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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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생물학

利用阴道灌洗的啮齿动物发情周期监测：没有正常周期这样的事情

Published: August 30, 2021
doi:
10.3791/62884
, , , , ,
1Department of Neurosurgery, Brain Injury Research Center,UCLA, David Geffen School of Medicine, 2Department of Psychology,Pepperdine University, Seaver College

Summary

本研究详细介绍了在涉及雌性大鼠的实验设计中需要考虑的关键因素。从更广泛的意义上说，这些数据有助于减少耻辱感，并有助于开发更具包容性的诊断和干预工具。

Abstract

目前的方法建立了一种可重复的，标准化的，具有成本效益的方法来监测雌性斯普拉格·道利（SD）青少年大鼠的发情周期。这项研究证明了荷尔蒙周期的复杂性以及构建可靠和有效的监测技术所需的广泛理解。通过对主要实验设计和程序要素的深入研究，这种对循环及其基本原理的描述为进一步理解和解构误解提供了一个框架，供将来复制。

除了使用阴道灌洗的样本收集过程的概述外，该程序还描述了数据分类为发情，发情，发情和发情的四阶段模型的机制。这些阶段的特征是一种新的方法，利用收集时阴道液状况，细胞类型，细胞排列和细胞数量的4个分类决定因素。每个阶段的变化，有利和不利的样本，周期性和非周期性之间的区别以及收集的分类组件的图形描述与数据的有效解释和组织实践一起呈现。总体而言，这些工具首次允许发布可量化的数据范围，从而在复制时实现分类因素的标准化。

Introduction

新颖的贡献
啮齿动物发情周期已被确定为健康的重要指标。然而，研究人员的无意识偏见和对女性身体的不准确解释阻碍了科学界的发展。“发情”这个词的词源本身就意味着一种自卑感和消极感。欧里庇得斯用这个词来描述“疯狂”或疯狂，荷马用来描述恐慌，柏拉图用来描述一种非理性的驱动力。这项研究强调了这些原始观点如何影响当前的科学界，并通过一种新颖的马赛克范式来解决这些问题 – 以前研究的方法的更新组合，扩大了范围，以更全面的方法。

首先，研究和使用这种技术是必要的，因为没有标准化和全面的监测技术，数据解释实践可能不清楚。其次，尽管发情周期特征取决于正在研究的个体大鼠，但它们通常是普遍存在的。第三，虽然荷尔蒙周期是常规和有益的过程，但它们被“翻译成人类”一节中探讨的危险污名所包围。本研究旨在以三种方式解决这三个问题 – （A）通过描述深入的发情周期监测技术并阐明如何解释结果，（B）通过概述保持每个周期的完整性和个性的方法，以及（C）通过提请人们注意使未经证实的做法永久化的误解。

这项研究的独特之处在于它关注青春期大鼠，这一时期的特点是关键的发育变化，揭示了成年期的各种行为，解剖学和生理表现1。建立一个标准化的实验设计来监测研究不足的人群中的荷尔蒙循环，同时解构常见的偏见，这将允许开发可靠和有效的荷尔蒙相关性2，3，4 和确定条件依赖性循环中断5，6，7，8，9，10.最终，这些新颖性有助于扩大各种健康问题的诊断标准，治疗方法和干预措施。

基本定义和用途
发情周期是响应于三种振荡的女性性类固醇激素而发生的动态生理过程的集合：雌二醇，致发光激素（LH）和黄体酮（图1A，B）。内分泌和中枢神经系统之间的相互作用调节周期，该周期通常持续4-5天，并从性成熟开始到生殖衰老和/或停止复发。它根据激素水平分为单独的类别 – 最常见的是发情期（DIE），发情期（PRO），发情期（EST）和发情期（MET）的4个阶段，它们以循环方式进行。分裂的数量可以从3阶段11 到13阶段12，具体取决于研究的性质13。较少的划分数量通常将MET排除为一个阶段，并将其归类为短期过渡期。较高的数字通常包括允许更仔细地检查诸如肿瘤发展或自发伪妊娠等现象的小节，即没有胚胎植入的怀孕的生理状态12，14，15。

在这项研究中，通过阴道管的成分鉴定分期，命名为3个分类决定因素 – 存在的细胞类型，细胞排列和细胞数量（图2A-D）。虽然本研究没有监测阴道液的状况，但建议将其作为第四种分类成分。有关检查阴道液的更多信息，请参见参考列表16。分类成分可以通过阴道灌洗提取细胞来检查，阴道灌洗是现代发情周期监测中推荐的主要技术。虽然每个阶段的深入生理过程不在本研究的范围之内，但更多信息可以在文献中找到17.

这种发情周期监测技术的使用和持续发展植根于性类固醇激素与身体系统功能之间的联系，例如心血管系统18，内分泌系统8和中枢神经系统19，20，21。同时，当涉及雌性啮齿动物22，23，24，25时，发情周期监测可能并不总是必要的。相反，重要的是首先考虑在特定研究领域是否报告了性别差异，这可以在已发表的评论22，23中进一步探讨。虽然发情周期监测在广泛的研究调查中至关重要，但它不应被视为将雌性啮齿动物纳入实验的障碍。虽然这种技术可能看起来很复杂且耗时，但根据研究者的不同，该过程本身可能需要不到15分钟的时间才能完成，并且具有成本效益。总体而言，将雌性啮齿动物纳入科学研究有利于了解身体系统，各种病症和病理以及一般健康，因为这些发展主要基于雄性身体模板。

啮齿动物的通用参数和自然变异性
为被视为“典型”的方面建立范围对于定义标准周期模式，设置用于比较和分析目的的参数以及检测异常和异常值是必要的。同时，同样重要的是要认识到每只大鼠的周期都是独一无二的，并且预计会根据动物品系，生理过程和环境条件出现偏差。事实上，发情周期最“正常”的方面之一是可变性。这在整个周期长度中可见，范围为3-38天26，27;性成熟年龄，范围从32-34天到多周28，29，30;什么被认为是非周期性的11，和分类行列式模式11，13。总体而言，发情周期没有通用的模板，将其转化为科学界和公众是实验过程的重要组成部分。

实验时间点和发育年龄
认识到这种可变性原则有助于建立可靠和有效的实验设计。例如，发情周期监测的开始依赖于大鼠的解剖学和生理学发育，这取决于环境和生理因素。在阴道开口（VO）的发展之前，监测无法开始，该阴道口是阴道外孔被通向阴道管内侧的外阴包围（图3A-D）。虽然VO通常在32至34天之间完全发展，但它仍然对每个受试者都是个体化的，并且关于该过程的许多内容仍然未知。这个开口已被用于识别性成熟的开始，这与雌二醇31的增加，下丘脑 – 垂体 – 卵巢轴32的成熟以及大鼠的第一次排卵17，33，34，35有关。然而，最近的出版物发现，它只是生殖发育的间接标志，因为它可以与不利环境中的荷尔蒙和发育事件脱钩31，并且可能代表雌二醇水平的变化而不是性成熟33。因此，建议不要仅仅依靠VO来确定发育年龄并作为发情周期监测36的限定符，而是利用第一EST阶段的外观和上皮细胞30的角化来标记性成熟的开始。

在30，37 的啮齿动物中，体重与青春期的发育年龄显着相关，因此也可以帮助确定该时期的发育年龄。与这种现象相关的拟议机制包括刺激生殖发育所必需的激素，例如生长激素，以及通过食欲调节器瘦素30抑制下丘脑 – 垂体肾上腺（HPA）轴。然而，不建议将这一措施作为发育年龄的唯一指标，因为不同物种和供应商之间的大鼠之间存在巨大差异38。在VO和体重的发展中看到的可变性说明了该概念在整个实验过程中的重要性。

对人类的翻译：文化和科学背景
动物与人类生殖研究的转化关系是双向的。基于动物的研究结果会影响如何评估，接近和分析人类过程39.对人类生殖系统及其相关过程的感知会影响动物的研究方式。事实上，该领域进一步研究的最响亮迹象之一源于与影响科学过程的荷尔蒙周期相关的偏见社会文化信仰。其中许多惯例源于对讨论月经的普遍文化厌恶，这导致了在充分证实的知识方面存在数据差距40，41。这会产生一系列后果，从轻微到致命 – 从货架高度和智能手机尺寸到警察防弹衣的安装和错过的癌症诊断42。

将月经描述为不卫生，破坏性和有毒 – 在受人尊敬的文本，媒体，字典和医学教义中看到 – 被科学出版物所保留。这是通过对荷尔蒙周期的不准确和有偏见的描述，生殖系统与神经内分泌对应物和环境影响的隔离，以及将周期完成视为“未能受孕”的还原论观点43，44。这导致产生不健全的实验实践，例如省略影响荷尔蒙周期的外部变量，仅根据解剖学发展确定开始和终点，以及以线性而不是循环方式测量周期进展。尽管社会文化因素与生物学后果之间存在直接相关性，但在科学文献中并不经常考虑这一点。通过检查更全面的出版物43，44，45，研究人员可以解构这些耻辱感，并创建更可靠和有效的实验设计。

Protocol

本协议中概述的所有处理和程序方法均符合美国国立卫生研究院（NIH）动物护理和使用指南，并已获得佩珀代因大学机构动物护理和使用委员会（IACUC）和加州大学洛杉矶分校校长动物研究委员会（ARC）的批准。 1. 动物护理和使用 获取雌性大鼠，根据功率分析以数量计，雄性大鼠促进惠顿效应或更一致的循环46。根据已知数据库中的研究目标确定菌?…

Representative Results

目前的数据反映了女性青少年SD国际遗传标准化计划（IGS）在男性SD大鼠存在的情况下的数据。作为合作研究的一部分，这些动物位于佩珀代因大学和加州大学洛杉矶分校的实验室。 图5 显示了4个循环阶段的多种变化。 图5A1 被鉴定为存在几种细胞类型的双发性样品。该示例表明，具有大量上皮细胞的样品在满足其他分类组分资格（例如 LEI…

Discussion

关键步骤和重要注意事项
所提供方案中的某些关键步骤需要强调，特别是在阴道细胞的收集中。在阴道液提取过程中，确保注射器插入的适当角度和深度是产生满意结果并最终防止对动物的刺激，伤害或宫颈刺激的关键。子宫颈的刺激可以是假性妊娠诱导的一个来源，表现为12-14天的白细胞纯阴道涂片11。在显微镜评估阶段，关注显示收集的阴道细胞的视觉平面至…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究是通过加州大学洛杉矶分校脑损伤研究中心（BIRC）之间NIH资助的合作进行的。

Materials

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Vaginal LavageRodent Estrous CycleCell CharacterizationEpithelial CellsLeukocytesTranslational ResearchLow-cost TechniqueComprehensive Data
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Robert, H., Ferguson, L., Reins, O., Greco, T., Prins, M. L., Folkerts, M. Rodent Estrous Cycle Monitoring Utilizing Vaginal Lavage: No Such Thing As a Normal Cycle. J. Vis. Exp. (174), e62884, doi:10.3791/62884 (2021).
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