summary, we have established a method to induce male sexual behavior in zebrafish in vivo. The method can be used to obtain fertilized eggs in zebrafish by simply adding agents into the water.
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  • 文章类型: Journal Article
    在精子竞争的研究中,特别是在外部肥料中,受精环境对敌对男性亲子关系的重要性往往被忽视。女性生殖液(FRF),由女性制作和发布,创造了精子在寻求受精时遇到的微环境,并可以通过影响精子竞争中的关键性状来产生亲子关系偏见。然而,FRF对精子性状的影响及其对竞争性受精动力学的影响之间是否存在直接联系仍有待探索。这里,使用斑马鱼Daniorerio,我们比较了在存在/不存在FRF的情况下,两名竞争男性之间的女性亲子关系份额以及受精成功的预测因素(即精子性状)。我们的结果明确地揭示了FRF对精子性状的影响的方向和大小与每个男性的竞争性受精成功的变化之间的直接联系。这项研究表明,FRF通过其对精子性能的不同影响直接介导交配后的雌性控制,而FRF对精子质量的影响足以预测适应性影响的大小。这些发现强调了需要考虑FRF在受精中的作用,避免由于只关注男性内在精子质量而产生的偏见。
    In studies of sperm competition, particularly in external fertilizers, the importance of the fertilization environment on the paternity share among rival males often goes overlooked. The female reproductive fluid (FRF), produced and released by females, creates the microenvironment that sperm encounter on their quest for fertilization and can generate paternity biases by affecting key traits in sperm competition. Yet, whether there is a direct link between FRF effects on sperm traits and its effect on competitive fertilization dynamics remains to be explored. Here, using the zebrafish Danio rerio, we compare within-female paternity share among two competing males and predictors of fertilization success (i.e. sperm traits) in the presence/absence of FRF. Our results unequivocally reveal a direct link between the direction and magnitude of the effect of FRF on sperm traits and the change in the competitive fertilization success of each male. This study demonstrates that the FRF directly mediates post-mating female control through its differential effect on sperm performance and that the FRF\'s effect on sperm quality alone is sufficient to predict the magnitude of the fitness effects. These findings highlight the need to consider the role of FRF in fertilization, avoiding biases resulting from an exclusive focus on male intrinsic sperm quality.
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  • 文章类型: Journal Article
    在大多数动物物种中,受精发生在卵母细胞减数分裂完成之前,并且精子内容物在小鼠和秀丽隐杆线虫的合子内移动长距离。如果掺入减数分裂纺锤体中,父系染色体可能被排入极体,导致致命的一元性。通过对秀丽隐杆线虫受精的实时成像,我们发现微管分解酶,katanin和kinesin-13限制了精子内容物的远距离运动,并且母体ataxin-2将父体DNA和父体线粒体保持为一起运动的粘性单位。katanin的消耗或驱动蛋白13和ataxin-2的双重消耗导致减数分裂纺锤体捕获精子内容物。因此,限制精子内容物的运动和保持精子内容物在合子内的凝聚力都有助于防止母本和父本基因组之间的过早相互作用。
    Fertilization occurs before the completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within the zygotes of mouse and C. elegans. If incorporated into the meiotic spindle, paternal chromosomes could be expelled into a polar body resulting in lethal monosomy. Through live imaging of fertilization in C. elegans, we found that the microtubule disassembling enzymes, katanin and kinesin-13 limit long-range movement of sperm contents and that maternal ataxin-2 maintains paternal DNA and paternal mitochondria as a cohesive unit that moves together. Depletion of katanin or double depletion of kinesin-13 and ataxin-2 resulted in the capture of the sperm contents by the meiotic spindle. Thus limiting movement of sperm contents and maintaining cohesion of sperm contents within the zygote both contribute to preventing premature interaction between maternal and paternal genomes.
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  • 文章类型: Journal Article
    钙是卵母细胞到胚胎转换(OET)和早期胚胎发生过程中的重要信号分子。雌雄同体线虫秀丽隐杆线虫为OET的研究提供了几个独特的优势,因为它是透明的,并且具有有序的性腺,在20°C下每〜23分钟产生一个成熟的卵母细胞。我们已经修改了基因编码的钙指示剂jGCaMP7,以荧光指示生物体内受精的时刻。我们将这个记者称为“CaFE”,用于线虫受精过程中的钙。CaFE记者被设计成一个安全的港口地点,对生理或繁殖力没有显著影响,并在受精时产生强大的信号。这里,提出了一系列方案,用于利用CaFE报告基因作为体内工具来解剖OET和胚胎发生。我们包括同步蠕虫的方法,检查RNAi敲低的效果,安装用于成像的蠕虫,并观察卵母细胞和胚胎中的钙。此外,我们提出了其他蠕虫菌株的产生,以帮助这种类型的分析。演示CaFE记者可视化受精时间的效用,我们报告说,当RNAi靶向ipp-5时,会发生双重排卵,并且只有第一个卵母细胞立即受精。此外,这里报道了早期胚胎发育过程中单细胞钙瞬变的发现,证明CaFE记者坚持早期发展。重要的是,蠕虫中的CaFE记者很简单,可以用于纳入基于课程的本科研究(CURE)实验室课程。CaFE记者,再加上蠕虫中有序的性腺和RNAi的简易性,促进对调节内部受精和早期胚胎发生所需的细胞-细胞动力学的研究。
    Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 °C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter \"CaFE\" for Calcium during Fertilization in C. elegans. The CaFE reporter was engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.
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