在小鼠和大鼠中确认青春期发作和执行生育力测定生殖能力
Summary
许多治疗和基因突变影响性成熟和生育的时间。本议定书描述了一种非侵入性的方法来评估小鼠和大鼠青春期发作之前, 建立一个生育研究在有性成熟的动物。
Abstract
对生殖能力的评估对于理解治疗或基因操作对生殖轴的影响至关重要, 也称为下丘脑-垂体-性腺轴。生殖轴是环境和内部输入的关键集成商, 适应生育条件, 有利于繁殖。在开始对小鼠和大鼠的生育研究之前, 性成熟度被评估为排除了观察到的生殖表型是由延迟或缺席青春期发作引起的可能性。本议定书描述了一种非侵入性的方法来评估男性青春期发作通过确定包皮分离, 并在女性通过阴道开放和第一次发情。在确认青春期完成和性成熟的成就后, 可以开始生育研究。该程序描述了小鼠和大鼠的最佳育种条件, 如何建立生育研究, 以及评估和确定治疗或基因删除是否影响生育能力的参数。
Introduction
通过青春期的过渡需要达到性成熟和生殖能力。成年期的青春期过渡和生育能力的维持由生殖轴调节, 也称为下丘脑-垂体-性腺轴 (图 1)。青春期开始和维持生育的时间是严格的内部和环境因素, 以增加后代和父母的生存几率1,2。该议定书提供了一种非侵入性的方法来确定小鼠和大鼠的青春期发作, 以确认在建立生育研究以评估生殖能力之前的性行为成熟度。
生育研究是在性成熟的动物进行的, 可以在动物经历青春期后开始。在青春期开始之前, 生殖轴是静止的, 并且性成熟的关键驱动因素, 促性腺激素释放激素 (促性腺素), 释放到垂体不足以启动青春期 (图 1)。青春期发作是一个复杂的过程, 导致增加促性腺激素释放在中位隆起。促性腺激素促进促黄体素 (LH) 和卵泡刺激激素 (FSH) 分泌从垂体, 两种激素对性腺成熟和生殖功能至关重要 (图 1)3,4,5.
对生殖轴的侮辱导致生育能力下降, 也可以促进或延缓青春期发作。已知影响青春期发作时间和生殖能力的条件包括接触内分泌干扰化学品6,7, 增加/减少体重1,8, 变化日长2,9和遗传突变10,11,12,13,14,15。
性成熟的开始是一个关键步骤, 需要在建立生育试验之前完成。通过包皮分离、阴道开放和第一次发情确定青春期发作的好处是这些程序的非侵入性特征, 因为它们不需要收集或牺牲动物16,17。
在青春期发作确定后, 正确地建立生育研究将提供有关生殖轴完整性的重要信息, 并且通常有生成实验动物的第二个优势进一步研究 (细化)18. 本议定书所述的生育率研究设置可以检测男性和女性生殖能力方面的次要和重大缺陷。评估的关键参数包括 1) 时间到第一个垃圾, 2) 在给定的时间段内生成的凋落物的数量和 3) 垃圾大小。最后, 还包括为确定生育障碍原因而进行的后续研究类型的建议。
所述的协议是指小鼠, 代表数据反映了转基因小鼠所做的工作。但是, 所有包含的协议在大鼠中同样有效。
Protocol
此处描述的所有方法均已通过密歇根州立大学的机构动物护理和使用委员会批准, 并按照实验室动物的护理和使用指南进行。 1. 确定青春期发作 遵循服装的制度准则, 至少, 有必要穿干净的实验室大衣和干净的手套。总是用干净的手套来处理老鼠。 通过在桌子上放置一个垫子来准备工作区域。 将干净的鼠标保持架顶部与网格朝向上朝上。 在工作?…
Representative Results
所提供的结果来自两个不同的转基因小鼠模型, 其中转录因子腹前同源盒 1 (Vax1) 已被删除在整个身体上的一个等位基因, 这里称为杂合子小鼠 (13), 或Vax1已有条件地删除在促性腺激素的神经元22, 这里称为条件 KO (cKO)。在建立生育研究之前, 确认所有小鼠青春期发作是很重要的。 ?…
Discussion
小鼠的整体健康对于成功的生育力测定21至关重要。在进行生育试验时, 不每天对老鼠进行身体检查是很重要的, 因为这会引起压力。进一步避免频繁的笼型变化, 因为这些也有压力。理想的笼型改变每周不超过1-2 次。暗相照射对夜间啮齿动物的繁殖有负面影响。在黑暗的时间里, 不要在繁殖室开灯。如果在天黑时需要进入房间, 请使用昏暗的红光照明。另一个压力源是在谢切诺夫…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我感谢提交人为本出版物的基础而作出的初步工作。多亏了 Aitor 老板, 珍娜. 瑞安和艾瑞卡 l. 舍勒帮助准备手稿。多亏了杰西卡. 苍利和奥斯汀下巴的技术援助手稿。H.M.H. 是由尤尼斯肯尼迪国家儿童健康 & 人类发展研究所的国家卫生研究院的奖励编号 R00HD084759 支持。
Materials
| Sterile Cotton Balls | Fisher | 22456885 | |
| Surface protector | Fisher | 1420637 | |
| Light meter | VWR | 21800-014 | |
| Methylene blue | Sigma-Aldrich | M9140 | |
| Microscope Slides | Genesee Scientific | 29-101 | |
| Optimouse rack with cages | AnimalCare systems | C89100 | |
| Water Bottle Basket | AnimalCare systems | C61011 | |
| Filtered Cage Tops | AnimalCare systems | C78210 | |
| Optimice Standard Feeder | AnimalCare systems | C40100SG | |
| Cage Card Holder | AnimalCare systems | C43251 | |
| Cage Cards | AnimalCare systems | M52010 | |
| Bottle Assambley | AnimalCare systems | C79122P | |
| Bed R'Nest Nesting | The Andersons | BRN4WSR | |
| 1/8" Corn Cob bedding | The Andersons | 8B | |
| Standard mouse chow | Teklad | 7904 (7004) | |
| Scale | VWR | 10205-004 | |
| Polypropylene Beaker | Fisher | 14-955-111F |
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Citazione di questo articolo
Hoffmann, H. M. Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats. J. Vis. Exp. (140), e58352, doi:10.3791/58352 (2018).