我们感谢MR Helmus的画外音，CM Popelier，S. Ypenburg和C Levesque的技术援助。这项研究是由荷兰科学研究组织（NWO），艺术与科学的荷兰皇家科学院（KNAW）和日本学生支援机构（JASSO）的财政支持。本出版物是由苏国的​​开放获取授予JMK支持。

1，养殖设施<ol><li>从实验室培养像一个在VU大学获得椎实螺stagnalis（L.）成虫标本。 </li><li>同时在养殖设施（molluscarium）和实验坦克，其中有连续的水体交换维持低铜水在20°C。 </li><li>将光/暗周期为12小时:12小时。 </li><li>对于养殖，饲料成年蜗牛无农药生菜自由采食 ，喂年轻的少年与TetraPhyll（鱼食）片。根据自己的年龄保持在单独的坦克蜗牛，从卵块这是一个24小时的时间内开始铺设。 </li><li>一旦蜗牛是从他们没有回来，以避免影响繁殖缸中删除已保存在稍有不同的条件以及防止pseudoreplication。 </li></ol> 2，解剖<ol><li>隔离蜗牛1周，并喂它们自由采食 ，以确保THA吨，他们有充分的精子和精液店。 </li><li>准备一个立体显微镜，解剖板管脚，小型手术剪，粗，细镊子，一个10毫升注射器装满一个50毫米MgCl 2溶液，注射针头（0.3毫米x 13毫米）和一些纸巾。 </li><li>通过注入氯化镁溶液（一般为3毫升以上的成年蜗牛31.6±2.1毫米，湿重为2.89±0.55 g的壳长）安乐死蜗牛。穿透脚与注射针呈45°角，轻轻施加压力持续，直到蜗牛放松并保持扩展（几秒钟内）注入。取出注射针，并检查蜗牛是否被安乐死（例如，通过检查触手撤回反射是否不存在）。 </li><li>用粗镊子以下曲率小心地取下外壳。半路，轻轻地从外壳用钳刮分离鼻小柱肌肉。轻轻扭动吨他蜗牛出它的外壳，继壳的卷绕方向。 </li><li>将蜗牛的解剖板和牵制脚的后端。然后，将针穿过头部，并把一些紧张的蜗牛，以方便清扫。 </li><li>本地化女生殖孔，这将稍后被解剖的参考点。 </li><li>用手术剪刀和细镊子，使第一个切口，刚下地幔的边缘和切向女生殖孔。当剪刀&#39;叶片中的一个下主体壁推压，并将其抬起一些，以确保只有体壁，并且没有内部器官的，被切断。继续切割朝向头部生殖孔上面和切割在一条直线上。 </li><li>本地化前列腺（后）和精囊（前）。仔细剖析这些出来，让他们在冰上。使用这些使含有精液和/或精子的测试解决方案。用生理椎实螺萨尔国家统计局（5.83 mmol / L的氯化钙2·2H 2 O，3.76 mmol / L的氯化镁2·6H 2 O，42.69毫摩尔/ L的NaCl，37.53 mmol / L的氯化钾）作为载体介质。 </li><li>收集精液是存在于前列腺的内腔，以及从精囊的精子。随后，通过轻轻地表达自己的内容，并用钳子取出器官组织在盐水中单独的小瓶暂停两者。保持在冰上，并使用相同的日子。后来，这些混合悬浮液以创建不同的治疗方案。 </li></ol> 3，阴道内注射<ol><li>开始所述阴道注射过程之前，准备一个硅壳模（由以适应外壳的前端部），1毫米注射器，10-12厘米的硅管，其直径为1毫米，钝注射针头（0.3毫米×13毫米），粗镊子和相同的10毫升注射器填充有50 mM的MgCl 2溶液配有一个尖锐的注射针。 </li><li>收集精子，需要增加在一些治疗精液（或个人精液的蛋白质）;生物相关的剂量等于前列腺和每次注射精囊的1/3。 注:在有代表性的结果部分（ 图1）的实验有三个不同的处理结果显示:对照（载体介质， 即生理盐水），Ovipostatin（已确定精液的蛋白质之一）单独Ovipostatin伴有精子。此外，估算生物相关剂量在其他物种，确定在重量那些最近交配和个人保持性隔离的16个个体的前列腺的差异。 </li><li>准备1毫升注射器每个准备测试解决方案。填写各注射器有一个解决方案，并确保不存在任何内部气泡。 </li><li>适合钝注射针头上的每个注射器。 </li><li>注射用1毫米直径的硅管与10cm的最小长度。 </li><li>小心地将硅管在注射针，确保不要刺破管，并轻轻地应用在注射器的压力从管取下空气。 </li><li>按照相同的协议在2.3点麻醉蜗牛。这一次，确保不注入超过2-3毫升50毫米MgCl 2。 </li><li>莱蜗牛的壳模使其保持在一个稳定的位置。 </li><li>定位雌生殖孔，这显然是由于在右侧的动物，前向右侧触手和雄性生殖孔的白点可见（此位置也适用于其它物种淡水蜗牛）。确保有畅通无阻的访问。 </li><li>使用镊子来支持硅管的端部，并轻轻插入约2-4毫米的硅管的入阴生殖孔。 </li><li>仔细施加压力注射器和注射0.03毫升供试品溶液。等待半分尤特以允许在管中的压力传播到阴道除去管之前。 </li><li>返回处理后的蜗牛其隔离容器，在那里它可以被监控，并允许它从镇静恢复在实验罐中。 </li></ol> 4，生物测定<ol><li>标记每个蜗牛识别并测量其壳长。是后者的大小，量化繁殖输出时，这是相关的一个良好指标。 </li><li>保持各蜗牛孤立在一个460毫升容器被穿孔，以允许为实验罐中的水交换。它把所有的容器在正确的方向，在水流的方向上穿孔，以允许高效的水交换是很重要的。 </li><li>在生物测定，定期喂养蜗牛生菜的标准化量。使用一个圆形，金属冲床切生菜。在这个协议中19.6 平方厘米生菜盘被使用，这近似于一个挂载它们可以最大限度地在一天内吃。 </li><li>使用刮刀的扁平侧收集卵块，并从容器壁刮的卵块。用勺子边收集卵块。 注:不透明的卵块刚刚被规定不能用于扫描（它们成为铺设后数小时内透明）。对于该模型的物种在这个协议中， 椎实螺stagnalis，检查卵块的每一天，因为它们产生2-3个卵块每星期。这可以在其他物种不同。 </li><li>扫描收集到的卵块数字，使用（笔记本）电脑含扫描仪驱动程序，平板扫描仪，一个标准的毫米级，锅铲，传输板和玻璃砖。 </li><li>将每个卵块上的传输板和重复，直到这些板块都满了。随后，轻拍卵块干的纸巾和他们在相同的顺序在传输板转印到玻璃砖。 </li><li>打开每瓦倒挂（在卵块会粘在玻璃瓦），并小心将其放置在平板扫描仪上，只需根据毫米刻度。适用的压力位，扁平化的卵块，这将分发鸡蛋均匀，使他们都是一样的聚焦平面内可见。适合用胶带缠绕两条边的标准化量的瓷砖，以确保所有的鸡蛋都同样夷为平地。 </li><li>调整扫描仪设置。重要的是要调整分辨率，其最大，做一个预览扫描，裁剪工作区，并调整亮度和对比度是非常重要的。扫描的卵块。 </li><li>将每个卵块在其自己的标记20ml玻璃容器的进一步发展和孵化。 </li><li>刷新在水中孵化小瓶隔日以保持其正常充氧。根据幼体数量和大小按以下顺序执行此操作:滗/浇灭（无幼体），溢出（孵化开始），并提取出用移液管（很多小海龟）的水。 </li></ol> &lt;P类="j​​ove_title"&gt; 5。测量和计数蛋<ol><li>安装可免费获得的图像分析软件ImageJ的（美国国立卫生研究院，美国）和细胞计数插件。 </li><li>打开ImageJ的还有一个电子表格程序将数据传输到。然后在ImageJ的打开卵块的扫描图片。 </li><li>通过使用缩放工具导航到毫米级的设置比例。放大和调整窗口1毫米到填满屏幕。使用绘制两条线，表明1毫米之间直线工具一行。导航到分析，并设置比例。在弹出菜单中测得的长度选项变为可见，调整已知的距离为1，单位长度为mm。选中该复选框全球，并关闭窗口。 </li><li>通过导航到分析设置首选的测量（长度，宽度，周长），并通过选择区域和费雷特直径（长度和宽度）设置测量。单击确定以确认并继续与图像分析。 </li><li>要测量蛋，ZOOM在第一个卵块，需要加以衡量的鸡蛋。建议的800％变焦。然后，使用椭圆工具绘制一个椭圆正是围绕着鸡蛋，按CTRL + D来绘制图像的周长（以防止测量同一鸡蛋的两倍）。然后按Ctrl + M来记录椭圆形（长，宽，周长）的性质的措施;一个弹出式屏幕打开，显示测量结果。 注意:使用椭圆工具来衡量鸡蛋并不适用于所有物种。物种与非均匀形鸡蛋，用写意的选择来代替。 </li><li>衡量尽可能多的鸡蛋，然后采取的措施另存为。XLS工作表。可选地，数据直接复制到工作表。 </li><li>随后算上蛋卵块内，使用柜台窗口，并把人工计数到工作表。 </li><li>另外，算上每卵块个别鸡蛋与细胞计数器插件的帮助。为此，导航到插件，然后，分析和选择单元格计数器。细胞计数窗口会弹出，选中"保持原有"框，然后按"初始化"。出现一个新的柜台窗口，从工具栏中选择十字线工具，并在细胞计数菜单中选择计数器类型。通过点击单个鸡蛋在细胞计数器计数窗个别鸡蛋。观察细胞计数菜单旁边的计数器类型的总数。手动复制这个数字到电子表格或按业绩获得的结果。 </li><li>通过添加计数器类型，如果需要计数多个卵块在一个计数事件。 </li></ol>

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作者什么都没有透露。

所提出的方案显示了如何收集精液，以及如何在生物学上有意义的方式来测试这一点。虽然在这里该程序使用的前列腺提取物被示出，一个单一纯化的精液蛋白的效果，也可以按照相同的程序进行测试。显然，在这样的测试中，它始终是至关重要的，以包括适当的对照组（假手术处理），以确保该程序和/或所述载体介质不会影响问题14,16的参数。使用这种方法时，它已经被证明是有一个单一的精液蛋白，即Ovipostatin，其介导在产蛋14,16的减少。这种蛋白质，产生在前列腺和沿与精子交配期间传输，表示在一个同步雌雄同体第一充分表征精液成分和不，到目前为止，没有发现任何的相似已知的蛋白质。 这一发现，Oviposta锡降低产蛋量产收件人证实了早期的工作表明，在精液中的物质可能会影响产蛋13-15。通过将生物相关的量（见3.2 16），一个可以令人信服地表明，它是一个精液蛋白，并没有精子的存在本身，其介导所观察到的减少产卵（ 图1）。考虑到通过自然交配多次收到精液每增加15蛋的投资，它现在变得高度相关的量化产蛋的其它参数。所提出的方案现在提供了必要的方法来这样做。虽然没有影响，发现Ovipostatin对孵化由收件人16下的蛋的成功，有可能是一致行动与该蛋白的其他精液蛋白。因此，需要进行量化在这方面的参数 - 旁铺设频率和产蛋数量 - 包括鸡蛋大小，HATC兴时，幼体发育和幼体大小。另外，也可以看看其他女性的进程比产蛋，如精子储存和使用情况，以及生殖资源对女性和男性功能14,17的分配效应。显然，该方法是有助于解决这些后续问题，无论是在椎实螺stagnalis和其他淡水螺。 总之，使用该协议显示实验，转精液的蛋白质会影响收件人在这些同时雌雄同体的女性生殖性能。由于很少有研究涉及这一点，它很可能是多在内部施肥雌雄同体比目前反映在科学文献中更常见。特别是当人们意识到预交配择偶和精子竞争已经显示出在这些雌雄同体14，发挥了重要作用的highlighTS以在该系统中通过性选择成雄性附腺的蛋白质的潜力。最后，本研究有助于支持显影概念，精液蛋白是用于同时雌雄同体同等的重要性，因为它们是独立的，性别鉴定物种。

转让时，精子通常伴随着由雄性附腺产生的精液。虽然一些精液成分的滋养和激活精子1发挥作用，影响其他女性生理为了增加精子捐赠者2-4受精成功。尤其是选择了这样的效果的，通过性选择，当一个物种是高度混杂和有效率的储存精子和精子消化。在这种情况下，精子捐赠者的卵子5的施肥严重竞争。虽然有丰富的相关性证据的精液物质对男性受精成功的重要性，直接的证据是比较罕见的，很少有研究钻研的生理变化和/或参与6种物质的详细信息。 在物种雌雄分开（ 即男性和女性; gonochorists）有一些良好的研究模型SPECI当涉及到精液成分和它们的影响上课。例子包括果蝇， 果蝇6，疟蚊， 冈比亚按蚊 7，赤拟谷盗， 赤拟谷盗 8。例如精液的研究更是少之又少，当涉及到那些同时雌雄同体（ 即个人在功能上是男性和女性的同时），即使复制这种模式是常见的整个动物王国9种。一个附属性腺物质转移的最显着的雌雄同体的例子可能就是所谓的爱飞镖土地蜗牛2,10，该物质是由捐精者通过皮下注射11,12， 即 ，不转移，但拍摄随着精子。但也有转移雄性附腺的产品以及它们的精子许多同时雌雄同体，如这里所使用的模型种类，伟大的池塘蜗牛椎实螺stagnalis。以前的研究已经证明，收据精液的影响该物种的13-15的女性生殖输出和几个参与精液的蛋白质已被确定16,17。 这个协议，这是报告中KOENE 等[16]的方法扩展的总体目标，是检验精液蛋白对产蛋的腹足软体动物各种参数的影响。这是由前列腺（ 即男性附属性腺产生的精液），从一个标准化的实验室培养分离出的性捐助蜗牛的解剖完成。二，捐赠者的精子来自精囊腺收集和不同的治疗方案而作出调整。随后，测试蜗牛麻醉和阴道内注射的试验溶液（例如，单独前列腺提取物，前列腺提取物与精子，精子单独，独自载体介质）。这些测试蜗牛，然后在接下来的几天监测到数周，在标准条件下，记录产卵。卵块收集，数字扫描计数和测量各种参数，然后放在小瓶单独孵化。大约两个星期后，幼体进行计数和数字扫描。所有的扫描无论卵块和幼体，使用可免费提供的图像分析软件包，然后进行分析。 对于这个协议的伟大池塘蜗牛， 椎实螺stagnalis，实验室菌株使用，这是孕育在VU大学在阿姆斯特丹。本种作为调查有关性选择和雌雄同体再现问题的模型系统。动物的比较大的尺寸使得它的实验非常方便。此外，因为它被用作模型物种在许多biological学科我们已经知道了很多关于这些动物的基本生物学。这种方法能帮助研究关于性选择那些一般通过精液蛋白介导的过程问题。此外，因为这些都是同步雌雄同体，有可能解决这样的蛋白质是否在相似和/或不同的方式行事，因为他们在那里的两性分开16,17物种。

<table><tbody><tr><td>Fish food flakes</td><td>TetraPhyll</td><td></td><td></td></tr><tr><td>Stereo microscope</td><td></td><td></td><td></td></tr><tr><td>Small surgical scissors</td><td>WPI</td><td></td><td></td></tr><tr><td>Coarse and fine forceps</td><td>WPI</td><td></td><td></td></tr><tr><td>10 ml syringe</td><td></td><td></td><td></td></tr><tr><td>Injection needles (0.3 mm x 13 mm)</td><td></td><td></td><td></td></tr><tr><td>50 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>5.83 mmol/L CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>3.76 mmol/L MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>42.69 mmol/L NaCl</td><td></td><td></td><td></td></tr><tr><td>37.53 mmol/L KCl</td><td></td><td></td><td></td></tr><tr><td>Two component Silicon</td><td>Sylgard</td><td></td><td></td></tr><tr><td>1 mm syringes</td><td></td><td></td><td></td></tr><tr><td>Silicon tubing (diameter 1 mm)</td><td></td><td></td><td></td></tr><tr><td>Blunted injection needles (0.3 mm x 13 mm)</td><td></td><td></td><td></td></tr><tr><td>Tubes</td><td>Eppendorff</td><td></td><td></td></tr><tr><td>460 ml perforated containers</td><td></td><td></td><td></td></tr><tr><td>Metal leaf puncher (19.6 cm&lt;sup&gt;2&lt;/sup&gt;)</td><td></td><td></td><td></td></tr><tr><td>Spatula/spoon</td><td></td><td></td><td></td></tr><tr><td>(Laptop) computer</td><td></td><td></td><td></td></tr><tr><td>Flatbed scanner</td><td></td><td></td><td></td></tr><tr><td>Millimeter scale</td><td></td><td></td><td></td></tr><tr><td>Transferring plates</td><td></td><td></td><td></td></tr><tr><td>Glass tiles</td><td></td><td></td><td></td></tr><tr><td>20 ml glass vials</td><td></td><td></td><td></td></tr></tbody></table>

effect of male accessory gland products on egg laying in gastropod molluscs

在内部施肥动物，精液通常添加到精子，共同形成了精液或精液。除了滋养和激活精子，在精液的成分也能影响女性生理，以增加捐赠者的精子受精成功。虽然许多研究报告中的物种有独立的男女这样的效果，很少有研究的同时雌雄同体的动物，解决了这个。这个视频协议提出来研究腹精液效果的方法，用一个雌雄同体的同时淡水螺，大池塘蜗牛椎实螺stagnalis，作为模式生物。而该过程是使用完整的前列腺提取物所示，各个部件的精液（ 即 ，蛋白质，肽，和其它化合物）可以以相同的方式进行测试。收据上产蛋精液成分的影响可以量化的产蛋频率方面和女性生殖性能，如每个卵块中卵的数字更微妙的估计。结果表明，精液中的蛋白质会影响在此同时雌雄同体雌性生殖输出，突出性选择的重要性。

图1示出的动物的阴道内注射或控制物质（盐水）后产卵的百分比，Ovipostatin，或Ovipostatin伴有精子。每组15对每个处理;看KOENE 等人 16的详细信息和统计数据（资料来源: 图3从KOENE 等人 ，2010年，PLoS ONE的）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51698/51698fig1highres.jpg" width="500" /> 图1。Ovipostatin对蛋批量生产椎实螺stagnalis的。该图显示了动物的阴道内注射或对照物质（盐水）后产卵的百分比，Ovipostatin，或Ovipostatin伴有精子。每组15对每个处理;看KOENE 等 2010的详细信息和统计信息。转载自KOENE 等人的许可，2010年PLoS ONE的5:E10117。

Watch this Scientific Journal Video about Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs at JoVE.com

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

男性附属性腺产品对腹足软体动物下蛋的影响

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and ...

effect-male-accessory-gland-products-on-egg-laying-gastropod

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.6K 次观看.  VU University. This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

视频: Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

一种非侵入性的方法<em&gt;原位</em&gt;交配成功的女美国龙虾​​（测定<em&gt;美洲螯龙虾</em&gt;）

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

科研

环境科学

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

生物学

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

This article reports a simple method to isolate the outer and inner perivitelline sublayers of chicken eggs while minimizing structural alteration and to optimize protein solubilization of each sublayer for proteomic analyses.

来自母鸡蛋的外层和内围线子层的机械分离和蛋白质溶解

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. ...

生物化学

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgf&#223; family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgf&#223; receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgf&#223; precursor proteins. RNA encoding Tgf&#223; precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of&#160;gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgf&#223; cleavage products under physiologic conditions.

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

When Transforming Growth Factor &#223; family precursor proteins are ectopically expressed in Xenopus laevis embryos, they dimerize, get cleaved and are secreted into the blastocoele, which begins at the late blastula to early gastrula stage. We describe a method for aspirating cleavage products from the blastocoele cavity for immunoblot analysis.

转变生长因子的分析 - 家族产品分泌到 异种胚胎 的爆炸

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, ...

发育生物学

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

高通量检测，以检查个体产蛋首<em&gt;果蝇</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

神经科学

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

两种类型的检测，检测青蛙精子Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

整个山<em&gt;原位</em&gt;对腹足软体动物杂交方法<em&gt;静水椎实螺</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater mussels are among the most imperiled organisms in the world, but information about early larval (glochidial) development and brooding periods is still lacking for many species. Previous studies have focused on the complex life history stage when female mussels are ready to parasitize host fish, but few studies have focused on the brooding period and timing of larval development. The protocol described here allows researchers to non-lethally evaluate the state of gravidity for female mussels. The results of this study show that this method does not affect a female mussel&rsquo;s ability to stay gravid or become gravid again after sampling has been performed. The advantage of this method may permit its use on federally threatened or endangered species or other populations of high conservation concern. This protocol can be adapted for use on both preserved or live individuals and was tested on a variety of mussel species. The database provided is a repository for a breadth of information on timing of reproductive habits and will facilitate future freshwater mussel research, conservation, and recovery efforts.

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels Bivalvia: Unionida

Freshwater mussel conservation is dependent on monitoring reproductive patterns and processes of species. This study standardizes a non-lethal protocol for sampling gill contents, characterizing larval development, and providing a digital repository for data collected. This protocol-database package will be an important tool for mussel researchers in recovery of imperiled species.

标准化一种非致命方法，以描述淡水贝类的生殖状况和幼虫发育（比瓦尔维亚:联合达）

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater ...

Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs

Mud crabs (Scylla spp.) are commercially important crustacean species that can be found throughout the Indo-West Pacific region. During culture, the induction of ovarian maturation is important to meet the consumer demand for mature mud crabs and hasten seed production. Eyestalk ablation is an effective tool to enhance ovarian maturation in mud crabs. However, there is no standard protocol for the eyestalk ablation of mud crabs. In this study, two eyestalk ablation techniques are described: cauterization (the use of hot metal to ablate the eyestalk of an anesthetized crab) and surgery (the removal of the eyestalk using surgical scissors). Before eyestalk ablation, sexually mature females (CW &#62; 86 mm) were anesthetized using an ice bag (&#8722;20 &#176;C) with seawater. When the water temperature reached 4 &#176;C, the ice bag was removed from the water. Flowing seawater (ambient temperature: 28 &#176;C) was used for recovery from the anesthesia immediately after eyestalk ablation. Mortality did not occur during or after the process of eyestalk ablation. The eyestalk ablation protocol presented here accelerated the ovarian maturation of the mud crabs.

Two eyestalk ablation protocols (i.e., cauterization and surgery&#160;approaches) were performed on anesthetized female crabs. The eyestalk ablation of mud crabs hastened the ripening of ovaries without decreasing the survival rate.

眼柄消融术可增加泥蟹卵巢成熟

Mud crabs (Scylla spp.) are commercially important crustacean species that can be found throughout the Indo-West Pacific region. During culture, the ...

Drosophila Male Accessory Gland Dissection: A Method to Isolate Secondary Cells

Source: Immarigeon, C., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/60218/" target="_blank">A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands</a>. J. Vis. Exp. (2019).
This video describes the dissection of the accessory glands from male flies and shows an example in which the isolated gland will be dissociated into a single cell suspension.

果蝇男性副腺解剖:一种分离次级细胞的方法

Source: Immarigeon, C., et al. A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands. J. Vis. Exp. (2019).
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男性附属性腺产品对腹足软体动物下蛋的影响

摘要

探索更多视频

此视频中的章节

一种非侵入性的方法

从非洲爪蟾女性获得卵子

来自母鸡蛋的外层和内围线子层的机械分离和蛋白质溶解

转变生长因子的分析 - 家族产品分泌到异种胚胎的爆炸

高通量检测，以检查个体产蛋首

两种类型的检测，检测青蛙精子Chemoattraction

整个山

标准化一种非致命方法，以描述淡水贝类的生殖状况和幼虫发育（比瓦尔维亚:联合达）

眼柄消融术可增加泥蟹卵巢成熟

果蝇男性副腺解剖:一种分离次级细胞的方法

男性附属性腺产品对腹足软体动物下蛋的影响

摘要

探索更多视频

此视频中的章节

一种非侵入性的方法

从非洲爪蟾女性获得卵子

来自母鸡蛋的外层和内围线子层的机械分离和蛋白质溶解

转变生长因子的分析 - 家族产品分泌到 异种胚胎 的爆炸

高通量检测，以检查个体产蛋首

两种类型的检测，检测青蛙精子Chemoattraction

整个山

标准化一种非致命方法，以描述淡水贝类的生殖状况和幼虫发育（比瓦尔维亚:联合达）

眼柄消融术可增加泥蟹卵巢成熟

果蝇男性副腺解剖:一种分离次级细胞的方法

转变生长因子的分析 - 家族产品分泌到异种胚胎的爆炸