Garrett W M, Guthrie H D
Germplasm and Gamete Physiology Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705, USA.
Biol Reprod. 1996 Nov;55(5):949-55. doi: 10.1095/biolreprod55.5.949.
Among the new antral follicles that develop after ovulation in pigs, the incidence of atresia, based on granulosa cell apoptosis, increases between Days 5 and 7 of the estrous cycle. The purpose of this study was to determine how follicular growth and atresia affected the expression of some key enzymes regulating follicular steroidogenesis and androgen receptor on Days 3, 5, and 7 after the onset of estrus. Ovaries were frozen in liquid propane for subsequent sectioning and immunohistochemical analysis. Ninety-six follicles were classified according to size as small (< 3 mm), medium (3-5 mm), or large (> 5 mm). Follicles in the active stages of the cell cycle were identified by the presence of the cell proliferation-associated nuclear antigen Ki-67 in granulosa cells. Follicles with apoptotic cells were identified by in situ 3'-end labeling of DNA. Staining intensity of antigens on sections was assigned a numeric value (0-3). Follicles assigned a value > 1 for 3'-end labeling in their granulosa cells were classified atretic. The percentage of atretic follicles increased (p < or = 0.05) from 5% on Days 3 and 5 to 41% on Day 7. Expression of Ki-67 in granulosa cells was more strongly (p < or = 0.05) associated with nonatretic follicles (98% expressing) than with atretic follicles (41% expressing). Aromatase cytochrome P450 (P450arom) was localized predominantly in granulosa cells of nonatretic follicles and was undetectable in atretic follicles. Androgen receptor in granulosa cells and expression of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) in theca interna were lower (p < or = 0.001) in atretic follicles than in nonatretic follicles. The expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was localized to the theca interna and was unaffected by follicle atresia. In nonatretic small follicles, the expression of P450arom and P450c17 decreased (p < 0.01) between Days 3 and 7 while expression of Ki-67 was unchanged. In nonatretic follicles, increased follicle size was associated with a decrease (p < 0.01) in androgen receptor expression and increases (p < 0.01) in P450arom, P450c17, and 3 beta HSD expression. In conclusion, increased expression of steroidogenic enzymes was associated with follicular growth. Loss of P450arom expression in vivo is an early event in atresia and is followed by decreased cell proliferation, and decreased expression of androgen receptor and P450c17.
在母猪排卵后发育的新窦卵泡中，基于颗粒细胞凋亡的闭锁发生率在发情周期的第5至7天增加。本研究的目的是确定卵泡生长和闭锁如何影响发情开始后第3、5和7天调节卵泡类固醇生成的一些关键酶以及雄激素受体的表达。将卵巢在液体丙烷中冷冻以便随后进行切片和免疫组织化学分析。96个卵泡根据大小分为小卵泡（<3毫米）、中卵泡（3 - 5毫米）或大卵泡（>5毫米）。通过颗粒细胞中存在与细胞增殖相关的核抗原Ki - 67来识别处于细胞周期活跃阶段的卵泡。通过DNA的原位3' - 末端标记来识别有凋亡细胞的卵泡。切片上抗原的染色强度被赋予一个数值（0 - 3）。颗粒细胞中3' - 末端标记值>1的卵泡被分类为闭锁卵泡。闭锁卵泡的百分比从第3天和第5天的5%增加到第7天的41%（p≤0.05）。颗粒细胞中Ki - 67的表达与非闭锁卵泡（98%表达）的相关性比与闭锁卵泡（41%表达）更强（p≤0.05）。芳香化酶细胞色素P450（P450arom）主要定位于非闭锁卵泡的颗粒细胞中，在闭锁卵泡中未检测到。闭锁卵泡中颗粒细胞中的雄激素受体以及内膜细胞中P450 17α - 羟化酶/C17 - 20裂解酶（P450c17）的表达低于非闭锁卵泡（p≤0.001）。3β - 羟类固醇脱氢酶（3βHSD）的表达定位于内膜细胞，且不受卵泡闭锁的影响。在非闭锁小卵泡中，第3天至第7天P450arom和P450c17的表达下降（p<0.01），而Ki - 67的表达未改变。在非闭锁卵泡中，卵泡大小增加与雄激素受体表达下降（p<0.01）以及P450arom、P450c17和3βHSD表达增加（p<0.01）相关。总之，类固醇生成酶表达的增加与卵泡生长相关。体内P450arom表达的丧失是闭锁的早期事件，随后是细胞增殖减少、雄激素受体和P450c17表达降低。
