Molecular Human Reproduction, Vol. 8, No. 3, 228-236,
March 2002
© 2002 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Cell death and its suppression in human ovarian tissue culture
1 Programme for Developmental and Reproductive Biology, Biomedicum Helsinki, and Hospital for Children and Adolescents, University of Helsinki, FIN-00290, 2 Infertility Clinic, The Family Federation of Finland, Kalevankatu 16, FIN-00100 and 3 The Department of Obstetrics and Gynecology, Helsinki University Central Hospital, FIN-00290, Helsinki, Finland
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Abstract |
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In women with premature ovarian failure, fertility may be preserved by ovarian tissue culture in vitro. However, techniques for tissue culture and follicle maturation have remained suboptimal. Our aim was to characterize ovarian tissue degeneration in cultures and to establish a model for cell death research in cultured ovarian tissue. Precise knowledge on the process resulting in cell death in cultured ovarian tissue will ultimately facilitate work aimed at improving long-term culture conditions. Ovarian tissue apoptosis was studied in a serum-free culture model in which nuclear DNA fragmentation was shown to occur within 24 h of the start of the culture. Activation of caspase-3 was detected in some stromal cells and a few oocytes. Since not all of the tissue exhibited signs of apoptosis and since DNA fragmentation increased over time, the tissue probably gradually dies by apoptosis. The antioxidant N-acetyl-L-cysteine (NAC; 25, 50 and 100 mmol/l) was found to inhibit this apoptosis. Thus, apoptosis appears to play a critical role in the degeneration of human ovarian cortical tissue cultures, and this cell death can be suppressed by NAC. The present tissue culture model can be used for identifying components capable of inhibiting cell death in vitro.
apoptosis/in-vitro culture/N-acetyl-L-cysteine/ovary
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Introduction |
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In attempts to sustain the fertility of women at risk of premature ovarian failure, cryopreservation of ovarian cortical tissue has, in recent years, become an alternative to cryopreserving embryos and oocytes (Bahadur and Steele, 1996
). Although long-term survival of young women with malignant diseases has improved and the need for fertility preservation has increased, there are no techniques that guarantee successful maturation of the follicles within the excised tissue. The follicles in in-vitro cultures have been reported to show signs of atresia after culture for only a few days (Wandji et al., 1996, 1997; Hovatta et al., 1997; Picton et al., 2000). So far, complete in-vitro development from a primordial follicle to a fully mature ovulatory follicle and offspring has been achieved only in mice (Eppig and O'Brien, 1996).
Follicular atresia in the ovary is a natural phenomenon, eventually leading to exhaustion of the pool of primordial follicles. The discovery that this process is due to apoptosis has led to extensive research on follicular cell death (Morita and Tilly, 1999). In addition to lethal physiological stimuli, apoptosis can be initiated by pathological stimuli such as cancer therapies or depletion of survival/growth factors. When the cell proceeds to apoptosis, the death cascade leads to activation of caspases, such as caspase-3, which break down the cell (Morita and Tilly, 1999). Although cell death pathways have previously been studied in isolated follicles, little research has been done on the overall survival of human ovarian cortical tissue. By improving the viability of the whole ovarian tissue in in-vitro cultures, it may be possible to bring follicles to a more advanced stage with a better chance of survival in further maturation processes. Increased tissue viability might also result in greater numbers of surviving follicles.
In in-vitro cultures, ovarian tissue (Hovatta et al., 1997) and isolated ovarian follicles (Tilly and Tilly, 1995) are usually kept under normoxic conditions, the oxygen concentration being considerably higher than in vivo. Thus, oxidative stress, i.e. the excessive production of reactive oxygen species (ROS), may contribute to the atresia observed in in-vitro cultures. All aerobic cells generate ROS as by-products of normal cellular metabolism. However, diverse stimuli are known to compromise the fine balance between intracellular oxidants and their defence mechanisms, an excess of ROS leading to oxidative stress and apoptosis or necrosis (Clutton, 1997; Mignotte and Vayssiere, 1998). The antioxidant N-acetyl-L-cysteine (NAC) is a compound that acts as a reductant and stimulates the synthesis of glutathione, the most abundant cellular thiol that removes intracellular peroxides (Mayer and Noble, 1994; Hall, 1999). If the viability of cultured slices of human ovarian tissue is impaired by oxidative stress, the survival of the tissue may be improved by adding such free radical scavengers to the culture. The objectives of the present study were to characterize the nature of cell death in human ovarian tissue culture and to investigate whether it could be inhibited by using NAC to reduce oxidative stress.
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Subjects and methods |
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Patients
Ovarian cortical tissue was obtained by biopsy from 41 women, aged 24–38 years, undergoing gynaecological operations for benign conditions. They had no diagnosis of ovarian dysfunction and they donated tissue after informed consent. The operations were performed at the Department of Obstetrics and Gynecology, Helsinki University Central Hospital, and the Helsinki City Maternity Hospital (Helsinki, Finland). The study protocol was approved by the ethics committees of the Department of Obstetrics and Gynecology, Helsinki University Central Hospital, and the City of Helsinki.
Long-term tissue culture
The ovarian biopsies were cut, under a preparation microscope, into slices of 0.3–0.5 mm on a Petri dish in tissue culture medium. The tissue was cultured for 1 or 3 weeks (long-term cultures) in Millicell CM inserts (6 mm diameter, 1.0 µm pore size; Becton Dickinson Labware, Bedford, MA, USA) precoated with 150 µl of extracellular matrix (Matrigel; Becton Dickinson Labware) and placed in 24 well plates (Becton Dickinson Labware) containing 500 µl of culture medium per well. The tissue culture medium contained Earle's balanced salt solution (EBSS; Gibco, Life Technologies Ltd, Paisley, Scotland), sodium pyruvate (Sigma, St Louis, MO, USA) and antibiotics (streptomycin, penicillin and amphotericin B; HyClone Laboratories Inc., Utah, USA), supplemented with 10% human serum (Finnish Red Cross), 0.5 IU/ml FSH (Organon, Cambridge, UK) and 33 ng/ml insulin (Sigma). The medium was changed every other day. After incubation, the tissue was fixed in Histochoise (Histochoise Tissue Fixative; Amresco, Solon, Ohio, USA).
Short-term tissue culture
For short-term cultures, the tissue was first handled as described above. The culture medium (nutrient mixture Ham's F-10, Life Technologies Inc., Europe, Paisley, UK) was supplemented with 0.1% human albumin (Sigma), 10 µg/ml gentamicin (Life Technologies), 0.5 IU/ml FSH (Organon) and 33 ng/ml insulin (Sigma). The 0 h control samples were immediately snap-frozen in liquid nitrogen or fixed in 4% formalin or Bouin's solution. The slices of tissue were transferred to 24 well culture plates (Nunclon, Roskilde, Denmark) containing 600 µl of the culture medium and cultured for 8, 24 or 48 h at 37°C in a humidified atmosphere containing 5% CO2. After the culture, the tissue was either snap-frozen in liquid nitrogen or fixed in 4% formalin or Bouin's solution.
Treatment with NAC
To study the effect of an antioxidant on the survival of ovarian tissue cultured in vitro, NAC (Sigma) was added to the short-term culture medium at final concentrations of 25, 50 and 100 mmol/l. The differences in the amount of apoptosis in tissues cultured with these concentrations of NAC were detected by Southern blot analysis of low molecular weight DNA fragmentation.
Histology
After fixation in 4% formalin or Bouin's solution, the tissue of the short-term cultures was dehydrated and embedded in paraffin blocks. The blocks were cut in 3 µm serial sections and mounted on slides coated with Vectabond (Vector Laboratories, Burlingame, CA, USA). Every sixth slide was stained with haematoxylin and eosin for histological evaluation. The remaining slides were stained with the in-situ end-labelling (ISEL, TUNEL) method and observed with a light microscope. The long-term cultures fixed in Histochoise were embedded in paraffin, cut into 7 µm serial sections and stained with haematoxylin–eosin in order to analyse the viability of the follicles. Sections (n = 16) from each tissue slice were analysed and classified as described previously (Gougeon, 1996).
Electron microscopy (EM)
The tissue was fixed in 2.5% glutaraldehyde in 0.1 mol/l phosphate buffer, dehydrated and embedded in epoxy resin. The samples were sectioned at 50 nm with an E Ultramicrotome (Reichert Jung, Vienna, Austria) and stained with uranyl acetate and lead citrate. Observations were made with a JEOL JEM 1200 EX transmission EM (JEOL, Tokyo, Japan).
Southern blot analysis of DNA fragmentation
Small pieces of tissue were snap-frozen in liquid nitrogen and stored at –80°C until DNA isolation. DNA was extracted using the Apoptotic DNA Ladder Kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions, with some modifications. Briefly, the pieces of ovarian cortical tissue were homogenized and incubated for 10 min at room temperature in binding/lysis buffer (6 mol/l guanidine-HCl, 10 mmol/l Tris-HCl, 10 mmol/l urea and 20% Triton X-100, pH 4.4). After quantitation, DNA samples were 3'-end labelled with digoxigenin-dideoxy-UTP (Dig-dd-UTP; Roche) by the terminal transferase reaction, subjected to electrophoresis on 2% agarose gels and blotted onto a nylon membrane overnight, as previously described (Erkkilä et al., 1997). The 3'-end labelled DNA fragments on the nylon membranes were detected with anti-digoxigenin antibody (Anti-Digoxigenin-AP, alkaline phosphatase conjugates; Roche) and the chemiluminescence reaction (CSPD; Roche), as described (Erkkilä et al., 1997). X-ray films exposed to luminescent membranes were scanned with a tabletop scanner (Hewlett Packard Scanjet 6300C). The optical density was transformed to pixels and the digital image was analysed with gel plot 2 macro for Scion Image beta 4.0.2 (Scion Corporation, Frederick, MD, USA) analysis software.
Non-radioactive in situ end labelling (ISEL) of apoptotic DNA
Formalin-fixed and paraffin-embedded sections were deparaffinized, rehydrated and washed twice for 5 min in distilled water. The sections were permeabilized with 10 µg/ml proteinase K (Promega, Madison, WI, USA) for 30 min in 37°C and washed twice in distilled water. After incubation for 10 min with terminal transferase reaction buffer [1 mol/l potassium cacodylate, 125 mmol/l Tris-HCl and 1.25 mg/ml bovine serum albumin (BSA), pH 6.6], the apoptotic DNA fragments were 3'-end labelled with Dig-dd-UTP (Roche) by the terminal transferase reaction for 1 h at 37°C. For the negative controls, the terminal transferase enzyme was replaced with the same amount of distilled water. The bound Dig-dd-UTP was detected with the antidigoxigenin antibody conjugated with horseradish peroxidase (Anti-Digoxigenin-POD; Roche). The antibody was localized by adding 0.05% diaminobenzidine substrate (Sigma). After light counterstaining with haematoxylin, the samples were dehydrated and mounted.
Immunohistochemistry for active caspase-3
To determine whether active caspase-3 could be found in the tissue, immunohistochemistry was performed using an antibody that recognizes the active caspase-3, i.e. the 17 kDa subunit of cleaved caspase-3, and thus, not the non-active pro-caspase-3 form of the enzyme. Ovarian tissue was frozen in Tissue-Tek O.C.T Compound (Sakura, Zoeterwoude, The Netherlands) and cut into 5 µm cryosections. The cryosections were dried at room temperature for 30 min and fixed in acetone for 10 min at –20°C. They were then washed three times for 5 min in phosphate-buffered saline (PBS) and blocked with blocking solution containing 5% normal goat serum in 3% BSA for at least 30 min at room temperature. Rabbit polyclonal antibody to active caspase-3 (Pharmingen, San Diego, CA, USA) was added to the preparations at 0.5 µg/ml in blocking reagent and incubation was performed overnight at 4°C. After incubation, the slides were washed three times for 5 min in PBS and the primary antibody was detected with a biotin-conjugated goat anti-rabbit immunoglobulin (Ig)G secondary antibody from the ABC-Elite Kit (Vector Laboratories), followed by incubation with ABC solution. The antibody was localized with 0.05% diaminobenzidine substrate (Sigma). After light counterstaining with haematoxylin, the samples were dehydrated and mounted. Control sections were stained without the first antibody or with affinity purified non-specific rabbit IgG (Sigma).
Statistical analysis
The experiments were repeated on at least three independent occasions. Quantitative data represent low molecular weight DNA (optical density from X-ray films) from Southern blot analysis of apoptotic DNA fragmentation. In the experiment for investigating nuclear apoptosis, the 0 h time point was set at 1.0 (100%), and the other time points (8, 24 and 48 h) were compared with it. In the experiments for studying the effects of NAC on the survival of the tissue, the 24 h time point cultured without NAC was set at 1.0 (100% apoptosis) and the data from the samples cultured for 1 day, treated with different concentrations of NAC, were compared with that. Data obtained from three to 14 replicate experiments (mean ± SEM) were analysed by the two-tailed paired t-test. A value of P < 0.05 was considered significant.
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Results |
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Cell death in cultured human ovarian tissue
To evaluate the survival of human ovarian cells during long-term in-vitro culture, we cultured ovarian tissue for 1 and 3 weeks in the presence of serum. In all of the fresh biopsies, the histology of the stroma appeared normal. Over 99% of the follicles in the fresh tissue were healthy. The proportion of primordial follicles was 81%. The rest of the follicles were mostly primary (18%) and only a few secondary (1%) or atretic follicles (<1%) were seen (Table I
). No antral follicles were observed in the biopsies used for this study. After 1 week of culture, light microscopic analysis of the cultured tissue showed several clearly atretic follicles undergoing apoptosis (Figure 1). The proportion of atretic follicles had increased to 36%, and the proportion of healthy follicles was 48% for the primary and 16% for the secondary follicles. No primordial follicles were found (Table I). Atresia of the follicles could be identified by condensing or highly condensed granulosa cells and distracted granulosa cell layers, eosinophilic ooplasm and vacuoles in the ooplasm, and also by the shrinkage and eventual disappearance of the follicle. In the tissues cultured for 3 weeks, the proportion of the atretic follicles had risen further to 61% (Table I). Most of the remaining healthy follicles were primary (37%) and only a few secondary (2%) follicles were found. It could be seen that the condensed granulosa cells disappeared first and, in the last stage of atresia, only traces of the eosinophilic oocyte could be seen.
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In fresh biopsies the interstitial tissue looked normal (~100% viable). After 1 week of culture, areas with condensed interstitial cell nuclei could be found. The number of condensed nuclei varied in different areas from only a few to numerous, and by approximation about half (50%, the mean proportion of 16 tissue sections per biopsy analysed from eight biopsies) of the tissue was viable. In the tissue cultured for 3 weeks, more areas with condensed nuclei were found. There were also some areas in which no visible cell structure could be observed and the tissue seemed totally degenerated. The proportion of healthy tissue had dropped to ~30% (as estimated from 12 biopsies).
In order to more closely study ovarian tissue apoptosis, the tissue was then cultured in shorter cultures which did not contain any serum. The cultures were terminated after 8, 24 or 48 h. Incubation of slices of ovarian cortical tissue under serum-free culture conditions induced rapid cell death. After culture for 8 h, DNA fragmentation in the samples varied from mild to moderate, whereas after 24 and 48 h every culture showed pronounced laddering (P < 0.001, 24 h of culture verus 0 h, uncultured tissue) (Figure 2). Incubation for 24 h increased DNA fragmentation relative to that at 0 h by ~3-fold (Figure 2).
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Identification of apoptotic cells by histology, ISEL and EM
The apoptotic nature of cell death in the serum-free, short-term cultures was further investigated by histology (Figure 3A,B) under a light microscope. The freshly fixed (uncultured) tissue looked healthy. In the short-term (8–48 h) cultures, fixed in formalin or Bouin solution, very few atretic follicles showing eosinophilia of the ooplasm and pyknosis of the granulosa cells were detectable. The connective tissue did not exhibit any clear morphological signs of apoptosis.
By ISEL, consistent with the morphological findings, little or no incorporation of digoxigenin-dd-UTP was found in uncultured ovarian tissue. However, tissue cultured for 24 h without serum often showed areas of positively stained interstitial cells, indicating apoptotic cell death. In some of the tissues, the positively stained stromal cells were individually located among the healthy cells. In some stromal cells, the condensed chromatin had assumed a concave shape, resembling a horseshoe or sickle, a feature typical of apoptotic cells. It is possible that not all the apoptotic cells could be localized or identified because nuclear pyknosis prevents the staining of some nuclei. Only a very few follicles stained positively for ISEL in the 24 h cultures (Figure 3C,D). Among the granulosa cells, there were some very small, condensed cells, which fulfilled the morphological criteria for pyknosis, but which were stained only with haematoxylin. In the negative controls, where the terminal transferase enzyme had been substituted for the same volume of distilled water, there was no ISEL positive staining.
The apoptotic nature of the cellular changes was confirmed by EM. Primordial, primary and secondary follicles were found in the tissue prepared for EM. The cell nuclei in freshly fixed interstitial tissue were elongated and the DNA was evenly distributed (Figure 4A). In contrast, the nuclei of the cells of the interstitial tissue cultured for 2 days under long-term culture conditions had often become more oval or rounded and DNA clumping and margination were apparent (Figure 4B). The granulosa cells in non-cultured, freshly fixed ovarian tissue were morphologically normal, with intact nuclear and cell membranes (Figure 4C). Moderate clumping of the chromatin is a feature typical for granulosa cells and does not by itself indicate apoptosis. Most of the granulosa cells in tissue cultured for 2 days were also normal. However, sporadic granulosa cells with an electron dense cytoplasm and nucleus could be detected among the morphologically normal ones in the primary and secondary follicles (Figure 4D). We suspect that these granulosa cells are apoptotic. The electron dense granulosa cells in the secondary follicles were usually observed close to or next to the oocyte. All of the granulosa cell mitochondria, in both uncultured and cultured tissue, looked morphologically normal (Figure 4E,F). In both the uncultured and cultured tissue, there were no signs of oocyte cell death (Figure 4G). Furthermore, no abnormalities in oocyte mitochondria morphology could be detected at either time point (Figure 4H,I). The mitochondria were spherical to ovoid in shape and contained few christae, which is a sign of quiescence.
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Caspase-3 activation in the culture
By immunohistochemical analyses for active caspase-3, scattered interstitial cells with clear caspase-3 activation in the cytoplasm were detected in the tissue cultured for 24 h without serum, whereas almost no positive cells could be found in fresh tissue. Some moderately stained and a few strongly stained oocytes could also be found after 24 h culture (Figure 3E,F). The granulosa cells very rarely showed any staining. Control sections stained without the primary antibody or with affinity-purified non-specific rabbit IgG as the primary antibody did not show any specific staining.
Inhibition of apoptosis by NAC
NAC was able to suppress programmed cell death at all of the concentrations tested (25, 50 and 100 mmol/l) (Figure 5) in the 24 h serum-free cultures. DNA fragmentation was suppressed by 28% at 25 mmol/l (P < 0.05), by 22% at 50 mmol/l (P < 0.05) and by 32% at an NAC concentration of 100 mmol/l (P < 0.05), as compared with samples cultured without NAC.
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Discussion |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionAcknowledgementsReferences |
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In the present study, we characterized the degeneration of human ovarian tissue in in-vitro culture. Cell death of the interstitial tissue in both long- and short-term cultures was shown to be apoptotic by morphological criteria and by detection of nuclear DNA fragmentation respectively. Furthermore, during the short-term cultures, caspase-3 was shown to be activated in the process of programmed cell death. This in-vitro ovarian apoptosis was suppressed by a thiol antioxidant, NAC. Thus, we have developed a short-term in-vitro culture model, in which ovarian apoptosis can be regulated and the survival of the tissue can be manipulated.
Cryopreservation of ovarian tissue with intact primordial follicles for later culture and maturation could be a powerful method when attempting to preserve the fertility of patients with prospective premature ovarian failure. In this method, the well-being of the entire tissue section, including all the cell types, is crucial. The in-vitro culture model described in this study has the advantage of preserving the integrity of the oocyte–granulosa–stroma interactions, which is important for the development of early follicles (Gougeon, 1996; McGee and Hsueh, 2000). Complete mouse oocyte development in vitro, beginning from the oocytes in the primordial follicles and resulting in a live pup, has been achieved by starting with an 8 day organ culture. Thereafter, the oocyte–granulosa cell complexes were isolated and further matured (Eppig and O'Brien, 1996). It is possible that a similar type of procedure, starting with pieces of the ovarian cortex, is needed for the maturation of human primordial follicles.
In our initial studies, human ovarian tissue was cultured for up to 3 weeks under conditions in which 10% serum was provided. Abundant follicular atresia was detected in the histological sections. The proportion of live follicles decreased dramatically from almost 100% at the start of the culture to 39% after 3 weeks of culture. This accords with previous studies, in which cultured ovarian tissue was found to show atresia and a decline in the total number of follicles (Wandji et al., 1996, 1997; Hovatta et al., 1997; Wright et al., 1999; Picton et al., 2000). Surprisingly, in the present study, we found that stromal cells showed clear nuclear changes after as little as 2 days of culture. Despite the stromal degradation, the follicles appeared to be viable. Only individual granulosa cells may have started the process of cell death and all of the oocytes appeared healthy.
Since serum contains various undetermined agents, we next developed more defined culture conditions without serum or extracellular matrix and with a reduced amount of albumin. FSH and insulin were provided. In the in-vitro cultures of the human ovarian cortex, FSH has been shown to reduce atresia and to increase the follicle diameter, suggesting anti-apoptotic as well as mitogenic effects for FSH in the tissue (Wright et al., 1999). Insulin, in turn, primarily affects glucose homeostasis, but it also promotes several other cellular events (Cheatham and Kahn, 1995) and improves the overall viability of follicles in long-term (2 weeks) ovarian cortical tissue cultures (Louhio et al., 2000). On the basis of these previous findings, FSH and insulin were added to the present cultures. In these well-defined short-term culture conditions, it became possible to study the effects of anti-apoptotic agents without the interference of unknown factors.
The apoptotic nature of cell degeneration during the short-term cultures was determined by Southern blot analysis of DNA fragmentation, ISEL and morphological findings. Southern blotting showed a variety of degrees of apoptosis after 8 h of culture, from weak laddering to a clear apoptotic ladder pattern. After 24 h, all the cultured tissues showed strong apoptosis. In a piece of cortical ovarian tissue, interstitial cells far outnumber follicular cells, and DNA laddering mainly reflects the condition of the stroma. Therefore, in addition to a morphological analysis, we performed ISEL to identify the apoptotic cells. In uncultured tissue, only sporadic interstitial cells stained positively and the follicles remained unstained. In the 24 h cultures, all of the interstitial cells stained positively in some areas. In other areas, only individual interstitial cells showed positive staining, whereas the majority of the interstitial cells were negative. The granulosa cells of only a few follicles showed moderate to strong staining, with most of the follicles being totally negative. The oocytes very rarely showed any detectable staining, which accords with their normal fine structural appearance in EM.
To evaluate whether caspase-3 activation plays a role in the apoptotic pathway induced in the ovarian tissue cultured for 24 h, we performed an immunohistochemical analysis of active caspase-3. Caspase-3 is a well-characterized protease that is involved in the effector phase of apoptosis (Cohen, 1997). Its expression has previously been demonstrated in human granulosa (Izawa et al., 1998), luteinized granulosa (Khan et al., 2000) and thecal cells (Krajewska et al., 1997). In the present study, caspase-3 activity was found in both ovarian somatic and germ cells in the 24 h cultures. Some of the interstitial cells showed clear caspase-3 activation, indicating that apoptosis in the in-vitro culture can be associated with its activation in the stroma. However, we found extremely little caspase-3 activation in the human granulosa cells, which accords with the EM results showing only a few potentially apoptotic granulosa cells. Interestingly, in contrast to the granulosa cells, several caspase-3-positive oocytes were found, although the EM morphology of the oocytes was normal. However, since the samples studied by EM were very small, the possibility of some apoptotic oocytes in the 24 h samples cannot be excluded. Consistent with our results, caspase-3 has been previously detected in female germ cells (Exley et al., 1999) and in some oocytes throughout late oogenesis, but there is no evidence that these oocytes are undergoing apoptosis (Reynaud and Driancourt, 2000). Therefore, the role of caspase-3 in oocyte apoptosis remains to be further clarified, for example by using specific caspase-3 inhibitors or by measuring the activity of caspase-3.
We also investigated whether ovarian tissue survival could be improved in the short-term cultures by suppressing apoptosis. We chose NAC, a thiol antioxidant, as a potential inhibitor of apoptosis, because (i) it is a well-established inhibitor of physiological cell death in several other systems and a compound known to act on granulosa cells as a survival factor (Tilly and Tilly, 1995), (ii) on the basis of our previous studies on human testis tissue (Erkkilä et al., 1998), we know that NAC penetrates the tissue very well and remains effective in the cultures, and (iii) it is a widely used, clinically safe drug (Moldeus et al., 1986; Burgunder et al., 1989; Roederer et al., 1992; Ahola et al., 1999). Interestingly, we found that cell death in cultured human ovarian cortex was suppressed by NAC. Our finding is consistent with previous studies showing that NAC inhibits apoptosis in cultured porcine ovarian primordial germ cells (Lee et al., 2000), in rat ovarian follicles (Tilly and Tilly, 1995), in rabbit corpus luteum cells (Dharmarajan et al., 1999) and in human male germ cells in vitro (Erkkilä et al., 1998). Thus, NAC appears to act as a survival factor in both male and female germ cells, and also in other cell types of the ovarian cortex.
NAC may suppress cell death by protecting the cells from increased levels of reactive oxygen species (ROS), which have been shown to lead to cell death (Chandra et al., 2000). NAC itself is a scavenger of free radicals. Since NAC is a cysteine analogue, it can increase the synthesis of glutathione, which has an important role in protecting the cells from ROS (Meister, 1983; Mayer and Noble, 1994). Interestingly, in a previous study, NAC was able to restore the fertility of mice lacking 
glutamyl transpeptidase, an enzyme important for glutathione synthesis (Kumar et al., 2000). Thus, NAC seems to be a survival factor in reproductive organs via its effects of redox control. However, since NAC may also have other targets of action, the exact mechanism by which NAC acts on the human ovarian tissue remains to be resolved.
Taken together, our results suggest that apoptosis plays a critical role in the degeneration of ovarian cortical tissue in vitro. According to our data the interstitial cells of the ovarian cortex are the first cells that start to die. Individual granulosa cells may follow the gradual cell death of the stroma, with oocytes persisting longest in the degenerating tissue. Survival of the tissue can be improved by manipulating the cascade leading to cell death. The thiol antioxidant NAC was shown to be an inhibitor of apoptosis in the serum-free cultures. The present short-term culture model can be used when striving to find ways to improve the viability of cultured ovarian tissue in order to grow follicles for subsequent IVF treatments.
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Acknowledgements |
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The authors thank Dr A.Jurisicova for her help with interpreting the electron micrographs and Dr T.Matikainen for valuable comments. We gratefully acknowledge the skilful assistance of Ms Virpi Ahokas, Ms Kaisa Alasalmi and Ms Sinikka Heikkilä. We also thank the staff of the Department of Obstetrics and Gynecology, Helsinki University Central Hospital, and Helsinki City Maternity Hospital for providing the ovarian biopsy samples. This study was supported by the Sigrid Juselius Foundation, Finland.
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Notes |
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4 To whom correspondence should be addressed at: Hospital for Children and Adolescents, University of Helsinki, P.O.Box 281,FIN-00029 Helsinki, Finland. E-mail: leo.dunkel{at}hus.fi
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Submitted on June 21, 2001; accepted on November 13, 2001.
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