我们要感谢山东省立医院动物实验室的技术支持。这项工作得到了国家自然科学基金（NSFC 82101645）、山东省自然科学基金（ZR2020QH088）和山东省高校青年创新科技支持计划（2021KJ051）的支持。

以下方案符合有关使用研究动物的所有机构伦理准则，并已获得中国山东省立医院动物伦理委员会的批准。所有手术操作均在深度麻醉下进行，动物在手术过程中的任何阶段都没有感到疼痛。
1. 术前准备
<ol><li>器械灭菌<ol><li>手术前在高压灭菌器（121°C，15分钟）中对手术器械进行蒸汽灭菌。准备足够的一次性缝合线和针头。</li>
	</ol></li>
	<li>手术平台设置<ol><li>在专门用于外科手术的房间进行手术。为手术分配至少 60 cm x 60 cm 的工作台区域。用75%酒精清洁该区域表面，并用一次性医用毛巾盖住，然后提前30分钟用紫外线辐射消毒（图1A）。</li>
	</ol></li>
	<li>动物准备<ol><li>将所有动物饲养在温度受控的房间（20-25°C）中，光照周期为12小时，黑暗循环为12小时。手术前将 8 周龄的雌性 C57BL/6 小鼠适应饲养设施 1 周。</li>
		<li>手术前称量小鼠。通过腹膜内注射三溴乙醇（280mg / kg）给予所有9周龄的雌性小鼠全身麻醉，以在手术过程中的任何阶段实现无痛。手术前约1小时皮下注射美洛昔康（2mg / kg）以缓解疼痛。</li>
		<li>在手术过程中涂抹眼药膏以防止角膜干燥。</li>
		<li>使用干净的棉签在背部涂抹脱毛化妆水。让化妆水在鼠标上静置3-5分钟，然后用纱布和棉签去除毛发。重复此步骤，直到从鼠标背面去除所有毛发。</li>
		<li>使用纱布和棉签用 75% 的酒精清洁皮肤。使用橡胶条或棉绳将鼠标固定在手术平台上（图1B），并使用碘伏溶液清洁背部。 注意:在卵巢切除术前通过脚趾捏住确认麻醉深度。</li>
	</ol></li>
</ol>2. 卵巢切除术
注意:三溴乙醇可以维持约 30 分钟，确保手术尽可能完成。
<ol><li>使用一次性手术刀从大腿根部向上纵向切开~1.0厘米的背侧切口，确保只切开皮肤和皮下筋膜，避免此时切入后腹膜。</li>
	<li>将切口向左拉，可以看到一个白色的脂肪垫。使用微型镊子和剪刀沿白色脂肪垫切割~0.5厘米，以暴露腹膜腔。</li>
	<li>切开腹膜后部后，用微镊子缓慢而轻柔地从腹膜腔中取出白色脂肪垫。立即用0.9%无菌盐水润湿浸泡的纱布外的白色脂肪组织。在腹腔外时，保持暴露的组织始终湿润。</li>
	<li>粉红色颗粒状物质，即卵巢，附着在白色脂肪垫的下部（图2A）。卵巢连接到细长的导管，即子宫。使用5-0可吸收缝合线结扎子宫的卵巢端并切除左卵巢（图2B）。</li>
	<li>切除卵巢时，尽可能保留周围的脂肪组织。避免手术器械与卵巢直接接触，防止卵巢组织腹腔内着床。</li>
	<li>小心地将白色脂肪垫放回腹膜腔内。用5-0可吸收缝合线在后腹膜上进行简单的间歇缝合（图2C）。缝合完成后，用 0.9% 无菌盐水浸泡的纱布清洁出血处。</li>
	<li>将皮肤切口向右拉，并使用相同的方法切除右侧卵巢。</li>
	<li>用4-0不可吸收缝合线进行间歇缝合，并用0.9%无菌盐水浸泡的纱布清洁任何出血（图2D）。</li>
	<li>完成两次缝合后，用碘伏溶液清洁伤口。腹腔注射广谱抗生素。</li>
</ol>3.术后观察
<ol><li>手术后将小鼠移至37°C恒温毯中。在老鼠可以自由移动之前，将动物放在各自的笼子里。不要让动物无人看管，直到它恢复足够的意识以保持胸骨卧位。</li>
	<li>术后24小时皮下注射美洛昔康（2mg / kg）以缓解疼痛。</li>
	<li>每天监测小鼠，以确保手术伤口正常愈合，没有任何并发症（裂开）的迹象。</li>
</ol>4.补充雌二醇
<ol><li>准备补充戊酸雌二醇的饲料。每 1 公斤饲料补充 2.6 毫克 β-雌二醇 17-戊酸酯。</li>
	<li>手术完成后3天，用戊酸雌二醇喂养小鼠。</li>
</ol>5. FSH注射
<ol><li>制备重组人FSH溶液。用0.9%无菌盐水溶解注射用重组人FSH粉末至100IU / mL。</li>
	<li>根据实验计划对小鼠进行分组，并通过腹腔注射给予溶剂或不同剂量的重组FSH2周。根据重组FSH的生物活性，在小鼠中使用FSH的注射剂量，相当于女性在绝经过渡期的血清FSH水平。 注:根据不同的处理，卵巢切除的补充雌激素的小鼠随机分为三组，溶剂（NS）组接受100μL /天溶剂，低剂量FSH（L-FSH）组每天接受15 IU / kg体重和高剂量FSH（H-FSH）组每天接受30 IU / kg体重。</li>
</ol>

开发 FSH 水平升高的卵巢切除小鼠模型

<ol>
	<li>Harlow, S. D., 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=Executive+summary+of+the+Stages+of+Reproductive+Aging+Workshop+++10:+addressing+the+unfinished+agenda+of+staging+reproductive+aging.">Executive summary of the Stages of Reproductive Aging Workshop + 10: addressing the unfinished agenda of staging reproductive aging.</a> Journal of Clinical Endocrinology &amp; Metabolism. 97 (4), 1159-1168 (2012).</li><li>Ulloa-Aguirre, A., Zari&#241;&#225;n, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Follitropin+Receptor:+Matching+Structure+and+Function.">The Follitropin Receptor: Matching Structure and Function.</a> Molecular Pharmacology. 90 (5), 596-608 (2016).</li><li>Franks, S., Stark, J., Hardy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicle+dynamics+and+anovulation+in+polycystic+ovary+syndrome.">Follicle dynamics and anovulation in polycystic ovary syndrome.</a> Human Reproduction Update. 14 (4), 367-378 (2008).</li><li>Guo, Y., 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=Blocking+FSH+inhibits+hepatic+cholesterol+biosynthesis+and+reduces+serum+cholesterol.">Blocking FSH inhibits hepatic cholesterol biosynthesis and reduces serum cholesterol.</a> Cell Research. 29 (2), 151-166 (2019).</li><li>Xiong, 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=FSH+blockade+improves+cognition+in+mice+with+Alzheimer's+disease.">FSH blockade improves cognition in mice with Alzheimer's disease.</a> Nature. 603 (7901), 470-476 (2022).</li><li>Sun, L., 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=FSH+Directly+Regulates+Bone+Mass.">FSH Directly Regulates Bone Mass.</a> Cell. 125 (2), 247-260 (2006).</li><li>Liu, X. M., 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=FSH+regulates+fat+accumulation+and+redistribution+in+aging+through+the+G&#945;i/Ca(2+)/CREB+pathway.">FSH regulates fat accumulation and redistribution in aging through the G&#945;i/Ca(2+)/CREB pathway.</a> Aging Cell. 14 (3), 409-420 (2015).</li><li>Maclellan, R. 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=Expression+of+Follicle-Stimulating+Hormone+Receptor+in+Vascular+Anomalies.">Expression of Follicle-Stimulating Hormone Receptor in Vascular Anomalies.</a> Plastic and Reconstructive Surgery. 133 (3), 344e-351en (2014).</li><li>Liu, P., 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=Blocking+FSH+induces+thermogenic+adipose+tissue+and+reduces+body+fat.">Blocking FSH induces thermogenic adipose tissue and reduces body fat.</a> Nature. 546 (7656), 107-112 (2017).</li><li>Ji, Y., 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=Epitope-specific+monoclonal+antibodies+to+FSH&#946;+increase+bone+mass.">Epitope-specific monoclonal antibodies to FSH&#946; increase bone mass.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (9), 2192-2197 (2018).</li><li>El Khoudary, S. R., 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=Trajectories+of+estradiol+and+follicle-stimulating+hormone+over+the+menopause+transition+and+early+markers+of+atherosclerosis+after+menopause.">Trajectories of estradiol and follicle-stimulating hormone over the menopause transition and early markers of atherosclerosis after menopause.</a> European Journal of Preventive Cardiology. 23 (7), 694-703 (2016).</li><li>Radu, 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=Expression+of+Follicle-Stimulating+Hormone+Receptor+in+Tumor+Blood+Vessels.">Expression of Follicle-Stimulating Hormone Receptor in Tumor Blood Vessels.</a> The New England Journal of Medicine. 363 (17), 1621-1630 (2010).</li><li>Sowers, M. R., 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=Endogenous+hormones+and+bone+turnover+markers+in+pre-+and+perimenopausal+women:+SWAN.">Endogenous hormones and bone turnover markers in pre- and perimenopausal women: SWAN.</a> Osteoporosis International. 14 (3), 191-197 (2003).</li><li>Kristensen, V. N., Kure, E. H., Erikstein, B., Harada, N., B&#248;rresen-Dale, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+susceptibility+and+environmental+estrogen-like+compounds.">Genetic susceptibility and environmental estrogen-like compounds.</a> Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 482 (1), 77-82 (2001).</li></ol>

作者没有什么可透露的。

在从生殖阶段到非生殖阶段（更年期）的过渡期间，许多女性经历了显着的生理和病理变化。FSH水平在绝经过渡期间升高1.新兴研究表明，各种性腺外组织和器官中的FSH在多种疾病的发病机制中至关重要，包括血脂异常4，阿尔茨海默病5，骨质疏松症9,10，动脉粥样硬化11，肥胖9和癌症12。因此，建立一个可以帮助研究FSH在体内独立作用的动物模型尤为重要。OVF小鼠模型模拟了更年期过渡的早期阶段，雌激素相对稳定，FSH水平升高，特别适合探索FSH性腺外作用的研究。
在这种方法中，卵巢切除术使用单个背侧切口进行，从大腿底部向上约1厘米（图1B）。使用锋利的解剖剪刀将皮肤几乎与背肌一起切割，从而进入腹膜腔。手术后，肌肉切口不需要缝合，皮肤伤口用一根猫肠缝合线双侧闭合（图2）。与其他方法相比，该操作在技术上更容易，耗时更少，对雌性小鼠的危害更小。
在手术过程中应注意的一些细节。首先，所有外科手术都应尽可能保持清洁和无菌，以降低术后感染的风险。其次，由于卵巢组织非常脆弱，手术器械在卵巢切除术时不能直接接触卵巢，避免腹腔内着床。第三，手术后，在恢复过程中将小鼠移至37°C恒温毯中，以防止术后体温过低导致死亡。
先前的一项研究证明，内源性雌激素在绝经前妇女的卵巢细胞或绝经后妇女乳房的脂肪基质细胞中合成，并在外周组织中少量合成14。卵巢切除小鼠的血清雌激素急剧下降，但不能消除（图3B）。然而，在性腺外组织中合成的内源性雌激素不会影响OVF模型中雌激素水平的稳定性（图4B）。
OVF 模型存在一些限制。一旦外科手术不小心，导致卵巢腹腔内着床，就可能导致模型失败。在这种情况下，血清雌激素不会急剧下降，并且在发情周期的不同阶段波动。外源性施用雌激素和FSH后，身体大约需要1周才能达到平衡。因此，在1周内发生的OVF模型的病理变化不能表明FSH的影响。
综上所述，OVF机型具有稳定、低成本、易于操作等优点。腹腔注射FSH后可观察到高水平FSH的全身效应;也就是说，OVF 模型适用于探索 FSH 性腺外作用的研究。然而，模型手术和腹膜内注射手术的要求相当高。如果资金充足，特定的基因敲除模型是最佳选择。

促卵泡激素 （FSH） 水平在绝经过渡期间升高（绝经过渡一词于 2011 年在生殖衰老研讨会 （STRAW） + 10 系统阶段定义）1。在更年期过渡期间，一个以 FSH 水平升高和雌激素1 相对稳定的时期为特征，女性会经历月经周期变化和涉及各种细胞和组织的显着生理变化。这些变化会严重影响妇女的生活质量和健康。探索FSH的影响可能会改善女性的生活质量和健康。
FSH 由垂体前叶的促性腺激素细胞分泌，在控制性腺功能和生殖方面至关重要2.FSH的功能是通过FSH受体（FSHR）介导的，FSHR属于G蛋白偶联受体（GPCR）3。FSHR通常在性腺中表达，即卵巢和睾丸。已经证明，FSHR在多种性腺外细胞和组织中普遍表达，包括肝脏4、海马体5、破骨细胞6、脂肪细胞7和内皮细胞8。新兴研究揭示了 FSH 的额外性腺作用及其在血脂异常4、阿尔茨海默病5、骨质疏松症 9,10、动脉粥样硬化11、肥胖 9 和癌症12 中的潜在临床相关性。因此，建立一个可以帮助研究FSH在体内独立作用的动物模型对于探索FSH在体内的作用尤为重要。
在协议中，我们介绍了建立具有相对稳定的雌激素和不断上升的FSH水平的小鼠模型的程序13。小鼠模型通过卵巢切除手术模拟绝经过渡，然后补充戊酸雌二醇和重组FSH。由于卵巢切除的小鼠补充了外源性雌激素以维持与假手术小鼠相似的雌激素水平，因此由于垂体的雌激素反馈，内源性FSH的水平是稳定的。在这种情况下，它可以通过施用外源性FSH来控制FSH水平，而不会改变雌激素水平。因此，OVF小鼠模型可以排除雌激素的影响，并观察FSH的性腺外生理和病理作用。我们相信，详细和可视化的程序有助于研究人员在实验室中建立OVF小鼠模型，并根据需要将其应用于研究更年期过渡期间的生理和病理变化。

<table><tbody><tr><td>beta-estradiol 17-valerate</td><td>Macklin</td><td>E829824</td><td></td></tr><tr><td>Estradiol sensitive ELISA</td><td>Demeditec</td><td>DE4399</td><td></td></tr><tr><td>Hematoxylin Staining Solution</td><td>Beyotime</td><td>C0107</td><td></td></tr><tr><td>Meloxicam</td><td>Aladdin</td><td>M129228</td><td></td></tr><tr><td>recombinant human Follicle-stimulating hormone</td><td>Merck Serono</td><td>N19Z8803G</td><td></td></tr><tr><td>Tribromoethanol</td><td>Sigma</td><td>T48402</td><td>Aliphatic name: 2,2,2-Tribromoethanol</td></tr></tbody></table>

exploring independent effects of follicle-stimulating hormone in vivo in a mouse model

在从生殖期到非生殖期（更年期）的过渡期间，许多女性经历了显着的生理和病理变化，包括骨量减少、血脂升高和内脏肥胖增加。促卵泡激素 （FSH） 水平在绝经过渡期间升高。许多研究表明，各种性腺外组织和器官中的FSH与多种疾病的发病机制有关。因此，建立一个可以帮助研究FSH 在体内 独立作用的动物模型尤为重要。本研究对C57BL/6雌性小鼠进行卵巢切除术，并补充戊酸雌二醇（OVX+E2），以消除下丘脑-垂体-性腺轴的影响。OVX + E2小鼠通过腹膜内注射接受溶剂（N.S.）或不同剂量的重组FSH，以创建小鼠模型（OVF），其特征是雌激素相对稳定，FSH水平升高。因此，我们成功地生成了一个实验小鼠模型来模拟更年期过渡的早期阶段，其特征是血清FSH水平升高。OVF模型具有稳定、低成本、易于操作等优点，适合研究FSH的性腺外作用。在这里，我们描述了小鼠OVF模型的详细协议。

OVF小鼠模型模拟了更年期过渡的早期阶段，雌激素相对稳定，FSH水平升高13。首先，对于卵巢切除手术，对9周龄的雌性C57BL / 6小鼠进行全身麻醉并进行假手术（Sham）或双侧卵巢切除术（OVX）。由于巴氏染色细胞的涂片图像清楚地识别了发情周期的前发情期、发情期、子宫期和发情期，OVX小鼠失去了发情周期（图3A），ELISA方法显示血清雌二醇（E2）水平显着降低（图3B）。其次，OVX小鼠补充β-雌二醇17-戊酸酯（OVX+E2），以维持血清雌激素与Sham组相同。第三，OVX + E2小鼠通过腹膜内注射接受溶剂（N.S.）或不同剂量的重组FSH，以创建小鼠模型（OVF），其特征是雌激素相对稳定，FSH水平升高（图4）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65665/65665fig01.jpg"> 图 1.手术环境和小鼠姿势。 （A） 至少 60 厘米 x 60 厘米的工作台区域用于手术。用75%酒精清洁该区域表面，并用一次性医用毛巾覆盖，然后提前30分钟用紫外线辐射消毒。（B） 使用橡皮条或棉绳将鼠标固定在手术平台上。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65665/65665fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65665/65665fig02.jpg"> 图2.外科手术的关键步骤。 （A）卵巢位置，（B）卵巢切除术，（C）缝合腹膜，（D）缝合皮肤切口。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65665/65665fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65665/65665fig03.jpg"> 图3.阴道细胞学检查。 阴道细胞学代表卵巢切除小鼠 （OVX） 和假手术小鼠（Sham;假手术组 n = 12;每个 OVX 组 n = 10）的发情周期和内源性雌激素的阶段。（A） 阴道细胞学根据白细胞、角质化上皮细胞和有核上皮细胞的相对存在来表示发情周期的各个阶段。发情期包括发情前期，有核上皮细胞占主导地位;发情，以去核角质化细胞为主;metestrus，白细胞的存在，角化和有核的上皮细胞;腹细胞，白细胞占主导地位。比例尺 = 100 μm。 （B） 卵巢切除小鼠 （OVX） 和假手术小鼠 （Sham） 中的内源性雌激素。数据显示为SEM±均值。 学生t检验用于统计分析。第 < 0.001。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65665/65665fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65665/65665fig04.jpg"> 图4.OVF 模型和血清激素水平。 （A） 流程图OVF模型。（B） 血清雌激素 （E2） 和 FSH 浓度的 ELISA 分析。数据表示为SEM±均值，采用单因素方差分析进行统计分析。* p< 0.05 和 ** p< 0.01。这个数字从4修改了。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65665/65665fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model at JoVE.com

作者聚焦:研究围绝经期女性 FSH 与病理生理变化之间的关系 - 来自小鼠模型的见解 

各种性腺外组织和器官中的卵泡刺激素（FSH）与多种疾病的发病机制有关。卵巢切除和 FSH 处理的小鼠模型 （OVF） 可用于探索 FSH 的性腺外作用。

在小鼠模型中探索卵泡刺激素 在体内 的独立作用

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

我们的研究重点是围绝经期妇女的生理和病理变化。在这项研究中，我们试图确定哪些病理生理学改变与 FHC 有关。绝经与血脂异常和心脑血管疾病风险增加有关。使用这种小鼠模型，我们发现与阿司匹林无关的促卵泡激素会增加血清胆固醇水平。此外，阻断FSH信号通路可以有效预防低胆固醇血症。该小鼠模型模拟了更年期过渡的早期阶段，其特征是血清FSH水平升高和雌激素水平相对稳定。该模型具有稳定、成本低、操作方便等优点，适合研究FSH的性腺外作用。

exploring-independent-effects-follicle-stimulating-hormone-vivo-mouse

Nanyang Technological University

Research

JoVE Journal

Biology

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

1.6K 次观看.  The Affiliated Hospital of Qingdao University. 我们的研究重点是围绝经期妇女的生理和病理变化。在这项研究中，我们试图确定哪些病理生理学改变与 FHC 有关。绝经与血脂异常和心脑血管疾病风险增加有关。使用这种小鼠模型，我们发现与阿司匹林无关的促卵泡激素会增加血清胆固醇水平。此外，阻断FSH信号通路可以有效预防低胆固醇血症。该小鼠模型模拟了更年期过渡的早期阶段，其特征是血清FSH水平升高和?...

视频: 作者聚焦:研究围绝经期女性 FSH 与病理生理变化之间的关系 - 来自小鼠模型的见解 

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化和共同培养小鼠的卵巢和卵巢卵泡

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

科研

发育生物学

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 ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

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 Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

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 ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

基于细胞基和细胞外矩阵的Murine睾丸器官生成

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

成人小鼠卵巢中的精确卵泡图解

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing factor. Single-point sampling measures are inadequate to fully understand the biological significance of the secretory pattern of pulsatile hormones either under normal physiologic conditions or during conditions of dysregulation. Luteinizing hormone (LH) is synthesized by the anterior pituitary gonadotrope&#160;cells and secreted in a pulsatile pattern which requires frequent collection of blood samples for pulse assessment. This has not been possible in mice until recently, due to the development of a high-sensitivity LH assay and advancement in a technique for frequent low-volume sample collection, initially described by Steyn and colleagues.1 Here we describe a protocol for the frequent peripheral blood sample collection from mice with sufficient handling acclimatization to detect pulsatile secretion of LH. The current protocol details an expanded acclimatization period that allows assessment of robust and continuous pulses of LH over multiple hours. In this protocol, the tip of the tail is clipped&#160;and blood is collected from the tail using a hand-held pipette. For assessment of pulsatile LH in gonadectomized mice, serial samples are collected every 5-6 min for 90-180 min. Importantly, the collection of blood and measurement of robust pulses of LH can be accomplished in awake, freely behaving mice, given adequate handling acclimatization and effort to minimize environmental stressors. Sufficient acclimatization can be achieved within 4-5 weeks prior to blood collection. This protocol highlights advances in the methodology to ensure collection of whole blood samples for assessment of pulsatile LH secretion patterns over multiple hours in the mouse, a powerful animal model for neuroendocrine research.

Here we present a tail-tip blood sampling protocol for frequent sample collection in unrestrained mice. This method is useful for assessing patterns of pulsatile luteinizing hormone secretion and could be adapted for analysis of other circulating factors.

小鼠经常尾端采血对脉动性促黄体激素分泌的评价

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing ...

神经科学

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

的效用舞台专用中旬至下旬<em&gt;果蝇</em&gt;卵泡隔离

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

生物学

DREADDs 技术和自动血液采样

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions.
This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

作者聚焦:自动输注和血液采样对清醒小鼠进行精确的激素分析

Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals.

远程神经元激活与自动血液采样相结合，以诱导和测量小鼠体内循环黄体生成素

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of ...

在小鼠模型中探索卵泡刺激素在体内的独立作用

副本

摘要

探索更多视频

此视频中的章节

文化和共同培养小鼠的卵巢和卵巢卵泡

体外模拟微重力下小鼠腔卵泡生长的研究

卵巢组织培养对小鼠卵巢现象的可视化

Hyperandrogenic 小鼠模型研究多囊卵巢综合征

基于细胞基和细胞外矩阵的Murine睾丸器官生成

成人小鼠卵巢中的精确卵泡图解

小鼠经常尾端采血对脉动性促黄体激素分泌的评价

的效用舞台专用中旬至下旬

远程神经元激活与自动血液采样相结合，以诱导和测量小鼠体内循环黄体生成素

远程神经元激活与自动血液采样相结合，以诱导和测量小鼠体内循环黄体生成素

在小鼠模型中探索卵泡刺激素 在体内 的独立作用

副本

摘要

探索更多视频

此视频中的章节

文化和共同培养小鼠的卵巢和卵巢卵泡

体外模拟微重力下小鼠腔卵泡生长的研究

卵巢组织培养对小鼠卵巢现象的可视化

Hyperandrogenic 小鼠模型研究多囊卵巢综合征

基于细胞基和细胞外矩阵的Murine睾丸器官生成

成人小鼠卵巢中的精确卵泡图解

小鼠经常尾端采血对脉动性促黄体激素分泌的评价

的效用舞台专用中旬至下旬

远程神经元激活与自动血液采样相结合，以诱导和测量小鼠体内循环黄体生成素

远程神经元激活与自动血液采样相结合，以诱导和测量小鼠体内循环黄体生成素

在小鼠模型中探索卵泡刺激素在体内的独立作用