Name: a neural network-based identification of developmentally competent or incompetent mouse fully-grown oocytes
Uploaded: 2018-03-03T13:00:00
Description: Watch this Scientific Journal Video about A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes at JoVE.com

这项工作使成为可能多亏了支持: 帕维亚大学悬浮角速度陀螺仪 2016;帕尔马大学 2014, 2016;并运动提供必要的 plasticware 进行这项研究。我们感谢夏恩温莎博士 (英国布里斯托尔大学工程学院) 提供 Cell_PIV 软件。

所有涉及动物的程序均由帕维亚大学的机构动物保育和使用及伦理委员会批准。动物在22°c 的情况下被维护了, 60% 空气湿气和一个光/黑暗的周期 12:12 h。
1.Ovary 隔离
<ol><li>注射腹腔2四-十一周老 CD1 雌性小鼠与 10 U 卵泡刺激激素与无菌1毫升胰岛素注射器。</li>
	<li>等46-48 小时。</li>
	<li>用肌肉注射50毫克/千克的 Zoletil (Tiletamina 和 Zolazepan cloridrate) 来称量老鼠和麻醉。弄死颈椎?...

<ol>
	<li>Patrizio, P., Fragouli, E., Bianchi, V., Borini, A., Wells, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+methods+for+selection+of+the+ideal+oocyte.">Molecular methods for selection of the ideal oocyte.</a> Reprod. Biomed. Online. 15 (3), 346-353 (2007).</li><li>Rienzi, L., Vajta, G., Ubaldi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+value+of+oocyte+morphology+in+human+IVF:+a+systematic+review+of+the+literature.">Predictive value of oocyte morphology in human IVF: a systematic review of the literature.</a> Human. Reprod. Update. 17 (1), 34-35 (2011).</li><li>Tan, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+configurations+in+the+germinal+vesicle+of+mammalian+oocytes.">Chromatin configurations in the germinal vesicle of mammalian oocytes.</a> Mol. Hum. Reprod. 15 (1), 1-9 (2009).</li><li>Vigone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+based+identification+of+mouse+cumulus+cell+markers+that+predict+the+developmental+competence+of+their+enclosed+antral+oocytes.">Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes.</a> BMC Genomics. 14, 380 (2013).</li><li>Bui, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+movement+profiles+of+mouse+surrounding+nucleolus+and+not-surrounding+nucleolus+antral+oocytes+during+meiotic+resumption.">Cytoplasmic movement profiles of mouse surrounding nucleolus and not-surrounding nucleolus antral oocytes during meiotic resumption.</a> Mol. Reprod. Dev. 84 (5), 356-362 (2017).</li><li>Zuccotti, M., Piccinelli, A., Giorgi Rossi, P., Garagna, S., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+organization+during+mouse+oocyte+growth.">Chromatin organization during mouse oocyte growth.</a> Mol. Reprod. Dev. 41 (4), 479-485 (1995).</li><li>Zuccotti, M., Garagna, S., Merico, V., Monti, M., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+organisation+and+nuclear+architecture+in+growing+mouse+oocytes.">Chromatin organisation and nuclear architecture in growing mouse oocytes.</a> Mol. Cell. Endocrinol. 234 (1-2), 11-17 (2005).</li><li>Zuccotti, M., Merico, V., Cecconi, S., Redi, C. A., Garagna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+it+take+to+make+a+developmentally+competent+mammalian+egg?.">What does it take to make a developmentally competent mammalian egg?.</a> Hum. Reprod. Update. 17 (4), 525-540 (2011).</li><li>Inoue, A., Nakajima, R., Nagata, M., Aoki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+oocyte+nucleus+and+cytoplasm+to+the+determination+of+meiotic+and+developmental+competence+in+mice.">Contribution of the oocyte nucleus and cytoplasm to the determination of meiotic and developmental competence in mice.</a> Hum. Reprod. 23 (6), 1377-1384 (2008).</li><li>Ajduk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythmic+actomyosin-driven+contractions+induced+by+sperm+entry+predict+mammalian+embryo+viability.">Rhythmic actomyosin-driven contractions induced by sperm entry predict mammalian embryo viability.</a> Nat. Commun. 2, 417 (2011).</li><li>Swann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C-&#950;-induced+Ca2++oscillations+cause+coincident+cytoplasmic+movements+in+human+oocytes+that+failed+to+fertilize+after+intracytoplasmic+sperm+injection.">Phospholipase C-&#950;-induced Ca2+ oscillations cause coincident cytoplasmic movements in human oocytes that failed to fertilize after intracytoplasmic sperm injection.</a> Fertil. Steril. 97 (3), 742-747 (2012).</li><li>Thakur, A., Mishra, V., Jain, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feed+forward+artificial+neural+network:+tool+for+early+detection+of+ovarian+cancer.">Feed forward artificial neural network: tool for early detection of ovarian cancer.</a> Sci. Pharm. 79 (3), 493-505 (2011).</li><li>Laudani, A., Lozito, G. M., Riganti Fulginei, F., Salvini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+Training+Efficiency+and+Computational+Costs+of+a+Feed+Forward+Neural+Network:+A+Review.">On Training Efficiency and Computational Costs of a Feed Forward Neural Network: A Review.</a> Comput. Intell. Neurosci. 2015, 818243 (2015).</li></ol>

有几个关键步骤, 你应该照顾, 同时执行此协议与鼠标卵母细胞以及其他物种。一旦从卵泡中分离出来, 卵母细胞就应该立即转移到记录滴中, 因为与伴卵丘细胞的分离触发了对产业转移的开始。对本议定书可能的修改可能是将 3-异丁基-1-甲基黄嘌呤 (IBMX) 添加到用于 COCs 隔离的 M2 介质中。IBMX 防止了即时触发的问-产业转移, 并允许同步的整个实验组的卵母细胞。
在我们的实验?...

女性不孕症是一种影响越来越多的妇女的病理学。根据世界卫生组织的数据, 大约20% 的夫妇是不孕, 40% 是由于女性不孕。此外, 在美国或意大利接受癌症治疗的妇女中, 有三人 (分别为30万年和3万岁) 发育过早卵巢衰竭。
预防癌症患者不孕症的策略是在肿瘤治疗前, 卵巢卵泡的分离和冷冻保存, 其次是 "体外" 成熟 (病媒) 在细胞产业部的阶段 (即对产业部的转变)。无创性的卵母细胞发育能力标志物的提供将改善受精和发育过程, 以及整个妊娠成功1,2。
根据 supravital fluorochrome 赫斯特33342的染色后观察到的染色质构型, 哺乳动物完全生长的卵母细胞被归类为被包围的核仁 (SN) 或未被包围的核仁 (诺基亚)3。除了不同的染色质组织, 这两种类型的卵母细胞显示许多形态学和功能差异3,4,5,6,7,8 ,9, 包括它们的减数分裂和发展能力。当从子房分离并成熟的体外时, 这两种卵母细胞都达到了产业部的阶段, 在精子受精后, 发展到2细胞阶段, 但只有有 SN 染色质组织的人才能发展到足月 9。虽然作为选择称职的vs不称职卵母细胞的分类方法是好的, 但主要的缺点是 fluorochrome 本身的诱变效应, 最重要的是用于其检测的紫外线光可能存在于细胞上。
由于所有这些原因, 我们寻找其他与 SN 或诺基亚的染色质构象相关的非侵入性标记, 可以替代赫斯特的使用, 同时保持相同的高分类精度。胞质运动速度 (CMVs) 的时间推移观察是细胞状态的一个显著特征。例如, 最近的研究表明, 在受精时记录的 CMVs 与小鼠和人类受精卵完成植入前和足月发育的能力之间的联系10,11。
基于这些早期的研究, 我们这里描述一个平台, 以识别发育能力或不称职的老鼠完全生长的卵母细胞5,6,7,8。该平台基于三主要步骤: 1) 从窦卵泡分离出的卵母细胞首先根据其染色质构型分类, 无论是被包围的核仁 (SN) 或未被包围的核仁 (诺基亚);2) 采用粒子图像测速仪 (PIV) 对各单卵母细胞的 CMVs 时间推移图像进行分析;3) 通过对盲分类12、13的前馈人工神经网络 (文芳), 分析了 PIV 获得的数据。我们详细介绍了为鼠标准备的程序中最关键的步骤, 使其可供测试并用于其他哺乳动物物种 (例如,牛、猴和人类)。

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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Folligon</td>
			<td style="width:239px;">Intervet</td>
			<td style="width:157px;">A201A02</td>
			<td style="width:499px;">Hormonal treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Hoechst 33342&#160;</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">B2261</td>
			<td>For oocyte heterochromatin staining</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:363px;">Cell culture Petri-dish 35 mm x 10 mm&#160;</td>
			<td style="width:239px;">Corning&#160;</td>
			<td style="width:157px;">430165</td>
			<td>For COCs isolation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">EmbryoMax M2 Medium (1X), Liquid, with phenol red</td>
			<td style="width:239px;">Merck-Millipore</td>
			<td style="width:157px;">MR-015-D</td>
			<td>For COCs isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Alpha medium (1X) + Glutamax&#160;</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">M4526</td>
			<td>For oocyte in vitro maturation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture Petri-dish 35 mm glass-bottom&#160;</td>
			<td style="width:239px;">WillCo&#160;</td>
			<td style="width:157px;">GWSt-3522</td>
			<td>For imaging experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">BioStation IM-LM&#160;</td>
			<td style="width:239px;">Nikon</td>
			<td style="width:157px;">MFA91001</td>
			<td>Live cell screening system&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Pasteur pipette</td>
			<td style="width:239px;">Delchimica Scientific Glassware</td>
			<td>6709230</td>
			<td>For follicles manipulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Mineral oil</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">M8410</td>
			<td>To prevent contamination and medium evaporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Penicillin / Streptomycin</td>
			<td style="width:239px;">Life Technologies</td>
			<td style="width:157px;">15070063</td>
			<td>To prevent medium contamination&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">ML16141079</td>
			<td>For making up &#945;MEM medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine&#160;</td>
			<td style="width:239px;">Life Technologies</td>
			<td style="width:157px;">25030</td>
			<td>For making up &#945;MEM medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">T0625</td>
			<td>For making up &#945;MEM medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">A3310</td>
			<td>For making up &#945;MEM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyruvate&#160;</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">&#160;P4562</td>
			<td>For making up &#945;MEM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zoletil (Tiletamina and Zolazepan cloridrate)</td>
			<td style="width:239px;">Virbac Srl</td>
			<td style="width:157px;">QN01AX9</td>
			<td>For mice anesthesia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:363px;">Cell_PIV sofware&#160;</td>
			<td style="width:239px;">Kindly provided by Dr. Shane Windsor, University of Bristol, UK</td>
			<td style="width:157px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;-</td>
			<td style="width:499px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;-</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MATLAB</td>
			<td style="width:239px;">The MathWorks, Natick, MA&#160;</td>
			<td style="width:157px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;-</td>
			<td>For multi-paradigm numerical computing</td>
		</tr>
	</tbody>
</table>

a neural network-based identification of developmentally competent or incompetent mouse fully-grown oocytes

不孕诊所将受益于选择发育能力的vs不称职的卵母细胞使用非侵入性程序, 从而改善整体妊娠结局。我们最近开发了一种基于显微活体观察的小鼠卵母细胞在他们的体外成熟从胚泡 (问) 到中期 II 阶段, 然后分析的细胞质运动在这段时间内发生。在这里, 我们提出了这个程序的详细协议。卵母细胞从完全生长的窦卵泡中分离出来, 在15小时内培养为37摄氏度和 5% CO2的时间失效分析显微镜。图片以8分钟的间隔拍摄。利用粒子图像测速 (PIV) 方法对图像进行分析, 计算每个卵母细胞在整个培养期内的细胞质运动速度 (CMVs) 剖面。最后, 通过一个数学分类工具 (前馈人工神经网络, 文芳) 来喂养每个卵母细胞的 CMVs, 预测配子发育能力或不称职的概率, 精度为91.03%。该协议, 为鼠标设置, 现在可以测试其他物种的卵母细胞, 包括人类。

图 2显示了一个代表性的发育能力和不称职的卵母细胞, 分别在开始 (在) 和在病媒治疗过程的末尾 (产业部)。全生长小鼠卵母细胞的病媒在 15 h 培养期间发生。时间推移观察记录减数分裂的进展, 检测主要的减数分裂事件, 包括 GVBD 和第一极性体的挤压。
200多名发育能力的vs不称职的卵母细胞的分析...

Watch this Scientific Journal Video about A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes at JoVE.com

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

在这里, 我们提出了一个非侵入性评估的卵母细胞发育能力在他们的体外成熟从生发泡到中期 II 阶段。该方法将时间推移成像与粒子图像测速 (PIV) 和神经网络分析相结合。

基于神经网络的发育能力或不称职小鼠全生长卵母细胞的识别

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Research

JoVE Journal

Developmental Biology

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

科研

发育生物学

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

基于核仁染色质组织分离鼠标窦卵母细胞与表征

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

分离和成人犬海马神经前体扩张

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

通过显微注射卵母细胞生成转基因小鼠

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

小鼠原发性颅神经冠细胞的解剖、培养与分析，用于神经冠细胞脱角和迁移研究

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

用于微CT成像的成人和早期产后小鼠肺的血管铸造

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...