Analysis of apoptotic cell death, Bcl-2, and p53 protein expression in freshly fixed and cryopreserved ovarian tissue after exposure to warm ischemia

Objective: To evaluate the effect of ischemia time on the expression of Bcl-2 and p53 proteins on freshly fixed and cryopreserved-thawed ovarian tissue. Design: Experimental study using porcine animal model. Setting: 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.