绵羊卵丘-卵母细胞复合体石蜡切片技术的改良

doi:10.3864/j.issn.0578-1752.2017.08.018

摘要/Abstract

摘要： 【目的】在绵羊有腔卵泡发育过程中，多层卵丘细胞紧密包绕着卵母细胞形成卵丘-卵母细胞复合体（cumulus-oocyte complex，COCs）特殊结构，存在于卵泡腔内。卵泡成熟后，绵羊COC从卵泡内排出，进入输卵管，等待受精。现有的卵丘-卵母细胞复合体蛋白鉴定技术，或者将包含各级卵泡的卵巢组织直接进行切片处理，或者将COCs从卵泡内分离，在完整卵丘-卵母细胞复合体水平上进行免疫染色。但这两种方法在检测绵羊有腔卵泡COCs中目的蛋白表达时，存在显著缺陷。研究旨在对常规石蜡组织切片技术进行改进，以期满足绵羊COCs蛋白鉴定需求。【方法】首先采用抽吸法从绵羊有腔卵泡内获得COCs，以石蜡包埋方式人为构建了一种可以进行切片处理的“包含多枚绵羊卵丘-卵母细胞复合体的仿组织结构”。然后以该特殊结构与健康绵羊卵巢组织为研究对象，分别采用改良与传统的石蜡组织免疫化学技术流程，检测了目的蛋白，尿激酶型纤溶酶原激活剂（urokinase-type plasminogen activator, uPA）与其受体（urokinase-type plasminogen activator recepter, uPAR），在绵羊COCs中的表达情况，比较了两种技术的免疫染色效果与染色流程差异。【结果】将所构建的绵羊COCs仿组织结构与正常绵羊卵泡组织，进行石蜡组织切片处理，经切片、贴片与脱蜡等步骤，分别形成了散在分布于载玻片上的COCs薄片与包含卵泡的卵巢组织薄层（厚度5µm）。经间接免疫染色处理后，结果显示：?目的蛋白在两种结构COCs中的表达是一致的，即uPAR在绵羊卵丘与卵母细胞上都有表达，而uPA只存在于绵羊卵丘细胞中；?在绵羊COCs薄片结构中，COCs整体形态完好，卵母细胞与外围卵丘细胞层次清晰，蛋白定位清楚，染色效果良好；在卵巢薄层结构中，绵羊卵泡内壁颗粒细胞与COCs等形态结构相对完整，卵母细胞蛋白定位清楚，但是卵丘细胞层次与蛋白表达较不清晰；?与绵羊卵巢组织石蜡切片技术相比较，绵羊COCs仿组织结构石蜡切片技术，在固定、脱水、透明、浸蜡以及脱蜡等步骤的处理时间都大大缩短，例如固定处理由24h缩短为2h；由逐级脱水的10h缩短为两级脱水的1h；透明由30min缩短为10min；软蜡与硬腊浸入由原来的6h缩短为1h；由逐级脱蜡的25min缩短为两级脱蜡的10min等。【结论】改进后的COCs仿组织石蜡切片染色技术，显示更优质的免疫定位与染色效果，不仅具有COCs获得率高、卵母与卵丘细胞形态层次清晰等优点，而且显著缩短了操作时间，简化了操作流程。该技术在保留COCs结构完整的基础上，有效检测了目的蛋白在绵羊卵丘-卵母细胞复合体中的表达模式，对于探明绵羊卵泡发育与卵母细胞成熟机制，具有重要意义。

关键词: 绵羊, 卵丘-卵母细胞复合体仿组织, 卵巢组织, 石蜡切片, 蛋白表达

Abstract: 【Objective】 During the ovine antral follicle development, the multilayer cumulus cells closely surround the oocyte to form a special structure named cumulus-oocyte complex (COC), existing in the follicular cavity. The ovine COC will be discharged tube from the mature follicle into the fallopian to be fertilized. The existing technologies for protein identification in COC choose the ovarian tissue containing follicles to be sliced up or to immunostain the whole COC after the COC isolated from the follicle. However, there are significant defects when using the two methods to detect protein expression in ovine COC from the antral follicle. In the present study, the conventional paraffin section technology was improved so as to meet the demand of ovine COC protein identification. 【Method】 The COCs were obtained from the antral follicles by aspiration, and the special structure containing multiple ovine COCs was created by paraffin embedding. Together with the healthy ovarian tissue, the target protein expression, urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR), were identified in the COCs structure and the immunostaining effects and procedures were compared between the modified and conventional paraffin immunohistochemical technologies. 【Result】 The samples of ovine COCs and ovaries were obtained by paraffin embedding. The single COC sheets distributed in glass slides and the thin ovarian layer (5µm) was formed following sectioning, plastering and dewaxing. The indirect immunostaining results showed that, ? The target protein expression was consistent in the two types of COCs, which revealed that uPAR was expressed in both bovine cumulus and oocyte, while uPA was only expressed in cumulus. ? In the COC section, a well-preserved bovine COC structure with well-defined layers and easily identified protein location in either the oocyte or cumulus was observed. In the ovarian section, the follicle remained intact, and the clear protein localization in oocyte could be observed; however, cumulus layers and its protein localization were unclear. ?A comparison of the staining procedures between the both methods described herein showed that ovine COCs paraffin-sectioning technique provided a more simplified process and significantly shorter handling for fixation, dehydration, transparent processing, waxing and dewaxing. For example, fixation was shortened from 24 h to 2 h; dehydration was converted from the granual steps of 10 h to the two-step of 1 h; transparent processing was shortened from 30 mins to 10mins; waxing and dewaxing were simplified from 6 h to 1 h, and from the granual steps of 25 mins to the two-step of 10 mins, respectively. 【Conclusion】 The modified COCs paraffin sectioning technique showed a better immunostaining effect and featured the higher generation rate for COCs, the clearer outline of oocyte and cumulus cells, and the more simplified operation process. Based on the complete COCs structure, this new technology effectively detected the target protein expression in ovine COCs, with great methodological significance to reveal the developing mechanism of ovine follicles.

Key words: sheep, cumulus-oocyte complexes-like tissue, ovarian tissue, paraffin section, protein expression

李海军，于泊洋，段云娇，刘星宇，翁娅政，杜晨光，王秀梅. 绵羊卵丘-卵母细胞复合体石蜡切片技术的改良[J]. 中国农业科学, 2017, 50(8): 1543-1550.

LI HaiJun, YU BoYang, DUAN YunJiao, LIU XingYu, WENG YaZheng, DU ChenGuang, WANG XiuMei. A Modified Paraffin-Section Technique for Ovine Cumulus-Oocyte Complexes[J]. Scientia Agricultura Sinica, 2017, 50(8): 1543-1550.

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