Name: collection of post-mating semen from the female reproductive tract and measurement of semen liquefaction in mice
Uploaded: 2017-11-18T14:00:00
Description: 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

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...

<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. (67), e4389 (2012).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab+Animal+Research.+Fundamentals+of+Breeding+and+Weaning.">Lab Animal Research. Fundamentals of Breeding and Weaning.</a> JoVE. , (2017).</li><li>Prassas, I., Eissa, A., Poda, G., 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=Unleashing+the+therapeutic+potential+of+human+kallikrein-related+serine+proteases.">Unleashing the therapeutic potential of human kallikrein-related serine proteases.</a> Nat Rev Drug Discov. 14 (3), 183-202 (2015).</li><li>Muytjens, C. M., Vasiliou, S. K., Oikonomopoulou, K., Prassas, I., 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=Putative+functions+of+tissue+kallikrein-related+peptidases+in+vaginal+fluid.">Putative functions of tissue kallikrein-related peptidases in vaginal fluid.</a> Nat Rev Urol. 13 (10), 596-607 (2016).</li></ol>

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。

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