Apoptosis and active caspase-3 expression in human granulosa cells

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1 Apoptosis and active caspase-3 expression in human granulosa cells Violeta Glamočlija, M.D., M.Sc., a Katarina Vilović, M.D., Ph.D., a Mirna Saraga-Babić, M.D., Ph.D., a Anamarija Baranović, M.Sc., b and Damir Sapunar, M.D., Ph.D. a a Department of Anatomy, Histology, and Embryology, and b Department of Obstetrics and Gynecology, School of Medicine, University of Split, Split, Croatia 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. (Fertil Steril 2005;83: by American Society for Reproductive Medicine.) Key Words: Apoptosis, human ovary, granulosa cells, caspase-3, DNA fragmentation Apoptosis is the cellular mechanism involved in the ovarian follicular atresia and luteal regression (1). In the early stage of follicular development (primordial, primary, and small preantral), atresia is initiated by oocyte apoptosis followed by death of the granulosa cells (2). Atresia of maturing (late preantral, antral) and mature (subordinate preovulatory) follicles is first demarcated by scattered granulosa cell apoptosis. As atresia progresses in these follicles, the number of dying granulosa cells increases dramatically, and large masses of apoptotic bodies are shed into the antral space (3). Granulosa cells from a population of follicles that exhibit a high rate of atresia (prehierarchal follicles, 9 mm diameter) are highly susceptible to apoptotic cell death (4). There Received February 9, 2004; revised and accepted June 22, Supported by the Croatian Ministry of Technology and Science grants no and Reprint requests: Violeta Glamočlija, M.D., M.Sc., Department of Anatomy, Histology, and Embryology, School of Medicine, University of Split, PAK, KB Split, Spinčićeva 1, Split, Croatia (FAX: ; vg@mefst.hr). is evidence suggesting that the granulosa cells of the dominant follicles maintain this apoptotic potential even after ovulation (5). Studies concerning follicular atresia have been difficult because of the number and variety of follicles present in the ovary at any given time (6, 7). Apoptosis is characterized by specific changes in cell surface and nuclear morphologic features caused mostly by caspase-3 (8). This executioner caspase is activated by an initiator caspase through the process of proteolytic cleavage at specific internal aspartate residue, causing separation of the large and small subunits of the mature caspase (8 10). In animals, expression of caspase-3 has been found in leukocytes, in granulosa cells of atretic follicles, theca cells of the healthy follicles, and luteal cells of healthy corpora lutea (11, 12). The theca interna and externa cells of secondary ovarian follicles are also caspase-3 immunopositive whereas the granulosa cells are less intensely immunostained (13). Caspase-3 is functionally required for the normal execution of apoptotic cell death in granulosa cells, but the finding that granulosa cells lacking caspase-3 do eventually die suggests 426 Fertility and Sterility Vol. 83, No. 2, February /05/$30.00 Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 that caspase-independent mechanisms of cell death may be activated or that other caspase family members are involved (14). The expression of caspase-1, caspase-3, DNA fragmentation factor, and apoptotic protease activating factor-1, which are intermediate molecules in phylogenetically conserved apoptotic pathways, were demonstrated in granulosa cells from patients undergoing in vitro fertilization (IVF). Moreover, proforms, but not activated enzymes, for both caspase-1 and caspase-3 were observed (15). However, expression of activated forms of the caspase-3 in human ovarian follicles has not been examined. Existence of proforms does not guarantee execution of the apoptotic process because its cleavage into activated form may require additional regulatory mechanisms. Caspase-3 conversion of cytoplasmic DNase into an active form results in characteristic internucleosomal cleavage of DNA (8 10). The existence of such endonuclease activity in differentiated granulosa cells has previously been demonstrated in animals (5). The endonuclease responsible for apoptotic DNA fragmentation in granulosa cells is DNase I (12). Detection of the DNA fragmentation has been the principal method used in identification of apoptotic cells in atretic follicles, during luteal regression, and in human granulosa cells (7, 12, 16). In the present study, we examined the expression of active caspase-3 in human ovarian tissue and in granulosa cells obtained from preovulatory human ovary follicles to determine whether the activation of caspase-3 might play a role in the occurrence of apoptotic cell death in those tissues. In addition, we analyzed the relationship between apoptotic morphologic features and DNA fragmentation in granulosa cells of the ovarian follicles. MATERIALS AND METHODS Materials Human ovarian tissue was collected from a total of eight women between the ages of 19 and 42 years who were undergoing a hysterectomy/ovariectomy for benign conditions. Apoptosis was investigated in 445 ovarian follicles (398 primordial, 19 primary, 2 secondary, 24 antral, and 2 dominant). More than 90% of primordial and dominant follicles were from younger women (age range: 19 to 34 years). Ovaries were fixed immediately in 10% neutralbuffered paraformaldehyde for 24 hours. After fixation, the tissue was washed, dehydrated, embedded in paraffin, serially sectioned (4 6 m) and mounted on silane-coated glass slides (Sigma Chemical Co., St. Louis, MO) for further immunohistochemical manipulations. Some sections were stained with hematoxylin and eosin, and Gomori silver stain to identify apoptotic cells using light microscope (17). Human granulosa cells were obtained from a pooled collection of follicular fluid from 15 women (age range: 31 to 40 years) who were undergoing oocyte retrieval for IVF at Split University Hospital. A total of 63 preovulatory follicles from 15 women were aspirated and flushed with Synvitro flush (MediCult Inc., Hopkinton, MA). Approximately 200 L of discarded follicular fluid, aspirated and pooled from several follicles per patient, was carefully layered onto 300- L Ficoll Paque (Biotechnology AB, Uppsala, Sweden) and centrifuged at 2000 g for 5 minutes at 20 C. The granulosa cells formed a band at the interface, and the red cells sedimented to the bottom of the tube. The granulosa cells were carefully transferred into a tube containing hyaluronidase (Sigma Chemical), 0.1% wt/vol, and pipetted thoroughly for 10 minutes. After separation, part of the cells was fixed in 4% neutralbuffered paraformaldehyde for future immunocytochemical procedures; remaining cells were placed on a glass slide and mixed with fluorescent vital dye (acridine-orange and ethidium bromide). Apoptotic granulosa cells were identified using fluorescence microscope (18). This process was completed within 1 hour after follicle aspiration to avoid postaspiration cell death. Informed consent was obtained from each woman undergoing IVF procedure. The study was approved by University of Split Medical School Ethics Committee review board. Immunochemistry Presence and distribution of the active caspase-3 was detected using a monoclonal antibody raised against human caspase-3 (anti-human/mouse caspase-3 Active AF835; R&D Systems, Minneapolis, MN). Antigen unmasking was performed by microwaving tissue sections for 10 to 30 minutes in sodium citrate. After cooling, tissue sections and cell smears were treated with 3% H 2 O 2 for 30 minutes, washed in phosphate-buffered saline (PBS), and then incubated with primary antibody for 18 hours at 4 C. After washing, the slides were incubated with biotinylated secondary antibody (Mouse and Rabbit UniTect ABC Kit; Oncogene, Boston, MA) for 30 minutes at room temperature then washed again. Localization of the primary antibody biotinylated second antibody complex was performed using a diaminobenzidine (DAB) reaction. To permit visualization of the tissue architecture, slides were lightly counterstained with hematoxylin before coverslipping. The control experiments included incubation of slides in the absence of primary antibody and replacement of primary antibody with normal serum (19). Under these conditions, no immunoreaction was observed. TUNEL Assay Localization of DNA fragmentation in fixed ovarian tissue sections and granulosa cells smears was performed using a nonradioactive DNA-labeling technique with the TdT FragEL Kit (Oncogene). Tissue was treated with proteinase K for 20 minutes, and cells were treated for 5 minutes at Fertility and Sterility 427

3 room temperature. Tissues and cells were treated with 3% H 2 O 2 for 5 minutes and labeled with biotin-d UTP by incubation with reaction buffer containing terminal deoxynucleotidyl transferase enzyme for 30 minutes at 37 C. Biotinylated nucleotides were detected with streptavidin-horseradish peroxidase conjugate and visualized with DAB. Finally, slides were counterstained with methylene green. Negative control slides were processed in the same manner except the labeling enzyme was omitted (20). Under these conditions no immunoreaction was observed. RESULTS No apoptotic cells or positive staining for caspase-3 were found in granulosa cells of primordial, primary, or dominant antral follicles (data not shown). The results of the active caspase-3 and TUNEL staining in apoptotic granulosa cells from atretic antral follicle are shown in Figure 1, A to D. Numerous apoptotic granulosa cells are shown in a large (11-mm) atretic antral follicle. Those cells are characterized by the inclusion of one or more black circular bodies, contained within a translucent circular surrounding. These inclusions of densely stained bodies are considered to be a condensed chromatin and are indicative of a late stage of apoptosis. Apoptotic bodies were seen as cell fragments containing condensed chromatin when examined under the light microscope. The large atretic antral follicle contained disorganized membrane granulosa with numerous apoptotic granulosa cells spreading inside the follicle (see Fig. 1A). Immunostaining for active caspase-3 was most intense in the apoptotic granulosa cells of those follicles. Caspase-3 was localized in the cytoplasm whereas the nucleus contained little or no staining for this enzyme. No staining for this enzyme was observed in the theca cells (see Fig. 1B). Occasionally, granulosa cells exhibited nuclear changes at late stage of apoptosis, positive immunostaining for caspase-3 and showed DNA fragmentation (see Fig. 1C). Different types of DNA fragmentation were found in apoptotic granulosa cells: relatively equal staining of the whole nucleus or strong positive staining granules within the nucleus (see Fig. 1D). Caspase-3 and TUNEL staining was performed in subsequent adjacent sections, so it was not possible to assess the co-localization of caspase-3 and apoptosis in individual cells. Apoptotic granulosa cells in preovulatory follicles, obtained from patients undergoing IVF, were characterized by the inclusion of more circular bodies in their cytoplasm when examined under the fluorescence microscope. A typical mass of circular bodies, which include fragmented and shrunken nuclei uniformly stained with light green and orange fluorescence are indicative of early and late stages of apoptosis (see Fig. 1E). Apoptotic granulosa cells obtained from preovulatory follicles expressed an active caspase-3 both in the cytoplasm and nuclei (see Fig. 1F). DISCUSSION The follicles in all growth stages, from primary to large antral follicles, undergo atresia. Moreover, this process occurs throughout life, from the fetal period to the onset of reproductive senescence (6). Initial studies of rat, avian, and porcine ovaries documented a role for apoptotic cell death in the loss of granulosa cells during follicular atresia (6, 21). The exact events that culminate in granulosa cells artesia are likely to differ between the various-sized follicles. We found that activation of caspase-3 does not occur in primordial and primary follicles, which concurs with previous findings in human ovary where DNA fragmentation primarily occurs in the granulosa cell layers of the early antral follicles (7). Furthermore, because activation of caspase-3 in granulosa cells occurs as early as the small antral stage, apoptosis may begin as early as the initial formation of the antrum. These findings support the hypothesis that follicles in the resting pool are on hold (7). The initial stages of follicular development, occurring before the cycle in which the follicle will ovulate, are probably independent of gonadotropin. Later, under gonadotropin stimulation, the follicle acquires antrum and theca layers, and begins to produce steroids (22, 23). It is known that gonadotropins maintain a suppression of apoptosis in granulosa cells of antral follicles (24). The presence of caspase-3 in granulosa cells of atretic but not healthy antral follicles also suggests that the expression of this enzyme is regulated by gonadotropins and may be up-regulated as a part of the apoptotic process in granulosa cells (12). In spite of advances in genetic and immunochemical techniques, the problem persists of recognizing apoptotic cells in tissues. In apoptosis, the nucleus is typically condensed and forms buds, frequently with a crescent shape of condensed chromatin. The fragments of cells containing condensed chromatin are often referred to as apoptotic bodies and are the morphologic hallmark of apoptosis (21). Silver stain, as described in our study, is selective by staining condensed chromatin and is useful as an additional method for assessing follicular apoptosis. In the final stage of cell death, necrotic cells eventually exhibit swelling of the nucleus, whereas apoptotic cells exhibit characteristic nuclear morphologic changes, including chromatin condensation and hypersegmentation of nuclear chromatin of irregular size (17). Also, nuclear DNA extracted from apoptotic cells is often degraded in an internucleosomal pattern (20). In situ localization of apoptosis-associated DNA strand breaks in fixed human and baboon ovarian tissue sections have indicated that apoptosis is essentially restricted to granulosa cells of atretic antral follicles (25). In one study, only a portion of all of the pyknotic nuclei in the membrana granulosa of atretic follicles were labeled by TUNEL (26). On the other hand, 3=-hydroxy nick end-labeling reactive cells were not observed in the follicular and luteal phases, indicating that apoptosis is not detectable through follicular and luteal regression (27). Therefore, it has been proposed that granulosa cell death may occur by more than a single pathway (28). 428 Glamočlija et al. Cell death in granulosa cells Vol. 83, No. 2, February 2005

4 FIGURE 1 Presence of apoptotic and caspase-3 expression in granulosa cells from human ovarian tissue (A D) and follicular fluid (E F). (A) Granulosa cells with characteristic morphologic features of apoptosis in the lumen of grossly atretic, 11-mm antral follicle after staining with the Gomori silver stain show a densely stained nucleus (arrows), multiple nuclear fragments (arrowheads), and apoptotic bodies (red arrow) ( 1000). (B) Intense brown staining of caspase-3 positive cells (arrows) is found in apoptotic granulosa cells of the same follicle as in A ( 200). (C, D) Few apoptotic granulosa cells show the extensive morphologic changes characteristic of apoptosis, and positive staining for caspase-3 also showed DNA fragmentation (arrows). DNA fragmentation was detected as staining of the whole nucleus (arrowhead) or as staining of the nuclear fragments (arrow) ( 200 and 1000, respectively). (E) Acridine-orange and ethidium bromide staining of granulosa cells from follicular fluid obtained from patients undergoing IVF show condensed nuclear chromatin masses characteristic for early apoptosis (arrow) and late apoptosis (arrowhead) ( 1000). (F) Expression of the active caspase-3 in the cytoplasm (arrow) and nuclei (arrowhead) of the granulosa cells from follicular fluid ( 1000). TF theca follicles, L lumen follicles. Glamočlija. Cell death in granulosa cells. Fertil Steril Fertility and Sterility 429

5 The TUNEL assay described in our study demonstrated DNA strand breaks only in a few apoptotic granulosa cells of the large antral follicle, but caspase-3 appeared to be distributed uniformly throughout the cytosol of most apoptotic granulosa cells. Caspase-3 resides primarily in the cytosol, and it must be transported into the nucleus during apoptosis to gain access to nuclear substrates (13). Negative TUNEL and positive caspase-3 staining suggested that caspase-3 may be activated in cytosol very early during the commitment steps of apoptosis, before DNA fragmentation occurs. Despite the limited numbers of follicles obtained from human ovaries and inherent limits of the methods used to detect apoptosis, our data also suggest a longer period for detection of caspase-3 than DNA fragmentation in apoptotic granulosa cells. Our study points out differences between marked nuclear morphologic changes in apoptosis and minor DNA fragmentation detectable by TUNEL techniques. This suggests that DNA fragmentation and nuclear morphologic changes may not necessarily be associated events. DNA fragmentation might be a secondary consequence, rather than an integral cause of apoptosis. The endonucleases released during cytoplasmic membrane lysis may be a process that occurs only after the final lytic event in the apoptotic sequence (29). Other investigators have proposed that the endonucleases, if activated, can be destroyed in the phagocytic process (28). Preovulatory follicles responding to the ovulatory LH surge seem to be rescued from the apoptotic pathway, as the number of corpora lutea roughly equals the number of preovulatory follicles. Granulosa cells profoundly change in their steroidogenic properties during the preovulatory period, producing more P after the LH surge (30). Although the reason for this is unclear, purified human granulosa cells obtained from preovulatory follicles of patients undergoing IVF apparently express more of the short or death-inducer form of bcl-x and display variable levels of DNA oligonucleosomes (16, 25). Nuclear features examined by fluorescence microscope in our study demonstrated apoptotic nuclear condensation in granulosa cells obtained from preovulatory follicles of patients undergoing IVF. The presence of apoptosis in human granulosa cells from follicular aspirates of patients undergoing IVF may be a normal physiologic process of the follicle during luteinization (16, 18). Our findings indicate expression of the active caspase-3 in granulosa cells from preovulatory follicles, which was absent in granulosa cells of healthy dominant follicle from ovarian tissue. This observation is based only on two dominant follicles from ovarian tissue and may be partly explained by manipulation during cell collection. The requirement for caspase-3 in the normal execution of granulosa cell death shown here may also be maintained as these cells transform into luteal cells (14). The activation of caspases works concurrently with changes in mitochondria during induced apoptosis in human luteinized granulosa cells (31). Thus, our previous proposal that cellular reorganization within the dominant follicle before, during, and after ovulation is associated with apoptosis may be further supported by the findings that the potential death-inducer is being upregulated in populations of granulosa cells during follicular rupture and the ensuing luteinization process (25). We do not yet have a satisfactory explanation of how the apoptotic process is selectively activated in the appropriate ovarian compartment and at the appropriate time (32). 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