Songsasen N, Thongkittidilok C, Yamamizu K, Wildt D E, Comizzoli P
Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, 22630, USA.
Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, 22630, USA.
Theriogenology. 2017 Mar 1;90:228-236. doi: 10.1016/j.theriogenology.2016.12.006. Epub 2016 Dec 9.
Using the domestic cat as a non-rodent, larger animal model, the objective was to determine the impact of a brief incubation in a hypertonic microenvironment on (1) ovarian follicle and oocyte growth in vitro, (2) developmental capacity of the resident oocyte, and (3) expression of aquaporin (AQP) genes in parallel with genes involved in regulation of folliculogenesis. In Study 1: Secondary or early antral follicles encapsulated in 0.5% alginate were allocated to one of three treatment groups: 1) culture in standard medium at 290 mOsm for 15 d (Control); 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for 15 d (Hypertonic-1h); or 3) incubation in 350 mOsm medium for 24 h followed by incubation in standard medium for additional 14 d (Hypertonic-24h). After measuring follicle and oocyte diameters on Day 15, in vitro-grown oocytes were incubated for 24 h before assessing nuclear status. In Study 2: secondary or early antral follicles were subjected to one of the three treatments: 1) culture in standard medium at 290 mOsm for 48 h; 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for additional 47 h; or 3) incubation in 350 mOsm medium for 24 h followed by culture in standard medium for additional 24 h. At the end of the culture period, all follicles were assessed for mRNA level of Cyp17a1, Cyp19a1, Star, Aqp1, 3, 5, 7 and 8 as well as Fshr using qPCR. Freshly collected follicles also were subjected to gene expression analysis and served as the 'Non-cultured control'. Hypertonic-24h follicles grew larger (P < 0.05) than the control, whereas those in Hypertonic-1h group exhibited intermediate growth, especially when the culture started at the early antral stage. Oocytes in the Hypertonic-24h group were larger and resumed meiosis at a higher rate than in the other treatments. In vitro culture affected (P < 0.05) mRNA expression of Cyp19a1, Star, Aqp1, and Aqp7 in both the secondary and early antral stage while Fshr was only affected in the former compared to the non-cultured control. Pre-incubating follicles in 350 mOsm medium for 24 h enhanced (P < 0.05) Star and Aqp7 while decreasing (P < 0.05) Aqp1 expression compared to the control in secondary follicles, but not in the early antral stage. In summary, short-term hypertonic exposure promoted cat follicle development in vitro (including the meiotic competence of the enclosed oocyte) possibly through a mechanism that does not involve water transport genes.
以家猫作为非啮齿类的大型动物模型，目的是确定在高渗微环境中短暂孵育对以下方面的影响：（1）体外卵巢卵泡和卵母细胞的生长；（2）驻留卵母细胞的发育能力；（3）水通道蛋白（AQP）基因的表达以及与卵泡发生调控相关基因的表达。在研究1中：将包裹在0.5%海藻酸盐中的次级或早期窦状卵泡分配到三个处理组之一：1）在290 mOsm的标准培养基中培养15天（对照组）；2）在350 mOsm培养基中孵育1小时，然后在标准培养基中培养15天（高渗-1小时组）；或3）在350 mOsm培养基中孵育24小时，然后在标准培养基中再孵育14天（高渗-24小时组）。在第15天测量卵泡和卵母细胞直径后，体外培养的卵母细胞在评估核状态前孵育24小时。在研究2中：次级或早期窦状卵泡接受三种处理之一：1）在290 mOsm的标准培养基中培养48小时；2）在350 mOsm培养基中孵育1小时，然后在标准培养基中再培养47小时；或3）在350 mOsm培养基中孵育24小时，然后在标准培养基中再培养24小时。在培养期结束时，使用qPCR评估所有卵泡中Cyp17a1、Cyp19a1、Star、Aqp1、3、5、7和8以及Fshr的mRNA水平。新鲜收集的卵泡也进行基因表达分析，并作为“未培养对照”。高渗-24小时组的卵泡比对照组生长得更大（P<0.05），而高渗-1小时组的卵泡生长处于中间水平，尤其是当培养从早期窦状阶段开始时。高渗-24小时组的卵母细胞比其他处理组的更大，并且减数分裂恢复率更高。体外培养影响（P<0.05）次级和早期窦状阶段卵泡中Cyp19a1、Star、Aqp1和Aqp7的mRNA表达，而与未培养对照相比，Fshr仅在前者中受到影响。与对照组相比，在350 mOsm培养基中预孵育卵泡24小时可增强（P<0.05）次级卵泡中Star和Aqp7的表达，同时降低（P<0.05）Aqp1的表达，但在早期窦状阶段则不然。总之，短期高渗暴露可能通过不涉及水转运基因的机制促进了猫卵泡的体外发育（包括封闭卵母细胞的减数分裂能力）。
