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.


METHODS
Experimental study using porcine animal model.


METHODS
Biological Resources Unit, Cleveland Clinic Foundation.


METHODS
Eight nonpregnant adult sows.


METHODS
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).


METHODS
Apoptosis (TUNEL assay) and Bcl-2 and p53 protein expression (immunoperoxidase method) were assessed.


RESULTS
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).


CONCLUSIONS
(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.
Hussein
Mahmoud R
MR
Department of Pathology, Assiut University School of Medicine, Assiut, Egypt.
Bedaiwy
Mohamed A
MA
Falcone
Tommaso
T
D000818:Animals; D017209:Apoptosis; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D005786:Gene Expression Regulation; D006358:Hot Temperature; D066298:In Vitro Techniques; D010053:Ovary / Q000378:metabolism* / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology*; D013552:Swine; D014018:Tissue Distribution; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53