Name: a hyperandrogenic mouse model to study polycystic ovary syndrome
Uploaded: 2018-10-02T14:30:00
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这项工作得到了国家卫生研究院 (赠款 R00-HD068130 白雪) 和巴尔的摩糖尿病研究中心的支持: 飞行员和可行性赠款 (白雪)。

在这里, 我们提出了氢睾酮颗粒的准备和插入, 以及生殖和新陈代谢测试的详细协议。在这项研究中使用的老鼠是一个混合的背景 (C57/B6, CD1, 129Sv), 并保持与食品和水广告随意在14/10 小时/暗循环24°c 在百老汇研究建筑动物设施在约翰霍普金斯大学学校医学。所有程序均经约翰霍普金斯大学动物保育和使用委员会批准。
1. 创建类似 PCOS 的小鼠模型
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氢睾酮颗?...

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Hyperandrogenism 是 PCOS 的一个重要特征。本协议所用的血清氢睾酮水平 (dht 小鼠的两倍以上) 比以前的研究中其他研究者报告的要低, 并且被校准成比例模拟的妇女与 PCOS5,19, 20,21。与其他模型不同的是, 这种2倍的 dht 模型不会改变体重和全身成分, 与无氢睾酮的小鼠相比, 3.5 月后, dht 插入23</s...

Hyperandrogenism 是多囊卵巢综合征 (PCOS) 的标志, 根据 NIH 标准和雄激素过剩和 pcos (AE pcos) 社会。患有 PCOS 的妇女很难怀孕, 并增加了妊娠并发症的风险1。即使他们怀孕了, 他们的雌性后代也会有不良的健康结局2,3。动物模型已经开发使用各种策略4,5,6,7,8,9,10,11,12并表现出多囊卵巢综合征 (停止排卵、葡萄糖和胰岛素耐受) 的特点, 体重增加和肥胖与脂肪细胞体积增大和脂肪细胞重量增加有关。生产用于研究 PCOS 的动物模型有两种主要的策略。一是治疗与高水平的雄激素直接 (外源性雄激素注射/插入) 或间接 (如阻断雄激素转化为雌激素与芳香化酶抑制剂) 出生后13。另一种是由胎儿 hyperexposure 的雄激素在妊娠期14,15学习的后代。例如, 恒河猴16、17、绵羊18的雌性后代和在宫内期间接触雄性雄激素水平的啮齿类动物, 在以后的生活中发育成 PCOS 样特征。这些模型大大增强了我们对雄激素效应的认识, 以及胎儿的编程和子宫环境的影响。然而, 这些模型有自己的局限性: 1) 动物发育肥胖, 因此很难将 hyperandrogenemia 的影响与肥胖引起的生殖和代谢功能障碍分开;2) 在怀孕前, 有 PCOS 的妇女已经表现出高水平的雄激素, 因此卵母细胞在受精前暴露在雄激素过剩的水平;3) 出生后或妊娠期间使用的睾酮 (T) 或 dihydrotestosterone (DHT) 的药理剂量可能不能反映 PCOS 的雄激素环境。在卵巢滤泡液和/或血清中测定睾酮和 dht 水平, 睾酮和 dht 水平在女性 PCOS5、19、20、21 的妇女中高出1.5 至3.9 倍. ,22,23与未受影响的妇女相比。我们创建了一个成年小鼠模型23,24,25 , 发展生殖和代谢功能障碍在两周内启动慢性双氢睾酮暴露从插入一个球团与4mm 长度的水晶 DHT 粉 (球团总长度为 8mm)。该模型产生的血清氢睾酮水平约2 倍以上 (称为 2xDHT) 比控制小鼠没有 DHT 治疗。2xDHT 小鼠不表现基底血清雌二醇、睾酮、LH 和不发育肥胖的变化, 并显示类似的卵巢重量, 血清胆固醇水平, 游离脂肪酸, 瘦素, TNFα和 IL-623,24, 25相对于控制, 甚至3.5 月后, DHT 插入23,24,25。此外, 通过交配雌性已经开发出 PCOS 的特点, 我们可以研究 hyperandrogenic 的产妇环境对后代的新陈代谢和生殖健康的影响15。
这种新范式 (与 NIH 和 AE-PCOS 社会标准有关) 通过生产相对较不受影响的妇女的雄激素水平与 2-到3倍高的睾酮或 DHT 水平较高的男性荷尔蒙的比例来模拟这种疾病。然而, 这一模型是由连续外源双氢睾酮, 而不是从编程内源 hyperandrogenism 一旦 DHT 被撤回。本文的总体目标是集中于 1) 如何使 DHT 颗粒;2) 如何生成像小鼠模型这样的瘦 PCOS;3) 评估来自这些水坝的雌性后代的战略。对表型的其他测量和评估在本手稿中没有提到, 但可以在5、15、23、24、25、26中找到。

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			<td height="26" style="height:26px;width:243px;">Crystalline 5&#945;-DHT powder&#160;</td>
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			<td height="21" style="height:21px;width:243px;">&#160;10 &#181;L pipette tips without filter</td>
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			<td height="21" style="height:21px;width:243px;">Microscope slide for smear</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Diff Quik for staining cells</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">NC9979740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#160; Lancet</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">NC9416572</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">3 mL Syring&#160;</td>
			<td style="width:135px;">Becton, Dickinson and Company (BD),</td>
			<td style="width:119px;">30985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#160;attached needle: 20G</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">305176</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">&#160;Ruler: any length than 10cm with milimeter scale.&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Xylazine&#160;</td>
			<td style="width:135px;">Vet one AnnSeA LA, MWI, Boise</td>
			<td style="width:119px;">NDC13985-704-10</td>
			<td>100mg/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Ketamine Hydrochloride</td>
			<td style="width:135px;">Hospira, Inc</td>
			<td style="width:119px;">NDC 0409-2051-05</td>
			<td>100mg/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">&#160;Surgical staple</td>
			<td style="width:135px;">&#160;AutoClip&#174; System, Fine Science Tool</td>
			<td style="width:119px;">12020-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#160;Insulin syringe</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">329461</td>
			<td>1/2 CC, low dose U-100 insulin syringe</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">&#160;Trochar&#160;</td>
			<td style="width:135px;">Innovative Research of America</td>
			<td style="width:119px;">MP-182</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Microscope</td>
			<td style="width:135px;">Carl Zeiss Primo Star</td>
			<td style="width:119px;">415500-0010-001</td>
			<td>Germany</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Ear punch</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">13-812-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Testosterone rat/mouse ELISA kit</td>
			<td style="width:135px;">IBL</td>
			<td style="width:119px;">B79174</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">DHT ELISA kit</td>
			<td style="width:135px;">Alpha Diagnostic International</td>
			<td style="width:119px;">1940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">One touch ultra glucometer</td>
			<td>Life Scan, Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">One touch ultra test stripes</td>
			<td>Life Scan, Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Eppendorf tube</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td><a href="https://www.fishersci.com/shop/products/eppendorf-snap-cap-microcentrifuge-safe-lock-tubes-24/0540218?keyword=true">05-402-18</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blade</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clidox</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td><a href="https://www.fishersci.com/shop/products/clidox-s-base-4x1gal/nc0089321#?keyword=true">NC0089321</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">surgical underpad</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>50587953</td>
			<td align="left"><img alt="Supplier Diversity Partner" height="13" src="file:///C:/Users/Tim/AppData/Local/Packages/oice_16_974fa576_32c1d314_e42/AC/Temp/msohtmlclip1/01/clip_image002.png" width="25" />
			<table cellpadding="0" cellspacing="0">
				<tbody>
					<tr>
						<td height="21" style="height:21px;width:412px;">Manufacturer:&#160; Andwin Scientific&#160;56616018</td>
					</tr>
				</tbody>
			</table>
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine Antiseptic Solution</td>
			<td style="width:135px;">Walgreens</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">3M Vetbond (n-butyl cyanoacrylate)</td>
			<td style="width:135px;">3M Science. Applied to Life</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Animal tattoo ink paste</td>
			<td style="width:135px;">Ketchum manufacturing Inc.</td>
			<td style="width:119px;"></td>
			<td>Brockville, Ontario, Canada</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scale</td>
			<td style="width:135px;">Ohaus Corporation&#160;</td>
			<td style="width:119px;">HH120D</td>
			<td>Pine Brook, NJ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electronic digital caliper</td>
			<td style="width:135px;">NEIKO Tools USA</td>
			<td style="width:119px;">01407A</td>
			<td>available from Amazon</td>
		</tr>
	</tbody>
</table>

a hyperandrogenic mouse model to study polycystic ovary syndrome

Hyperandrogenemia 在女性生殖和代谢功能中起着至关重要的作用, 是多囊卵巢综合征的标志。开发一种瘦的 pcos 样小鼠模型, 模仿妇女与 PCOS 是临床意义。在本协议中, 我们描述了这样一个模型。通过插入4毫米的氢睾酮 (dihydrotestosterone) 晶体粉末颗粒 (总长度的颗粒是8毫米), 并在每月更换, 我们能够产生一个 PCOS 样的小鼠模型与血清氢睾酮水平2倍高于未植入 dht (无氢睾酮) 的小鼠。我们观察到生殖和代谢功能障碍, 而不改变体重和身体组成。在表现出高度不孕症的同时, 这些 PCOS 样雌性小鼠的一小部分可以怀孕, 他们的后代显示延迟青春期和增加睾酮作为成人。这种 pcos 样瘦老鼠模型是一个有用的工具, 研究 pcos 的病理生理学和这些 pcos 样水坝的后代。

血清双氢睾酮水平与葡萄糖耐受试验
用 ELISA 法和 LC-MS 根据1.24–1.25 和2.9、3.0 测定血清中的双氢睾酮水平。双氢睾酮的绝对值在质谱和 ELISA 上是不同的, 但是, dht 的相对折叠 (大约2倍) 与无氢睾酮的插入是相似的从测试和横跨实验15,23,24 (图 2A<...

Watch this Scientific Journal Video about A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome at JoVE.com

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

我们描述了一个瘦的 pcos 样小鼠模型的发展与 dihydrotestosterone 丸研究 pcos 的病理生理学和这些 pcos 样水坝的后代。

Hyperandrogenic 小鼠模型研究多囊卵巢综合征

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

这种方法可以帮助回答生殖领域的关键问题。和内分泌学。并适用于多囊卵巢综合征或PCOS和超生激素的研究。这种技术的优点是，它促进一致，均匀，长期释放激素在昂贵的有效方式。J朱斯治疗这个程序将是平雪，一个技术员，从我的实验室。对于二氢酮或 DHT 颗粒制备，请使用剃刀刀片切割 15 毫米的硅胶管。并使用装有钝化 20 量针的三毫升注射器将 2 到 5 毫米医用粘合剂硅胶注射到管子的一端。让管道干燥过夜。并检查密封端的气泡。穿着适当的个人防护装备，将 DHT 粉末倒入流动中的塑料快艇中。将胶粘管的开端印象成粉末。使用大拉直的回形针，将粉末向下插入管中，直到 DHT 达到适当的实验深度。然后用硅胶密封管的开路侧过夜。第二天早上，切割每个密封的一侧，以获得两毫米长度的硅胶两侧的DHT和没有DHT控制颗粒。然后在室温下将用铝箔包裹的50毫升圆锥管中储存颗粒长达三个月。对于 PCOS 小鼠模型生成平衡高达 20 DHT 或控制颗粒在单独的 50 毫升锥形管中，包含 30 毫升 0.9% 盐水，在 37 摄氏度下 24 小时。第二天，确认对麻醉两个月大的雌性小鼠的"手趾捏"反应不足，并使用无菌纱布对手术区进行连续的波维酮碘和70%的乙醇磨砂。接下来，通过颈部附近的皮肤做一个五毫米的切口。并使用十轨小车在旋转方向上建造一条 15 毫米的隧道。使用小车将颗粒粘入隧道，并使用手术粘合剂密封切口。手动用钳子近似伤口边缘，并使用柔和的刷冲，将三层液体粘合剂涂抹到对对伤口边缘两侧一厘米外。然后将鼠标单独放在热垫上的笼子中，进行监控，直到完全恢复。每四周更换一次颗粒，以保持恒定的地激素暴露水平。颗粒插入三天后，使用 p10 移液器将十微升无菌盐水喷入阴道腔，并使用移液器尖端立即恢复盐水。将阴道细胞盐水样本铺到贴有标签的幻灯片上。让盐水在室温下干燥。将干滑在 100% 乙醇的容器中固定至少五分钟。然后在缓冲器 B 中染色幻灯片一分钟，从 dif 快速染色套件中染色 C。在缓冲液结束时，C 孵育用自来水清洗幻灯片三秒钟，并在室温下干燥。在光学显微镜的十倍目标下确定最圆环视图的幻灯片。计算每个阶段的天数，并除以总天数，以计算每个阶段的百分比时间。为了测试葡萄糖耐受性，20天DHT插入，禁食小鼠过夜。第二天早上称体重老鼠，并使用血糖仪和测试条测量尾静脉血的罗勒葡萄糖水平。给老鼠注射每公斤2克的葡萄糖，重量为20%。然后在葡萄糖后15、30、60、90、120分钟测量尾静脉血样的葡萄糖水平。要从 DHT 插入的母子获得雌性后代，在颗粒插入后，将雌性小鼠移至笼子中，在经过证明的肥沃雄性小鼠中进行测试。每周记录一次大人的体重，以确定老鼠是否怀孕。产后第七天，将长矛尖浸入纹身膏中，并在脚趾上用纹身标记每只小狗。每七天称体重一次，直到70天。在产后日期12至16之间，耳朵打小鼠，根据系统对它们进行编号，如图所示。要区分性别，检查每个标记的小鼠的腹侧。雌性将有可见的奶嘴。收集血液样本进行各种下游分析，使用长矛收集九至十上午间血液的近似实验量，通过亚曼多利静脉出血。并离心样品，以允许血清收集。然后将血清样本储存在零下80摄氏度的1.5毫升微离心管中，直到分析。为了评估DHT雌性后代的青春期，每天在产后第21天下奶后，通过目视检查检查阴道开口。并使用口径测量无生殖器距离。虽然质谱法和Elisa分析血清DHT的绝对值不同，但DHT与无DHT插入的相对民间变化在测定和实验中都是相似的。DHT水平在DHT和没有DHT小鼠的孕前到怀孕显著增加。然而，DHT小鼠的DHT水平比在孕前和妊娠时间点没有DHT小鼠高两倍。成年DHT女儿在生殖功能上表现出差异。例如，与成人相比，被破坏的循环性没有DHT女儿经历明显更长的时间在甲体死亡和更少的时间在亲酯和酯。在尝试此程序时，重要的是要记住在颗粒用于均匀释放激素之前孵育这些颗粒 24 小时。按照这个程序，也可以检查DHT接触的生殖和代谢后果。在它的发展之后，这一技术为内序和母体胎儿相互作用领域的研究人员研究PCOS和低水平高激素症对雌性小鼠的影响铺平了道路。不要忘记，使用 DHT 可能非常危险，执行此过程时应始终采取预防措施，如戴手套、戴口罩和护目镜，以及在生物安全罩中工作。

Research

JoVE Journal

Developmental Biology

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

科研

发育生物学

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

原发性大鼠瓣膜间质细胞的分离与鉴定: 主动脉瓣钙化的新模型研究

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

成人耳皮肤全安装共焦显微术: 一种研究神经血管分支形态发生和免疫细胞分布的模型系统

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

卵巢组织培养对小鼠卵巢现象的可视化

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

A Mouse Distraction Osteogenesis Model

小鼠牵张成骨模型

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

应用造血干细胞移植评估骨髓增生异常综合征的起源

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

平行板灌注系统的体外模型研究了细菌对移植组织的粘附性

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

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

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

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 ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...