Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing-thawing of ovarian cortex in sheep

Abstract

Objective: To evaluate DNA fragmentation in the oocyte of primordial and primary follicles and morphology of these follicles after freezing and thawing of ovarian cortex in sheep using two freezing protocols. Design: Fragmentation of DNA was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique. Setting: Fertility clinic in a large university hospital. Animals: Five- to 6-month-old lambs. Intervention(s): Two-millimeter-thick slices of hemi-ovary cortex were prepared. Main Outcome Measure(s): Histological structure and DNA fragmentation. Result(s): In the frozen fragments, the percentage of morphologically normal follicles was significantly lower for both protocols compared with the case of the control group of fresh fragments. There was no significant difference between the two types of freezing protocols (60.4% ± 13.2% vs. 68.4% ± 13.7%). However, the distribution of abnormalities (nucleus, cytoplasm, and nucleus and cytoplasm) was dissimilar. The results of the TUNEL technique for the three groups showed no significant difference, but the percentage of the TUNEL-positive follicles was slightly lower for the frozen fragments for both protocols with respect to the control group. Conclusion(s): The freezing and thawing process of the ovarian cortex does not induce fragmentation of the DNA on the oocyte of primary and primordial follicles.