这项工作得到了兽医学院 (立) 启动基金对 WW 的支持。作者感谢塞拉奥尔森 (立) 对这篇手稿的批判性阅读。

本研究中使用的所有动物和程序都是根据华盛顿州立大学 (立) 动物保育和使用委员会的指导方针, 并遵照立批准的动物议定书 #4702 和 #4735 处理的。
1. 小鼠
<ol><li>
使用标准 C57BL/6J 成年雄性小鼠或其他有兴趣的品系。在温度和湿度控制的环境下维护动物, 使用ad 随意获取水和食物。
	<ol>
<li>使用雄性饲养者, 从8周大到10月大不等。在实验中只使用经过验证的育种者。</li>
	</ol>
</li>
	<li>
使用具有组织选择性删除生殖道雌激素受体α (由Esr1基因编码) 的成年雌性小鼠在上皮细胞中10 (Wnt7a.在本研究中, Esr1f/f, 称为挖空或 "KO") 和控制窝 (Esr1f/f, 称为 "CTRL")。
	<ol>
<li>使用8周大的女性。女性的年龄根据兴趣实验而变化。</li>
	</ol>
</li>
	<li>确定在早上08:00h 的动情周期的阶段。只在周期的发情阶段使用雌性。有关阴道涂抹和细胞学分析的详细方法, 请参阅以前发布的过程11。</li>
</ol>2. 交配和评估交配插头
<ol><li>在发情的那天 (16:00h), 把雌性放在雄性笼子里。</li>
	<li>第二天早上 (08:00h), 把雌鼠和雄性老鼠分开。</li>
	<li>使用以前发布的方法12的改编来访问阴道或交配插头的存在或缺失作为交配的指示。</li>
	<li>将母鼠从尾部的底座中抱起, 以短暂地抬起后肢。</li>
	<li>找到交配插头的存在, 一个白色的精物在阴道开口使用牙签。如果交配插头存在, 牙签将无法插入到阴道管。</li>
</ol>3. 组织收集前的准备
<ol><li>准备10毫升的莱博维茨的 L-15 培养基, 辅以1% 胎牛血清 (称为 L15 培养基 + 1% FBS) 在15毫升管。在37° c 的培养基上孵育15分钟的水。</li>
	<li>为了保持组织和精子在下游应用的可行性, 分2毫升的 5ml L15 培养基 + 1% FBS 在2毫升离心管组织收集。</li>
	<li>温暖的阶段显微镜到37° c。将热块设置为37° c, 并在块中放置一个空的 1.5 mL 离心管。</li>
	<li>如果下游应用需要成像的精子运动, 温暖的玻璃幻灯片到37° c。</li>
</ol>4. 组织收集
<ol><li>带 5ml L15 媒体 + 1% FBS 分到组织收集区。</li>
	<li>安乐女性 (~ 08:30h) 使用二氧化碳窒息, 其次是颈椎脱位。 注意: 这是大约 8 h 在交配之后, 假设交配发生在午夜。</li>
	<li>将动物置于仰卧位以暴露腹部区域。</li>
	<li>在后肢附近的腹部喷洒70% 酒精。</li>
	<li>使用解剖剪刀在下腹部的皮肤上做一个小切口 (~ 1 厘米)。</li>
	<li>使用占优势的手拉尾巴 (和后肢, 如果有必要) 而非手拉皮肤相反的方向 (向头部) 暴露腹部肌肉。这种方法是用来减少头发污染的内脏器官的数量。</li>
	<li>将腹部底部的 V 型肌肉切开, 靠近阴道, 露出内脏器官。</li>
	<li>将肠道和腹部脂肪移到侧面以暴露生殖道。</li>
	<li>在阴道区域上方修剪膀胱和其他组织。</li>
	<li>切开耻骨联合以获得阴道组织。</li>
	<li>在阴道开口处提起阴道组织, 用剪刀将阴道导管从直肠组织中分离出来。</li>
	<li>用弹簧剪刀仔细修剪子宫两侧的肠系膜脂肪。 注意: 子宫很脆弱, 里面有精子。注意不要刺穿子宫, 否则精子会丢失。</li>
	<li>抬起较低的生殖道, 并切断两侧的卵巢脂肪垫。</li>
	<li>将整个生殖道转移到5ml 莱博维茨的 L-15 培养基 + 1% FBS。</li>
</ol>5. 从子宫含量采集中测定精子液化 (黏度)
<ol><li>在显微镜的加热阶段, 将组织转化为35毫米无菌组织培养皿, 填充3.5 毫升 5ml L-15 培养基 + 1% FBS, 以清洁血液和其他污染物的组织。 注意: 清洗时, 用脂肪, 而不是子宫来抓组织。此时, 精子有望在子宫内完好无损。</li>
	<li>使用实验室抹布涂抹过量介质的组织。</li>
	<li>在阴道末端放置组织, 同时将卵巢末端置于预热离心管中。在保持阴道末端的同时, 切下子宫底部的子宫角, 允许子宫角落入离心管。</li>
	<li>让精子从子宫角流出管。用无牙钳将子宫角从卵巢末端轻轻挤压到阴道末端, 排出残余的精子。</li>
	<li>丢弃生物危害袋中的组织并焚烧。</li>
	<li>简单地在 minicentrifuge (1-2 秒) 内将精子向下旋转。</li>
	<li>放置管, 平行于加热阶段-精子不会溢出。</li>
	<li>准备好计时器并设置为 "计数"。</li>
	<li>在一个180°角 (平行于管壁) 上插入一个25µL 毛细管进入精子样品。这是为了尽量减少各调查人员之间的差异, 从不同角度持有毛细血管管。</li>
	<li>当毛细管与精子接触时, 立即按下定时器。</li>
	<li>记录精子到达25µL 线所需的时间。</li>
	<li>当测量完成后, 立即把含有精子的管子放回干热块中进行进一步的功能分析, 如对精子运动的视频成像。</li>
	<li>在10% 漂白剂溶液中浸泡20分钟, 并将毛细管放在锋利的容器中, 对含有精子的毛细管进行消毒。</li>
</ol>6. 精子运动的影像学
<ol><li>打开显微镜和视频成像软件。</li>
	<li>吸管20µL 的剩余的精子的5ml 玻璃幻灯片, 而玻璃幻灯片被置于加热阶段。</li>
	<li>盖上玻璃片。</li>
	<li>将滑动片侧放在显微镜下。</li>
	<li>使用20X 物镜来查找感兴趣的区域。</li>
	<li>更改为视频成像的100X 物镜。</li>
	<li>录制的视频在12帧/秒 (fps) 为三十年代。</li>
	<li>随机记录每个动物至少3不同区域的视频。</li>
	<li>快速进行成像, 并尽量减少光线照射到幻灯片, 以保持精子的活力和活力。 注: 此成像程序应在2-3 分钟内完成。</li>
	<li>使用 ImageJ 软件执行数据分析, 如前所述8。</li>
</ol>

<ol>
	<li>Suarez, S. S., Pacey, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+transport+in+the+female+reproductive+tract.">Sperm transport in the female reproductive tract.</a> Hum Reprod Update. 12 (1), 23-37 (2006).</li><li>Bedford, J. M., Yanagimachi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+sperm+motility+after+mating+in+the+rat+and+hamster.">Initiation of sperm motility after mating in the rat and hamster.</a> J Androl. 13 (5), 444-449 (1992).</li><li>Lilja, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+semenogelin.">Cell biology of semenogelin.</a> Andrologia. 22 Suppl 1, 132-141 (1990).</li><li>Lilja, H., Abrahamsson, P. A., Lundwall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semenogelin,+the+predominant+protein+in+human+semen.+Primary+structure+and+identification+of+closely+related+proteins+in+the+male+accessory+sex+glands+and+on+the+spermatozoa.">Semenogelin, the predominant protein in human semen. Primary structure and identification of closely related proteins in the male accessory sex glands and on the spermatozoa.</a> J Biol Chem. 264 (3), 1894-1900 (1989).</li><li>Veveris-Lowe, T. L., Kruger, S. J., Walsh, T., Gardiner, R. A., Clements, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seminal+fluid+characterization+for+male+fertility+and+prostate+cancer:+kallikrein-related+serine+proteases+and+whole+proteome+approaches.">Seminal fluid characterization for male fertility and prostate cancer: kallikrein-related serine proteases and whole proteome approaches.</a> Semin Thromb Hemost. (33), 87-99 (2007).</li><li>Emami, N., Deperthes, D., Malm, J., Diamandis, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+role+of+human+KLK14+in+seminal+clot+liquefaction.">Major role of human KLK14 in seminal clot liquefaction.</a> J Biol Chem. 283 (28), 19561-19569 (2008).</li><li>Gerhard, I., Roth, B., Eggert-Kruse, W., Runnebaum, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+kallikrein+on+sperm+motility,+capillary+tube+test,+and+pregnancy+rate+in+an+AIH+program.">Effects of kallikrein on sperm motility, capillary tube test, and pregnancy rate in an AIH program.</a> Arch Androl. 24 (2), 129-145 (1990).</li><li>Li, S., Garcia, M., Gewiss, R. L., Winuthayanon, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+role+of+estrogen+for+the+mammalian+female+in+regulating+semen+coagulation+and+liquefaction+in+vivo.">Crucial role of estrogen for the mammalian female in regulating semen coagulation and liquefaction in vivo.</a> PLoS Genet. 13 (4), e1006743 (2017).</li><li>Elia, J., 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=Human+semen+hyperviscosity:+prevalence,+pathogenesis+and+therapeutic+aspects.">Human semen hyperviscosity: prevalence, pathogenesis and therapeutic aspects.</a> Asian J Androl. 11 (5), 609-615 (2009).</li><li>Winuthayanon, W., Hewitt, S. C., Orvis, G. D., Behringer, R. R., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uterine+epithelial+estrogen+receptor+alpha+is+dispensable+for+proliferation+but+essential+for+complete+biological+and+biochemical+responses.">Uterine epithelial estrogen receptor alpha is dispensable for proliferation but essential for complete biological and biochemical responses.</a> Proc Natl Acad Sci U S A. 107 (45), 19272-19277 (2010).</li><li>McLean, A. C., Valenzuela, N., Fai, S., Bennett, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+vaginal+lavage,+crystal+violet+staining,+and+vaginal+cytological+evaluation+for+mouse+estrous+cycle+staging+identification.">Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification.</a> J Vis Exp. 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作者没有什么可透露的。

从小鼠子宫收集精子可以提供优于精子分析的优势体外, 因为前者可以说明精子与女性生殖道分泌物之间的生理相互作用.在这项研究中提出的技术使研究人员能够确定精子的质量, 以及在理想的生理条件下的活力和活力。此外, 还有各种各样的下游分析, 可以使用从子宫收集的精子, 例如, 精子可以计数, 以确定实际精子数进入子宫和精子也可以成像的运动化验。理想的时间点收集精子?...

雌性生殖道由上部和下层组成。上生殖道包括输卵管、子宫和宫颈。下生殖道包括 ectocervix 和阴道。在人类, 被射出的精子沉积在阴道前壁, 毗邻 ectocervix1。然而, 在小鼠体内, 精子在交配2后几分钟内就会被扫入子宫。
精子含有精凝胶, 在射精的几秒钟内凝固, 导致精子被诱捕并固定在3。液化释放的固定精子通过改变精子从凝胶状到水样的一致性。这一过程对精子的运动和哺乳动物的繁殖至关重要。关于精子液化的知识主要是基于体外研究, 其中精子在培养皿中液化。在人类中, 前列腺特异抗原或 PSA (也称为激肽释放酶相关的肽3或 KLK3) 的酵素活性是通过水解 semenogelins (在精子中存在凝胶形成的蛋白质) 来液化的主要贡献者4 ,5,6。降解 semenogelins 释放精子从精物, 增加精子的流动性。自由精子游向输卵管, 使卵子受精。液化 (或精子粘度) 试验是标准的初步筛查精子分析的生殖诊所, 以评估男性生育能力7。
最近发表的一篇文章表明 , 从雌性小鼠生殖道 post - mating 中收集到的的分析可以用来说明液化的不足 , 从而导致生育缺陷8。有缺陷的液化, 如精子高, 导致 11.8-32.3% 的男性不育症患者的9, 表明正常的液化是必不可少的哺乳动物繁殖。然而, 对液化缺陷的研究和处理完全集中在男性7。女性生殖道在精子液化中发挥作用的可能性尚未得到探讨。因此, 收集和测定液化时间的方法将为研究人员提供一种新的诊断工具, 以确定液化是否可能是模型机体中不育的原因之一。

<table border="0" cellpadding="0" cellspacing="0" width="571">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Toothpick</td>
			<td style="width:71px;">Diamond</td>
			<td style="width:119px;">750-Flat</td>
			<td style="width:167px;">Autoclaved sterile, use blunted toothpick. Do not use point-ended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Dissecting scissors</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">08-940</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Spring scissors</td>
			<td style="width:71px;">Roboz</td>
			<td style="width:119px;">RS-5600</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fine forceps</td>
			<td style="width:71px;">Roboz</td>
			<td style="width:119px;">RS-5045</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">25-&#956;L glass capillary tube</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">53432-761</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Leibovitz&#8217;s L-15 media</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:119px;">11415064</td>
			<td style="width:167px;">Supplement with 1% FBS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Fetal bovine serum (FBS)</td>
			<td style="width:71px;">Gemini Bio-Products</td>
			<td style="width:119px;">100-106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">1.5-mL microcentrifuge tube</td>
			<td style="width:71px;">Axygen</td>
			<td style="width:119px;">MCT-150-C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">2-mL microcentrifuge tube</td>
			<td style="width:71px;">Axygen</td>
			<td style="width:119px;">MCT-200-A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:215px;">Digital dry block heater</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">13259-052</td>
			<td style="width:167px;">For warming the microcentrifuge tube prior to semen collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Minicentrifuge</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">LSE 6765</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">35-mm culture dish</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">10861-656</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">15-mL polypropylene centrifuge tube</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">10025-686</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Waterbath</td>
			<td style="width:71px;">Precision</td>
			<td style="width:119px;">TSGP05</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Heated stage stereomicroscope</td>
			<td style="width:71px;">Leica Microsystems</td>
			<td style="width:119px;">MZ10F</td>
			<td style="width:167px;">To keep the tissue warm prior to semen collection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Brightfield microscope</td>
			<td style="width:71px;">Leica Microsystems</td>
			<td style="width:119px;">DMi8</td>
			<td style="width:167px;">Optional. For video imaging of the sperm motility</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Camera</td>
			<td style="width:71px;">Leica Microsystems</td>
			<td style="width:119px;">DMC2900</td>
			<td style="width:167px;">Optional. For video imaging of the sperm motility</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Glass slides</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">12-552-3</td>
			<td style="width:167px;">Optional. For video imaging of the sperm motility</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Coverslip</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48393-059</td>
			<td style="width:167px;">Optional. For video imaging of the sperm motility</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:215px;">Bleach</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Dilute in dH2O to 10% concentration</td>
		</tr>
	</tbody>
</table>

collection of post-mating semen from the female reproductive tract and measurement of semen liquefaction in mice

在小鼠体内, 被射出的精子在子宫内沉积。射精后, 精子的一致性从凝胶状变为水样, 这一过程称为液化。在这项研究中, 我们展示了如何收集 post-ejaculated 精子从女性生殖道在一个小鼠模型。首先, 发情阶段的成年雌性老鼠在一夜之间被安置在一个雄性笼子里。第二天早上, 交配被证实的交配插头在阴道开口。交配插头的雌性小鼠被安乐死, 每个生殖道都被收集成一个整体 (阴道、子宫、输卵管、卵巢), 确保一个封闭的系统来控制精子。生殖道被放置在1.5 毫升离心管, 阴道被切断, 以释放的精子进入管。为了保证最大的精子量进行分析, 用无牙钳将子宫角从卵巢端挤压到阴道末端, 排出残余的精子。整个生殖道被丢弃。该精子管被短暂剥离。25μ l 毛细管吸管被放置在一个180°角 (平行于管壁) 管。记录了在25μ l 线上填充毛细管所用的时间。从一个成熟的雄性饲养者的精子通常需要大约60-180 秒来填满25μ l 毛细管。这种精子收集技术也可用于其他下游应用, 如精象和运动分析。

在与肥沃的 ctrl 雄性交配后, 从 ctrl 和高雌鱼身上收集了大约8小时的精子。液化 (或精子粘度) 是量化的时间采取填补25μ l 毛细管与精子。从 CTRL 子宫收集的精子采取了2.06 ±0.12 分钟 (mean±东南亚 M, n = 5) 填充毛细管 (图 1)。然而, 从高子宫采集的精子的试验时间超过60分钟 (60.0 ±0.0, mean±东南亚米, n = 5)。这些数据表明, 与高子宫相比, 在 CTRL 的时候, 精子的液化程...

Watch this Scientific Journal Video about Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice at JoVE.com

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

雌性生殖道交配后精子的收集及对小鼠精子液化的测定

精子的液化需要从精凝胶中解放出来。本研究提供了从雌性生殖道 post-coitus 中收集精子, 以及测定精子液化时间的程序。

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

collection-post-mating-semen-from-female-reproductive-tract

Research

JoVE Journal

Developmental Biology

16.3K 次观看.  Washington State University. 精子的液化需要从精凝胶中解放出来。本研究提供了从雌性生殖道 post-coitus 中收集精子, 以及测定精子液化时间的程序。

视频: Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

科研

发育生物学

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

原位卵巢移植术对雌性小鼠卵巢衰老的影响及寿命及健康范围研究

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning.

Trichostatin a 和维生素 C 的组合治疗提高体细胞细胞核移植克隆小鼠的效率

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

人子宫内膜基质细胞的体外分离蜕膜化

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

小鼠血睾丸屏障完整性的一种活体研究方法

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

线虫生殖道内精子引导和运动的测定

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

牛早期蚁卵泡卵母细胞的体外培养策略

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Transuterine Fetal Tracheal Occlusion Model in Mice

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Various animal models of congenital diaphragmatic hernia and fetal tracheal occlusion present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and availability of genetic tools. This model provides a new tool to study the impact of both tracheal occlusion and increased luminal pressure on lung development.

小鼠的转宫胎儿气管闭塞模型

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). ...