Vitrification preserves murine ovarian follicular cell transcriptome in a 3D encapsulated in vitro follicle growth system.
Vitrification preserves murine ovarian follicular cell transcriptome in a 3D encapsulated in vitro follicle growth system.
复制标题
玻璃化冷冻在 3D 封装的体外卵泡生长系统中保存小鼠卵巢卵泡细胞转录组。
DOI：
10.1093/biolre/ioab185
复制
发表时间：
2021
影响因子：
3.6
通讯作者：
Xiao,Shuo
中科院分区：
文献类型：
--
作者：
Wang,Yingzheng;Drake,RileyS;Russo,DanielaD;Pattarawat,Pawat;Zhang,Qiang;Zelinski,MaryB;Shalek,AlexK;Goods,BrittanyA;Xiao,Shuo
尊敬的编辑，玻璃化冷冻是一种用于长期生物样品冷冻保存的方法，通过冷却将细胞转化为玻璃状状态，而不会导致细胞内和细胞外的冰形成，这是细胞冷冻损伤的主要驱动因素。与另一种传统的冷冻保存方法缓慢冷冻相比，玻璃化冷冻简单，成本效益高，不需要复杂的可编程冷冻机[1]。在辅助生殖技术（ART）中，玻璃化冷冻越来越多地用于冷冻保存配子和胚胎以保持生育力[2]。此外，玻璃化冷冻单个卵泡，然后进行体外成熟（IVM）已成为一种新的生育力保存方法，特别是对于没有成熟卵母细胞可供收获的儿童癌症患者以及由于冷冻保存后无法接受卵巢组织移植的患者。重新引入恶性细胞的风险[3]。然而，单个卵泡的玻璃化冷冻一直具有挑战性，因为完整的卵泡具有比单个卵母细胞或早期胚胎更复杂的结构和更大的尺寸。传统的卵母细胞/胚胎玻璃化冷冻方法对于单个卵泡并不优化，并且已经显示会损害卵泡或卵母细胞的质量，部分原因是破坏卵泡细胞之间的差距连接或卵母细胞与卵丘细胞之间的跨带突起（TZP）[4]。在我们以前的研究中，我们开发了一种用于冷冻保存卵巢组织的封闭玻璃化方法[5]，该方法针对单个卵泡进行了修改[6]。此外，使用藻酸盐水凝胶包封的体外卵泡生长（eIVFG）系统，我们最近证明，与新鲜收获的卵泡相比，玻璃化卵泡具有正常的卵泡和卵母细胞生殖结果，以及促性腺激素依赖性卵泡发生和卵子发生所必需的几种基因的表达水平相当[6]。然而，目前尚不清楚玻璃化冷冻是否在整个转录组水平上保留了卵泡发生的分子特征，这是本研究的主要研究重点。如我们先前的研究[6]所述，从16日龄CD-1雌性小鼠中机械分离直径为130-160 μm的多层次级卵泡，并使用封闭玻璃化冷冻方法进行玻璃化冷冻。在液氮中储存2周后，将玻璃化冷冻的卵泡加温，并与新鲜收获的卵泡一起使用eIVFG方法培养8天[7-9]。与我们之前的结果一致[6]，玻璃化冷冻卵泡具有与新鲜卵泡相当的发育模式，并且能够从第0天的次级阶段生长到第8天的窦期以达到成熟（补充图1A）。第8天，玻璃化冷冻和新鲜卵泡的卵泡存活率分别为92.5%和93.1%，卵泡直径分别为358.9±24.3和350.6±68.8 μm（补充图1B和1C）。接下来，
Dear editor, Vitrification is a method for long-term biological sample cryopreservation that transforms cells into a glass-like state by cooling without causing intra-and extra-cellular ice formation, which is a major driver of cell cryoinjury. Compared to slow freezing, another conventional cryopreservation method, vitrification is simple, costeffective and does not require a complex programmable freezer [1]. Vitrification has been increasingly used to cryopreserve gametes and embryos for fertility preservation in assisted reproductive technology (ART)[2]. Moreover, vitrification of individual follicles followed by in vitro maturation (IVM) has emerged as a new fertility preservation method, particularly for childhood cancer patients who have no mature oocytes available for harvesting and for patients who cannot undergo ovarian tissue transplantation after cryopreservation because of the risk of reintroducing malignant cells [3]. However, vitrification of individual follicles has been challenging because intact follicles have a more complex structure and larger size than individual oocytes or early embryos. Traditional oocyte/embryo vitrification methods are not optimized for individual follicles, and have been shown to compromise the qualities of follicles or oocytes, partly by damaging the gap junction between follicular cells or the transzonal projections (TZP) between the oocyte and cumulus cells [4]. In our previous studies, we developed a closed vitrification method for cryopreserving ovarian tissues [5] that was modified for individual follicles [6]. Furthermore, using an alginate hydrogel encapsulated in vitro follicle growth (eIVFG) system, we have recently demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes as well as comparable expression levels of several genes that are essential for gonadotropin-dependent folliculogenesis and oogenesis [6]. However, it is unknown whether vitrification preserves the molecular signatures of folliculogenesis at the whole transcriptomic level, which is the primary research focus in this study.As described in our previous studies [6], multilayered secondary follicles with a diameter of 130-160 μm were mechanically isolated from 16-day-old CD-1 female mice and vitrified using the closed vitrification method. After a 2-week storage in liquid nitrogen, vitrified follicles were warmed, and together with freshlyharvested follicles, cultured for 8 days using the eIVFG method [7–9]. Consistent with our previous results [6], vitrified follicles had a comparable development pattern to fresh follicles and were able to grow from the secondary stage on day 0 to the antral stage on day 8 to reach maturity (Supplemental Figure 1A). On day 8, the follicle survival rates were 92.5% and 93.1% and the follicle diameters were 358.9±24.3 and 350.6±68.8 μm for vitrified and fresh follicles, respectively (Supplemental Figures 1B and 1C). Next,