Blondin P, Dufour M, Sirard M A
Département des sciences animales, Université Laval Ste-Foy, Québec, Canada.
Biol Reprod. 1996 Mar;54(3):631-7. doi: 10.1095/biolreprod54.3.631.
This study was performed in order to validate flow cytometry as an acceptable method for analyzing follicular atresia in bovine granulosa cells by comparing it to two other techniques, histology and ELISA. Ovaries from 35 nontreated cows, all at different times of their estrous cycle, and 12 superovulated cows were collected. Superovulation treatments began between Days 9 and 12 (Day 0 = estrous), and animals were administered 8 doses of FSH-P (32 or 20 mg) at 12-h intervals over 4 days with or without the addition of 1 mg of prostaglandin s.c. on the third day. Animals were slaughtered after the last FSH-P injection. Granulosa cells from 133 follicles from non-treated cows and 85 follicles from superovulated cows were analyzed. Follicular diameters ranged from 2 to 20 and 2 to 16 mm, respectively. Because of the ample amounts of cells collected, it was possible to perform more than one technique for each follicle. Flow cytometry detected in most follicles a subpopulation of cells that possessed less DNA than normal, viable cells (referred to as -G1 cells). Histological classes used (established in previous work) were nonatretic (< or = 5% picnotic nuclei), slightly atretic (> 5 to < 15% picnotic nuclei), and atretic (> or = 15% picnotic nuclei). A strong linear correlation existed between the percentage of picnotic nuclei and the percentage of -G1 cells (R = 0.86; p < 0.001) with granulosa cells from follicles from nontreated cows. In some cases, flow cytometry detected a certain percentage of cells with reduced DNA content while histology revealed very few picnotic nuclei, indicating a higher sensitivity of flow cytometry. Superovulation decreased considerably the percentage of atretic cells seen with both techniques. The linear correlation was not as strong because follicles from superovulated animals represent a very homogenous population (R = 0.54; p < 0.001). The ELISA technique coincided with flow cytometry as seen in the strong correlation between the two techniques (R = 0.91; p < 0.001). Flow cytometry appeared to be very effective and rapid in evaluating the atretic states of follicles from nontreated and superovulated cows. Strong correlations existed between this method and histology and ELISA.
本研究旨在通过将流式细胞术与另外两种技术（组织学和酶联免疫吸附测定法）进行比较，验证其作为分析牛颗粒细胞卵泡闭锁的可接受方法。收集了35头未经处理的奶牛（处于发情周期的不同阶段）和12头经超数排卵处理奶牛的卵巢。超数排卵处理在第9至12天（第0天=发情期）开始，动物在4天内每隔12小时注射8剂促卵泡素-P（32或20毫克），第三天可选择皮下注射1毫克前列腺素。在最后一次注射促卵泡素-P后屠宰动物。分析了来自未经处理奶牛的133个卵泡和经超数排卵处理奶牛的85个卵泡的颗粒细胞。卵泡直径分别为2至20毫米和2至16毫米。由于收集到的细胞数量充足，每个卵泡可采用不止一种技术进行分析。流式细胞术在大多数卵泡中检测到一群DNA含量低于正常活细胞的亚群细胞（称为-G1细胞）。所使用的组织学分类（在之前的研究中确定）为非闭锁卵泡（≤5%固缩核）、轻度闭锁卵泡（>5%至<15%固缩核）和闭锁卵泡（≥15%固缩核）。未经处理奶牛卵泡的颗粒细胞中，固缩核百分比与-G1细胞百分比之间存在很强的线性相关性（R = 0.86；p < 0.001）。在某些情况下，流式细胞术检测到一定比例的DNA含量降低的细胞，而组织学显示固缩核很少，这表明流式细胞术具有更高的灵敏度。超数排卵显著降低了两种技术所观察到的闭锁细胞百分比。线性相关性没有那么强，因为经超数排卵处理动物的卵泡代表了一个非常同质的群体（R = 0.54；p < 0.001）。酶联免疫吸附测定法与流式细胞术结果一致，两种技术之间存在很强的相关性（R = 0.91；p < 0.001）。流式细胞术在评估未经处理和经超数排卵处理奶牛卵泡的闭锁状态方面似乎非常有效且快速。该方法与组织学和酶联免疫吸附测定法之间存在很强的相关性。
