Hum. Reprod. Advance Access originally published online on May 2, 2006
Human Reproduction 2006 21(8):1974-1980; doi:10.1093/humrep/del109
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Caspase-3, TUNEL and ultrastructural studies of small follicles in adult human ovarian biopsies
Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, Dunedin, New Zealand
1 To whom correspondence should be addressed at: Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand. E-mail: peter.hurst{at}stonebow.otago.ac.nz
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Abstract |
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BACKGROUND: The aim of this study was to investigate evidence for cell death by apoptosis in small unilaminar ovarian follicles of adult humans. METHODS: Cortical biopsies from 13 healthy donors were either frozen and protein extracted for western blots or fixed for immunohistochemistry (IH) to localize procaspase-3 and active-caspase-3, to detect DNA fragmentation in situ and undertake routine transmission electron microscopy (TEM). RESULTS: Blots identified the presence of the inactive pro-form of caspase-3, and IH localized this in all follicles studied. In contrast, the active form of caspase-3, a major effector of apoptosis, was only detected in large antral follicles that also had microscopic signs of atresia. Active caspase-3 was not detected in primordial (n = 87), primary (n = 8) or secondary follicles. The atretic follicles were also the only ovarian structures with positive evidence of DNA fragmentation after terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) treatment. Confocal microscopy showed dual labelling for both active caspase-3 and TUNEL in individual granulosa cells in large atretic follicles, but no such labelling was evident in any other follicles. No apoptotic bodies were seen by TEM in sections of 39 small follicles from seven patients. CONCLUSION: This study found evidence for TUNEL and active caspase-3 only in human ovarian antral follicles.
Key words: apoptosis/caspase-3/follicles/human/ovary
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Early estimates (Block, 1952
) from a range of human samples indicated that at the time of puberty ovaries have a population of over 250 000 follicles. During adulthood, only 400–500 of these develop to the point where ovulation occurs, but by the time of reproductive senescence, ovaries contain few if any primordial, primary or growing follicles (Block, 1952; Gougeon and Chainy, 1987; Gougeon, 1998; Westhoff and Murphy, 2000). The fate of the vast majority of follicles is degeneration (follicular atresia). The mechanism of this degeneration includes apoptosis—a form of programmed cell death—that occurs principally in constituent granulosa cells and is well defined morphologically by nuclear pycnosis in antral follicles (reviewed in Gougeon, 1996; Hussein, 2005), but the evidence for this in primordial and primary follicles is equivocal. Possible approaches to investigate apoptosis in discrete cellular structures of tissue samples obtained from human biopsy specimens include histological and immunohistochemical approaches. These describe morphological features and the presence of antigens specific to apoptotic pathways respectively (Hussein, 2005). Histological criteria include observation of nuclear condensation and pycnotic nuclear profiles that are discernible by both light and electron microscopy of tissue sections (Kerr et al., 1972). With transmission electron microscopy (TEM), the identification of small areas of dense, granular masses at nuclear margins and discrete nuclear fragments are determinants of apoptosis (Wyllie et al., 1980; Yuste et al., 2005), and this is evident in the granulosa cells of antral follicles in many species (Inoue et al., 2000) including human (Makrigiannakis et al., 1999). In contrast, there was no clear evidence of such morphological features of apoptosis in primordial or primary follicles. In a recent TEM study of human biopsies (de Bruin et al., 2002), qualitative and morphometric analysis of over 150 small follicles indicated the presence of necrotic structures in some follicles, but no morphological indications of apoptosis were reported. This was the case in primordial, transitory and possibly primary follicles, regarded collectively as a pool of resting follicles (Gougeon, 1996).
Cysteine proteases including some members of the caspase family of enzymes are known to be involved as initiators and effectors (executioners) of apoptosis (Shi, 2002). Caspase-3 is a major effector present in many cells as the inactive zymogen, procaspase-3, that upon activation forms two subunits that complex into a tetramer with a range of cellular substrates and specifically induces caspase-activated DNase to cleave DNA and subsequent nuclear fragmentation. In human ovary, procaspase-3 (as CPP32) has been detected by western blot and in theca and granulosa of ‘secondary’ follicles by IH (Krajewska et al., 1997). Active caspase-3 has been localized to atretic antral follicles in four human biopsies (Matikainen et al., 2001) and also in mice (Matikainen et al., 2001; Fenwick and Hurst, 2002). DNA fragmentation can be shown histochemically with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL). This approach has shown apoptotic granulosa degeneration in ovary sections in a range of species including mice (Matikainen et al., 2001) and also in a human study in which primordial and primary follicles were regarded as apoptotic (Depalo et al., 2003). In contrast, however, no such human follicles had either caspase-3 or TUNEL activity in the Matikainen et al. (2001) study. Other studies concerning apoptosis in adult human ovaries found TUNEL activity in granulosa cells of antral follicles and a small proportion of secondary follicles (Vaskivuo et al., 2001). In the same study, however, no TUNEL activity was observed in primordial or primary follicles. Makrigiannakis et al. (1999) also used TUNEL staining and did not ‘demonstrate any apoptosis’ in cells of pre-antral follicles. Consequently, the notion that primordial and primary follicles are not normally apoptotic requires clarification. We describe a number of detailed microscopic studies designed to determine further if there is evidence for caspase-3 and its activation, TUNEL-positive cells as well as morphological features of apoptosis in ovarian follicles in a range of adult human biopsy specimens. Western blots and IH were used to detect procaspase-3 and dual labelling with a confocal microscope (Berardinelli et al., 2004) to simultaneously detect active-caspase-3 and DNA fragmentation using TUNEL procedures with strict regard to protocol controls and specificity. Some biopsy specimens were also processed for routine TEM.
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Materials and methods |
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Ovarian biopsies
Human ovarian cortical biopsies measuring approximately 0.5 cm3 were collected from consenting healthy individuals (n = 13, age range 25–36 years) undergoing sterilization at the Dunedin Public Hospital (Otago Ethics approval no. 99/12/113). For IH, tissue was immediately fixed in 25 ml of freshly prepared 4% (w/v) paraformaldehyde in 0.2 M phosphate buffer (PB). After 4 h of fixation, tissue was changed into 70% ethanol, followed by two more changes into fresh 70% ethanol at approximately 6 h intervals. Tissue was processed through various ethanol solutions and xylene before embedding in paraffin wax. Samples for electron microscopy were dissected to approximately 0.1 cm3 and fixed for 2 h in freshly prepared 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M PB. Portions of tissue destined for Western blotting were frozen in liquid nitrogen and stored at –80°C.
Follicle classification
The following microscopic criteria were used to define follicle stages: Primordial: a single layer of predominantly flat or squamous granulosa cells; Primary: a single layer of cuboidal-shaped granulosa cells; Secondary: two to three layers of granulosa cells surrounded by a thecal tissue, but no evidence of an antrum and Antral: where an antrum was present within the granulosa cell layer.
Western blotting for procaspase-3
Two separate patient samples of frozen ovarian tissue were crushed under liquid nitrogen and homogenized in phosphate-buffered saline (PBS) containing ‘complete’ protease inhibitor cocktail tablets (Boehringer Mannheim, No. 1697498, Roche Diagnostics, Mannheim, Germany). Tissue was sonicated and centrifuged at 14 700 g for 15 min at 4°C. Protein concentration of the supernatant was measured using a BCA protein assay kit (Pierce No. 23225). Known concentrations of protein were mixed with loading buffer [1.5 M Tris–HCl l–1 pH 6.8, 10% (v/v) glycerol, 10% (w/v) SDS, 1 M dithiothreitol l–1, 0.16% (w/v) bromophenol blue] and boiled for 5 min. Proteins were loaded and separated according to size on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked overnight in PBS/0.02% Tween-20 containing 5% non-fat milk powder and incubated for 1 h at room temperature with 1 : 200 polyclonal anti procaspase-3 (Santa Cruz Biotechnology, H-277, Santa Cruz, California, USA). Membranes were washed with PBS/0.02% Tween-20 and incubated with horseradish peroxidase-conjugated donkey anti-rabbit secondary immunoglobulin (Ig)G, diluted 1 : 10 000 in PBS for 2 h at room temperature. Membranes were washed before the detection of bound antibody using enhanced chemiluminescence reagent (Amersham Biosciences, Bucks, England) for 1 min and exposed to hyperfilm (Amersham). As positive control, mouse ovarian samples (Fenwick and Hurst, 2002) were treated and loaded onto gels in identical conditions.
Immunohistochemistry
Procaspase-3 and active caspase-3 were both studied in 4-µm sections from paraffin-embedded tissue from six patients. A further four patient samples were prepared for procaspase-3 IH. Sections were placed onto superfrost plus-coated slides (BDH), baked for 1 h at 60°C and kept overnight at 37°C. Sections were dewaxed and equilibrated in 0.1 M Tris–HCl buffer with 5% urea (pH 10.0). Antigen retrieval was undertaken in 0.1 M Tris–HCl buffer (pH 10.0) for 23 min in a domestic microwave on high setting and cooled for 15 min. Endogenous peroxidases were blocked with 0.3% (v/v) hydrogen peroxide in methanol and proteins blocked using donkey serum (Sigma-Aldrich Corp. St. Louis, Missouri, USA) 1 : 20 in PBS. Sections were incubated with either polyclonal antibody to active caspase-3 (R and D Systems, Minneapolis, Minnesota, USA. No. AF835, dilution of 1 : 500) or anti procaspase-3 (Santa Cruz Biotechnology, H-277, dilution of 1 : 50) in diluting buffer (1% bovine serum albumin, 0.1% Tween-20 in PBS) overnight at 4°C. Sections were incubated with biotinylated donkey anti-rabbit secondary antibody (Amersham) 1 : 200 in PBS for 30 min and streptavidin biotinylated HRP (Amersham) 1 : 100 in PBS for a further 30 min. Detection was achieved using 3-amino-9-ethylcarbazole for 12 min and lightly counterstained with Gills Haematoxylin.
Sections of mouse ovary were used as a positive control, as previous experiments showed both procaspase-3 and active-caspase-3 in granulosa cells of atretic antral follicles (Fenwick and Hurst, 2002). Negative control slides were incubated with an equivalent volume of diluting buffer only (no antibody) or rabbit IgG serum (Sigma-Aldrich).
TUNEL
Paraffin-embedded tissue from all 13 patients was sectioned at 4 µm and placed onto slides coated with 3-aminopropyltriethoxylane, dried at 42°C for 45 min and left at 37°C overnight. Sections were dewaxed and rehydrated in diethyl pyrocarbonate (0.05%)-treated solutions to reduce nuclease activity, followed by antigen retrieval in 20 µg ml–1 of proteinase K for 20 min at room temperature. Permeabilization involved incubation in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice, followed by TUNEL for 1 h at 37°C (using Boehringer Mannheim TUNEL enzyme and TUNEL label), with an enzyme-to-label ratio of 1 : 9. Sections were counterstained using Hoechst stain (Molecular Probes, Eugene, Oregon, USA), mounted with Vectashield (Vector Laboratories, Burlington, Ontario, Canada) and fluorescence viewed in a Zeiss confocal laser scanning microscope (488 nm and 543 nm wavelengths).
Positive control sections received the same treatment but were pretreated with DNase I (Promega Corp. Madison, Wisconsin, USA) for 30 min at 37°C prior to TUNEL. For negative controls, sections were incubated with TUNEL label only.
TUNEL/active caspase-3 dual labelling and confocal microscopy
Sections from eight patients were mounted onto superfrost plus-coated slides (BDH) as above, dewaxed and hydrated in DEPC-treated solutions before microwaving in 0.1 M Tris–Urea buffer (pH 10.0) for 25 min. Slides were incubated in ice-cold permeabilization solution, and proteins blocked with donkey serum (Sigma) 1 : 20 in PBS for 10 min at room temperature. Sections were incubated in 1 µg ml–1 of anti-active caspase-3 (R and D Systems No.AF835) for 24 h at 4°C. TUNEL solution (Roche reagents, Roche, Mannheim, Germany) of enzyme and label, 1 : 9 respectively, was applied to sections for 1 h at 37°C. Incubation with biotinylated donkey anti-rabbit IgG (Sigma) 1 : 200 in PBS for 30 min, followed by 30 min in streptavidin-conjugated-Texas Red (Vector Laboratories) (20 µg ml–1) in PBS. Slides were mounted using Vectashield anti-fade mountant (Vector Laboratories) and viewed in a Zeiss confocal laser scanning microscope (488 nm and 543 nm wavelengths). TUNEL-positive control sections received the same treatment but were pretreated with DNase I as above.
Electron microscopy
Following fixation, samples were washed in PB and postfixed in 1% osmium tetroxide for 1 h before washing in sodium maleate buffer (0.05 M, pH 5.2), block staining in uranyl acetate in maleate buffer, dehydrated in an ethanol series and embedding in Agar 100 resin. Semi-thin sections were screened by light microscopy for the presence of primordial and primary follicles. Subsequently, examples from seven patients were trimmed and ultra-thin sections stained with uranyl acetate and lead citrate and examined in a Philips CM100 TEM.
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Results |
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Extracted protein samples from both human and mouse ovary showed a strong positive band at approximately 32 kDa when blotted with the procaspase-3 antibody. The band was detected with a loading of 25 or 50-µg human protein. No blots were detected at 11 or 17 kDa that would have suggested cross reactivity with active caspase-3. (Figure 1).
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A variety of follicle stages were observed in light microscopic sections from the 13 biopsies with the number ranging from 1 to 37 per sample; mean ± SD. = 12.7 ± 11.3. Of these, 151 were primordial or primary, four were secondary and 14 antral follicles.
In IH studies, procaspase-3 was detected in all specimens with labelling localized to granulosa cell layer and to a lesser extent thecal tissues in both healthy and atretic antral follicles, and also in granulosa and oocytes of primordial, primary and secondary staged follicles (Figure 2a-c). No staining was observed in control sections when primary antibody was omitted from the incubation medium and also in seven biopsy specimens where rabbit IgG replaced the antibody (Figure 2d-f). In the other three examples, some, but not all, oocytes had light cytoplasmic staining after IgG treatment.
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In sections treated with the antibody to active caspase-3, labelling was only detected in granulosa of six antral follicles that also had morphological criteria of atresia, namely the presence of fragmented, pycnotic nuclei, separated clumps of granulosa in the antral aspect of the follicle and uneven layering of the follicle wall (Figure 3). The majority of staining was noted in the granulosa aggregates but was also distinct in some separate mural granulosa cells. Five healthy antral follicles and all primordial, primary and secondary follicles (Table I and Figure 4) had no immunoreactivity for active caspase-3.
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In the 13 biopsies studied by TUNEL, eight atretic antral follicles were observed, in which granulosa cells had pycnotic nuclear fragments that emitted well-defined fluorescent signal with the Hoechst stain, and all of these were TUNEL positive (Figure 5). In these same follicles, other non-pycnotic granulosa nuclei were also TUNEL positive, but thecal tissue was only sparsely positive with only one or two nuclei showing labelling per follicle profile. No TUNEL was detected in any of 151 small follicles, the four secondary follicles or in six large healthy antral follicles (Table I). In each case, control sections treated with DNase prior to TUNEL procedure showed widespread nuclear labelling, including vascular and stromal fibroblastic cells. In sections in which TUNEL enzyme was omitted, no labelling of any cells was observed.
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Only atretic antral follicles showed labelling with confocal microscopy after dual labelling to co-localize TUNEL and active caspase-3. Individual granulosa cells were either caspase positive, TUNEL positive or showed positivity for both signals. Groups of aggregated granulosa cells adjacent to the antrum exhibited the strongest signals (Figure 6). Some positive caspase granulosa cells were located within the follicle wall although most of these were TUNEL negative. Thecal tissues of these follicles had only occasionally one or two cells stained for active caspase-3. No primordial, primary or secondary follicles had TUNEL or active caspase-3-labelled cells.
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By TEM, profiles of 24 and 15 follicles containing oocytes with predominantly flattened granulosa (primordial follicles) and cuboidal-shaped granulosa (primary follicles) respectively were studied for cellular integrity, organelle swelling and membrane integrity. All granulosa and oocyte nuclear profiles surveyed had intact nuclear membranes and no evidence of marginal, dense-staining chromatin indicative of apoptotic body formation. Prominent multivesicular bodies and annulate lamellae forming Balbiani’s vitelline body were characteristic features of oocytes (Figure 7). In some samples of both flattened and cuboidal granulosa, individual cells had convoluted nuclear profiles and moderately dilated cisternae of endoplasmic reticula. In all of these, the cells were joined to otherwise healthy cells by desmosome and tight-junctional complexes, and hemidesmosome-like junctions abutting the basement membrane were found frequently (Figure 8). One example of a grossly degenerate small follicle was detected (Figure 9). Here, the follicle outline was convoluted, and cytoplasmic vacuolation was widespread in all constituent granulosa and the oocyte, neither of which had clear organelle nor membrane definition. A basement membrane was evident, but no apoptotic nuclear structures were seen.
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Discussion |
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Antral follicles with pycnotic nuclei have consistently shown active caspase-3 and TUNEL in their granulosa tissue, and this feature provides a positive control in histochemical approaches to investigate apoptosis in other ovarian structures including primordial and primary staged follicles. In contrast to the report of Depalo et al. (2003)
, no TUNEL was detected, nor was there any other morphological evidence for apoptosis in primordial and primary staged follicles in specimens that had TUNEL-positive large atretic follicles in adjacent sections. Furthermore, it was also evident that many, if not all, follicles contained procaspase-3, but the active form of caspase-3 was only detected in atretic antral follicles. This finding is in agreement with Matikainen et al. (2001), in which non-atretic follicles had neither active caspase-3 nor TUNEL staining. Consequently, it is probable that a low level of constitutive production of procaspase-3 occurs in many follicles at all stages of development, but signals and factors for activation are normally only effective and/or present in antral follicles. The pale oocyte staining seen after IgG control treatment in some sections suggests that some non-specific binding of Igs may occur during IH, and further work is needed to clarify whether some or all oocytes express procaspase-3 protein. Only a small number of secondary follicles were observed in the current study, and further samples need to be investigated to clarify the extent of any caspase-3 activation and TUNEL positivity. In this context, Vaskivuo et al. (2001) reported occasional TUNEL-positive cells in two of 22 secondary follicles in adult whole ovary samples.
In preliminary studies using TUNEL procedures on formalin-fixed tissues and detection with chromogens such as diaminobenzidine, instances of false-positive results included labelling of stromal cell and vascular nuclei and some primordial and primary follicle cells. Critical approaches to using TUNEL have been reported as a result of concerns regarding sensitivity and specificity (Labat-Moleur et al., 1998). We have found that using freshly prepared paraformaldehyde for fixation, proteinase K treatment, fluorescent detection of dUTP incorporation (rather than chromogens such as diaminobenzidine) and treatment of all water solvent with diethyl pyrocarbonate to reduce nuclease activity were essential procedures to ensure high sensitivity and elimination of non-specific labelling. The inclusion of DNase treatment and no TUNEL enzyme controls were also important to provide specificity of detected DNA fragmentation. The dual labelling studies provide further support that co-localization of active caspase-3 and TUNEL was confined to granulosa within atretic follicles as reported also in isolated pig atretic follicles (Berardinelli et al., 2004). The finding of mural granulosa showing caspase-3 only as well as cells with both caspase-3 and TUNEL suggests cytoplasmic activation of caspase-3 is initiated before the appearance of more dense aggregates of TUNEL-positive pycnotic nuclei in the antral aspect of the follicles. The lower levels or absence of caspase-3 activation and TUNEL in thecal tissue as compared with granulosa also corroborates the observations of Matikainen et al. (2001), in which caspase-3 activity in thecal cells was only discerned in grossly atretic follicles.
The TEM studies showed many of the features of primordial and primary human follicles as described previously by Baca and Zamboni (1967), Hertig and Adams (1967), Hertig (1968) and de Bruin et al., 2002). The single follicle regarded as grossly degenerate was not characterized by cell separation, nuclear chromatin margination or fragmentation—these being major morphological features of advanced apoptosis (Wyllie et al., 1980). In all other follicles, the absence of cell-surface blebbing, chromatin condensation and nuclear fragmentation in both granulosa and oocytes is strong support for the notion that primordial and primary follicles do not show morphological evidence of apoptosis. The finding of occasional vacuolation, in the absence of nuclear condensation, is not characteristic of apoptotic cells. These features could be explained by fixation artefact as indicated by Hertig and Adams (1967), where oocyte mitochondrial morphology varied considerably with changes to fixatives. These features could also be a consequence of cellular necrosis, as suggested by de Bruin et al (2002). Further studies are required to address the notion of necrosis in otherwise normal ovaries. It is possible that the pool of primordial and primary follicles has very slow rates of cell death by a form of necrosis in individual granulosa but also low rates of compensatory proliferation that would provide a means of maintaining cellular constituency and thus follicle health and patency for many years.
This study has shown that whilst procaspase-3 is present in many types of ovarian follicles, the signals and factors for caspase-3 activation and subsequent cell death are normally only effective and present in antral follicles.
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Acknowledgements |
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The excellent electron microscopic skills of Bronwyn Smaill are gratefully acknowledged. The authors thank Associate Professor W.R. Gillett, and Drs A. Mekhail and S. Tout for their clinical support and provision of biopsies. This study was supported by a grant from the New Zealand Lottery Health Board.
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References |
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Submitted on December 15, 2005; resubmitted on March 8, 2006; accepted on March 16, 2006.
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