Apoptosis and active caspase-3 expression in human granulosa cells.
OBJECTIVE
To document the expression of activated forms of caspase-3 in human granulosa cells.


METHODS
Laboratory study.


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


METHODS
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.


METHODS
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.


METHODS
Microscopic evaluation to assess the presence and cellular co-localization of active caspase-3 and TUNEL-positive cells.


RESULTS
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.


CONCLUSIONS
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.
Glamoclija
Violeta
V
Department of Anatomy, Histology, and Embryology, School of Medicine, University of Split, Split, Croatia. vg@mefst.hr
Vilović
Katarina
K
Saraga-Babić
Mirna
M
Baranović
Anamarija
A
Sapunar
Damir
D
D000328:Adult; D053148:Caspase 3; D020169:Caspases / Q000235:genetics*; D053938:DNA Fragmentation; D005260:Female; D005307:Fertilization in Vitro; D015971:Gene Expression Regulation, Enzymologic; D006107:Granulosa Cells / Q000166:cytology* / Q000201:enzymology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques
C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases