Regeneration of meiotic spindle during oocyte vitrification

描述（由申请人提供）：卵母细胞的冷冻保存对于女性的生育能力保护至关重要。由于癌症治疗（如化疗和放疗）即将失去卵巢功能的癌症患者可以保存未受损的卵母细胞供日后使用。由于职业或婚姻状况而希望在老年时生孩子的女性可以通过使用保存的年轻卵母细胞来实现这一目标，这些卵母细胞可能具有完整的染色体。在过去的几年里，许多婴儿出生来自冷冻保存的卵母细胞。尽管如此，卵母细胞冷冻保存仍然被认为是一个实验性的程序，成功率是不同的诊所。因此，需要通过在细胞和分子水平上仔细检查玻璃化过程来进一步研究其安全性和稳健性。这些方法对于预测卵母细胞的任何潜在损伤特别重要，这可能会影响它们发育成健康个体。本研究的重点是利用快速冷冻法冷冻保存卵母细胞，探讨玻璃化冷冻过程中纺锤体再生的机制。减数分裂纺锤体是将正确数目的染色体分离到卵母细胞中的关键装置。纺锤体的损伤可能导致染色体不分离，这是发育失败和出生缺陷的主要原因。已知卵母细胞在冷冻过程中减数分裂纺锤体退化，但在解冻和加温过程中它再生。然而，纺锤体如何再生以及没有纺锤体时染色体排列是否稳定还没有得到充分的研究。在这方面，我正在使用小鼠和人类卵母细胞追求以下三个具体目标。在具体目标1中，将在玻璃化过程中检查几种纺锤形成核组分（如g-微管蛋白、围中心蛋白和NEDD 1）的定位。我的假设是，这些成核成分在卵母细胞冷冻过程中不受干扰，因此它们可以作为解冻后纺锤体再生的组织中心。在具体目标2中，我将真实的监控纺锤体再生的动态过程。将使用表达绿色荧光蛋白标记的b-微管蛋白的活卵母细胞，通过荧光延时电影摄影术检查再生过程。这种分析将产生洞察纺锤体再生的模式在空间和时间上的方式。在具体目标3中，将研究不存在纺锤体时中期染色体的稳定性。由于卵母细胞玻璃化会导致减数分裂纺锤体解聚，它可能会破坏染色体排列的稳定性，增加不分离的机会。我将确定各种玻璃化条件，特别是初始平衡步骤，对中期平板染色体比对的影响。这些信息将帮助我们改进玻璃化冷冻方案，以建立更一致和更安全的卵母细胞冷冻保存。

DESCRIPTION (provided by applicant): Cryopreservation of oocytes is becoming crucial for fertility preservation in women. Cancer patients who are about to lose their ovarian function because of cancer treatments, like chemotherapy and radiotherapy, can preserve their undamaged oocytes for later use. Women who desire to have a child at older ages due to their career or marital status can do so by using their preserved young oocytes that are likely to have intact chromosomes. In the past several years, a number of babies were born that were derived from cryopreserved oocytes. Nonetheless, oocyte cryopreservation is still considered as an experimental procedure, and success rates are variable among clinics. Thus, the process of vitrification needs to be further investigated for its safety and robustness by examining it carefully at the cellular and molecular levels. Such approaches are particularly critical to predict any potential damage to oocytes, which may affect their development into healthy individuals. The focus of this proposal is on the mechanisms of meiotic spindle regeneration during vitrification, which utilizes the fast-freezing method to cryopreserve oocytes. The meiotic spindle is a crucial apparatus to segregate the correct number of chromosomes into an oocyte. Damages to the spindle potentially result in chromosome nondisjunction, which is a major cause of developmental failure and birth defects. It is known that meiotic spindle degenerates during oocyte freezing but it regenerates during thawing and warming. However, how the spindle regenerates and whether chromosome alignment is stable without the spindle have not been fully investigated. In that respect, I am pursuing the following three Specific Aims, using the mouse as well as human oocytes. In Specific Aim 1, the localization of several spindle-nucleating components, such as g-tubulin, pericentrin, and NEDD1, will be examined during the vitrification process. My hypothesis is that these nucleating components are undisturbed during oocyte freezing so that they can serve as the organizing center for spindle regeneration after thawing. In Specific Aim 2, I will monitor the dynamic process of spindle regeneration in real time. The regeneration process will be examined by fluorescence time-lapse cinematography using live oocytes that express Green Fluorescent Protein-tagged b-tubulin. This analysis will generate insight into the mode of spindle regeneration in a spatial and temporal manner. In Specific Aim 3, the stability of metaphase chromosomes in the absence of spindle will be investigated. Because oocyte vitrification causes depolymerization of the meiotic spindle, it may destabilize alignment of chromosomes and increase the chance of nondisjunction. I will determine the effect of various vitrification conditions, particularly of the initial equilibration step, on chromosome alignment at the metaphase plate. This information will help us improve the vitrification protocol to establish a more consistent and safer cryopreservation of oocytes.