Apoptosis and active caspase-3 expression in human granulosa cells

Abstract

Objective: To document the expression of activated forms of caspase-3 in human granulosa cells.

Design: Laboratory study.

Setting: In vitro fertilization (IVF) laboratory of the Split University Hospital and laboratory of the Department of Anatomy, Histology, and Embryology.

Patient(s): Ovarian tissues were obtained from women undergoing hysterectomy/ovariectomy for benign conditions and human granulosa cells were obtained from women undergoing oocyte retrieval for IVF.

Intervention(s): Immunostaining of tissue sections and cell smears using antibody to active caspase-3 and terminal deoxynucleotidyl transferase (TdT) assay (TUNEL) for detection of internucleosomal DNA fragmentation.

Main outcome measure(s): Microscopic evaluation to assess the presence and cellular co-localization of active caspase-3 and TUNEL-positive cells.

Result(s): In human ovarian tissue, no apoptosis was observed in primordial and primary follicles. Apoptosis in granulosa cells was detected only in atretic antral follicles. Granulosa cells classified as apoptotic on the basis of their morphologic features contained a single condensed nucleus, multiple nuclear fragments, or apoptotic bodies. All apoptotic granulosa cells expressed active caspase-3, but only few contained fragmented DNA detected with the TUNEL method. The expression of active caspase-3 was also demonstrated in human granulosa cells of preovulatory follicles obtained from patients undergoing IVF.

Conclusion(s): Caspase-3 dependent apoptosis occurs in human granulosa cells and activates when follicles begin to leave the resting pool. After initial formation of the antrum, activation of caspase-3 is a normal physiologic process of the follicle during atresia and luteinization. Higher numbers of granulosa cells positive with caspase-3 than cells positive with TUNEL suggest an earlier activation of caspase-3 compared with the DNA fragmentation detected by TUNEL assay and also a longer detection period of caspase-3 than DNA fragmentation in apoptotic granulosa cells.