Zucker R M, Keshaviah A P, Price O T, Goldman J M
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, North Carolina, USA.
J Histochem Cytochem. 2000 Jun;48(6):781-91. doi: 10.1177/002215540004800607.
This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pro, LysoTracker Red, hydroethidine, ethidium bromide, and 7-aminoactinomycin-d) were applied either to fresh tissue or to tissue that had been fixed with glutaraldehyde or paraformaldehyde. After fixation and staining, the tissue was dehydrated with MEOH and cleared with benzyl alcohol/benzyl aldehyde. CLSM was then used with the appropriate laser excitation, dichroics, and bandpass filters to acquire images of oocytes contained in follicles. Analysis of the data revealed three principal findings. First, a rapid increase in oocyte size occurred in the preantral stages of follicle development. In the antral stage of follicle development, there was a rapid increase in follicle size without any substantial increase in oocyte size. Second, accompanying these changes in oocyte and follicle growth was a differential staining pattern in which the nucleus stained more than the cytoplasm in a young follicle, but stained less than the cytoplasm as the follicle enlarged into the late antral stage. Lastly, using CLSM, atretic follicles showed increased LysoTracker Red staining in the granulosa region of the antral follicle, suggestive of cell death.
本研究使用共聚焦激光扫描显微镜（CLSM）来研究大鼠卵巢毫米切片中的卵泡发育。要使用该技术，在使用共聚焦显微镜进行激光激发之前对组织进行染色至关重要。将各种荧光染料（Yo-Pro、Bo-Pro、溶酶体示踪剂红、氢乙锭、溴化乙锭和7-氨基放线菌素-D）应用于新鲜组织或已用戊二醛或多聚甲醛固定的组织。固定和染色后，组织用甲醇脱水，并用苯甲醇/苯甲醛透明。然后使用CLSM，通过适当的激光激发、二向色镜和带通滤光片来获取卵泡中卵母细胞的图像。数据分析揭示了三个主要发现。第一，在卵泡发育的窦前期，卵母细胞大小迅速增加。在卵泡发育的窦期，卵泡大小迅速增加，而卵母细胞大小没有任何实质性增加。第二，伴随着卵母细胞和卵泡生长的这些变化，存在一种差异染色模式，即年轻卵泡中细胞核染色比细胞质多，但随着卵泡发育至晚期窦期，细胞核染色比细胞质少。最后，使用CLSM观察到，闭锁卵泡在窦卵泡的颗粒细胞区域显示溶酶体示踪剂红染色增加，提示细胞死亡。
