Winship Amy L, Sarma Urooza C, Alesi Lauren R, Hutt Karla J
Development and Stem Cells Program, Monash Biomedicine Discovery Institute, and Department of Anatomy and Developmental Biology, Monash University.
Development and Stem Cells Program, Monash Biomedicine Discovery Institute, and Department of Anatomy and Developmental Biology, Monash University;
J Vis Exp. 2020 Oct 16(164). doi: 10.3791/61782.
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.
有性繁殖的雌性哺乳动物在出生时就拥有其一生所需的全部卵母细胞。未成熟的静止卵母细胞存在于原始卵泡中，原始卵泡是雌性生殖系的储存单位。它们不可再生，因此出生时的数量以及随后的损失率在很大程度上决定了雌性的生育寿命。准确量化女性和动物体内的原始卵泡数量对于确定药物和毒物对卵巢储备的影响至关重要。对于评估现有和新兴的生育力保存技术的必要性和成功率而言，这也是必要的。目前，尚无方法能够准确测量构成女性卵巢储备的原始卵泡数量。此外，从大型动物或濒危物种获取卵巢组织进行实验通常不可行。因此，小鼠已成为此类研究的重要模型，而评估整个小鼠卵巢中原始卵泡数量的能力是一项关键工具。然而，文献中关于小鼠卵巢中卵泡绝对数量的报道差异很大，使得数据难以比较和/或重复。这是由多种因素造成的，包括品系、年龄、治疗组以及所采用计数方法的技术差异。在本文中，我们提供了一份分步指导指南，介绍如何使用两种不同方法制备组织切片并计数小鼠卵巢中的原始卵泡：[1] 体视学，它依赖于分数法/光学分割器技术；[2] 直接计数法。每种方法的一些关键优点和缺点将被突出强调，以便提高该领域的可重复性，并使研究人员能够为其研究选择最合适的方法。
