Hussein Mahmoud R, Bedaiwy Mohamed A, Falcone Tommaso
Department of Pathology, Assiut University School of Medicine, Assiut, Egypt.
Fertil Steril. 2006 Apr;85 Suppl 1:1082-92. doi: 10.1016/j.fertnstert.2005.10.020.
To evaluate the effect of ischemia time on the expression of Bcl-2 and p53 proteins on freshly fixed and cryopreserved-thawed ovarian tissue.
Experimental study using porcine animal model.
Biological Resources Unit, Cleveland Clinic Foundation.
ANIMAL(S): Eight nonpregnant adult sows.
INTERVENTION(S): Bilateral oophorectomy was performed in eight sows and the ovaries were subjected to time-dependent (1, 10, 20, and 30 min) warm ischemia (room temperature). Each specimen was divided into two parts, One was fixed as a fresh tissue (freshly fixed) and the other was subjected to cryopreservation, thawing, and fixation (cryopreserved).
MAIN OUTCOME MEASURE(S): Apoptosis (TUNEL assay) and Bcl-2 and p53 protein expression (immunoperoxidase method) were assessed.
RESULT(S): At 1, 10, 20, 30 min of warm ischemia the apoptotic indices were: 1) statistically significantly higher in the atretic (1.36 +/- 0.20, 1.59 +/- 0.20, 1.67 +/- 0.22, and 1.67 +/- 0.24, respectively) than in the nonatretic follicles (0.69 +/- 0.06, 0.69 +/- 0.06, 0.76 +/-, and 0.06, 0.71 +/- 0.06, respectively; P<.05); 2) not significantly different between freshly fixed and cryopreserved tissues; and 3) nonsignificantly higher with the increased duration of ischemia. Bcl-2 expression was seen in the granulosa but not in the theca cells of most of the healthy and occasional atretic follicles. p53 expression was seen only in few atretic follicles. Increased duration of ischemia was associated with insignificant incremental rise of the number of follicles with Bcl-2 expression (1.82 +/- 0.30, 2.01 +/- 0.44, 2.02 +/- and 0.35, 2.05 +/- 0.42 for healthy follicles at 1, 10, 20, and 30 min, respectively; P=.99).
CONCLUSION(S): (1) Apoptosis is involved in follicular atresia; (2) Bcl-2 is induced by warm ischemia; and (3) cryopreservation insult does not alter the apoptotic signals with short tissue preparation time.
评估缺血时间对新鲜固定及冷冻解冻后的卵巢组织中Bcl-2和p53蛋白表达的影响。
使用猪动物模型的实验研究。
克利夫兰诊所基金会生物资源部。
八只未怀孕的成年母猪。
对八只母猪进行双侧卵巢切除术，卵巢进行时间依赖性（1、10、20和30分钟）的温缺血（室温）处理。每个标本分为两部分，一部分作为新鲜组织固定（新鲜固定），另一部分进行冷冻保存、解冻和固定（冷冻保存）。
评估凋亡（TUNEL检测）以及Bcl-2和p53蛋白表达（免疫过氧化物酶法）。
在温缺血1、10、20、30分钟时，凋亡指数如下：1）闭锁卵泡中的凋亡指数（分别为1.36±0.20、1.59±0.20、1.67±0.22和1.67±0.24）在统计学上显著高于非闭锁卵泡（分别为0.69±0.06、0.69±0.06、0.76±0.06和0.71±0.06；P<0.05）；2）新鲜固定组织和冷冻保存组织之间无显著差异；3）随着缺血时间延长，凋亡指数无显著升高。在大多数健康卵泡和偶见的闭锁卵泡的颗粒细胞中可见Bcl-2表达，但在卵泡膜细胞中未见。仅在少数闭锁卵泡中可见p53表达。随着缺血时间延长，Bcl-2表达阳性的卵泡数量有不显著的增加（健康卵泡在1、10、20和30分钟时分别为1.82±0.30、2.01±0.44、2.02±0.35和2.05±0.42；P = 0.99）。
（1）凋亡参与卵泡闭锁；（2）温缺血可诱导Bcl-2表达；（3）在短组织制备时间内，冷冻保存损伤不会改变凋亡信号。
