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Hospital for Children and Adolescents, University of Helsinki (K.E., V.P., L.D.), FIN-00029 Helsinki, Finland; Helsinki Bioenergetics Group, Department of Medical Chemistry, Institute of Biomedical Sciences, University of Helsinki (M.W.), FIN-00014 Helsinki, Finland; and the Department of Anatomy, University of Turku (M.P.), FIN-20520 Turku, Finland
Address all correspondence and requests for reprints to: Dr. Krista Erkkilä, Hospital for Children and Adolescents, University of Helsinki, P.O. Box 281, Stenbäckinkatu 11, FIN-00029, HYKS (Helsinki), Finland. E-mail: krista.erkkila{at}huch.fi
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Even during normal spermatogenesis, a number of germ cells die via apoptosis or programmed cell death (PCD), a process that is sensitive to hormonal control (8, 9, 10). To study human germ cell apoptosis, we have recently developed an in vitro tissue culture model in which germ cell PCD is induced by incubating segments of seminiferous tubules without survival factors, i.e. without serum or hormones (9). Testosterone (a testicular survival factor) prevents the PCD of spermatocytes and spermatids, thus demonstrating the physiological nature of the organ culture (9). The mechanisms by which hormonal signals or the lack of them are transduced within these cells to direct their life and death are unclear, however (11, 12, 13).
Regardless of the PCD-inducing signal (e.g. withdrawal of hormones), oxidative changes and reactive oxygen species (ROS) have been associated with apoptosis in many cell types (13, 14, 15, 16, 17, 18, 19, 20). PCD in response to withdrawal of growth factors has been suggested to down-regulate antioxidant defenses and thereby increase the sensitivity of the cells to ROS (21). Furthermore, apoptosis has been shown to be suppressed by antioxidants or antioxidative enzymes (16, 17, 18, 22). However, some data indicate that ROS are neither common nor obligatory mediators of PCD in general (14, 23). Moreover, the role of oxygen, a necessary substrate for ROS production, is not clear either (13, 19, 24, 25, 26, 27).
Oxygen is consumed mainly by cell respiration in mitochondria, which are also the main site of ROS production. Recent evidence indicates that in several cell types, the apoptotic pathways converge to mitochondria, which then play an essential role in the execution of cell death (14, 15, 20, 28, 29, 30, 31, 32, 33, 34). Crucial roles have been proposed for alterations that decrease mitochondrial energy transduction and enhance production of ROS (20, 33), and particularly for the induction of the mitochondrial permeability transition (PT) (15, 28, 29, 30, 31, 32). PT, in turn, can be inhibited by several factors, including cyclosporin A (CsA) and its nonimmunosuppressive derivative N-methyl-Val4-CsA (14, 15, 30, 31, 32, 34, 35). Interestingly, recent evidence suggests that morphological changes in mitochondria during apoptosis vary with the type of cell and the inducer of the apoptotic pathway (36).
The aim of the present study was to investigate the mechanisms underlying human testicular germ cell apoptosis by evaluating whether, in our in vitro model, this process is regulated by oxygen or by inhibition of PT. Thus, germ cell death was induced by incubating segments of seminiferous tubules in the absence of survival factors (i.e. serum and hormones). The effects of different oxygen tensions on this apoptosis were tested. Furthermore, the role of CsA or N-methyl-Val4-CsA, which block the mitochondrial PT, was studied.
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Subjects and Methods |
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Testis tissues were obtained from 17 adult men undergoing orchidectomy as hormonal treatment for prostate cancer. The operations were performed between December 1997 and August 1998 at the Surgical Unit of Helsinki City Health Department (Helsinki, Finland). The patients had received neither hormonal nor chemotherapeutic medication, nor had they received radiotherapy before the operation; they had no endocrine disease, and none of them had suffered from cryptorchidism. Their age range was 64–84 yr. The ethics committees of the Departments of Pediatrics and Urology, University of Helsinki, approved the study protocol.
Tissue preparation
The tissue was prepared as recently described (9). The testis tissue was microdissected under a transillumination stereomicroscope in a petri dish containing PBS. Segments of seminiferous tubules, 3–5 mm in length, were isolated and transferred to a culture plate. For squash preparations, segments of seminiferous tubules were placed in 10 µL medium on a microscope slide and gently squashed under a coverslip to enable the cells to migrate and produce a monolayer. The preparations were then fixed as previously described (9, 22). For Southern blot analysis of DNA fragmentation, for in situ detection of apoptotic DNA, and for morphological analysis by electron microscopy, the samples were processed as described below.
Culture
Samples were incubated under serum-free conditions in tissue culture medium (nutrient mixture Ham’s F-10, Life Technologies, Europe, Paisley, UK) supplemented with 0.1% human serum albumin (Sigma Chemical Co., St. Louis, MO), and 10 µg/mL gentamicin (Life Technologies, Inc.). The final concentrations of CsA (Sigma Chemical Co.) were 10, 5, and 0.5 µmol/L, and that of N-Met4-Val-CsA (provided by Dr. Ove Eriksson) was 10 µmol/L. Incubations were performed at 34 C in a humidified chamber under continuous gas flow from gas bottles (Aga, Espoo, Finland) containing 5% of CO2 and different concentrations of O2; the remainder was nitrogen.
Southern blot analysis of apoptotic DNA fragmentation
Testis tissue samples were snap-frozen in liquid nitrogen. Genomic DNA was extracted as previously described (9, 22). After isolation and quantitation, DNA samples were 3'-end labeled with digoxigenin-dideoxy-UTP (Dig-dd-UTP; Roche Molecular Biochemicals GmbH, Mannheim, Germany), using the terminal transferase (TdT; Roche Molecular Biochemicals) reaction, fractionated through 2% agarose gels, and blotted onto a nylon membrane. Dig-dd-UTP 3'-end-labeled DNA on the nylon membrane was detected with an antibody reaction (anti-DIG-AB, AFOS-conjugated; Roche Molecular Biochemicals) as recently described (9). The luminescence reaction was performed in CSPD solution (Roche Molecular Biochemicals) as previously described (9). The membrane was then exposed to x-ray film. The information (optical density) given by the x-ray films was digitized by a tabletop scanner (Microtec ScanMaker, Microtec International, Inc., Taiwan), and the data were analyzed with NIH Image (1.61) analysis software (NIH, Bethesda, MD). A labeled DNA marker was used to identify low molecular weight DNA fractions (<1.3 kb). The pixel number of the low molecular weight DNA fraction of the 0 h sample was set at 1.0 (100%), and other lanes of the same Southern blot were analyzed by dividing the pixel number by that of the 0 h sample. Thus, the results are expressed in relation to zero time.
Nonradioactive in situ end labeling of DNA (ISEL)
Preparations were squashed and fixed as previously described (9, 22). ISEL of the squash preparations was performed as described previously (9, 22) with some modifications. After rehydration and preincubation with terminal transferase reaction buffer (1 mol/L potassium cacodylate, 125 mmol/L Tris-HCl, and 1.25 mg/mL BSA, pH 6.6), apoptotic DNA of the samples was 3'-end labeled with Dig-dd-UTP (Roche Molecular Biochemicals) by the TdT (Roche Molecular Biochemicals) reaction for 1 h at 37 C. Antidigoxigenin antibody conjugated to horseradish peroxidase (Anti-Digoxigenin-POD, Roche Molecular Biochemicals) and diaminobenzidine (Sigma Chemical Co.) were used to detect Dig-dd-UTP-labeled DNA. The slides were weakly counterstained with hematoxylin, after which the samples were dehydrated. For the negative controls, the TdT enzyme was replaced with the same volume of distilled water.
Electron microscopy
Segments of seminiferous tubules were microdissected under a transillumination microscope and cultured as described above. They were fixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.2, postfixed with 1% osmium tetroxide in 0.1 mol/L phosphate buffer, dehydrated, and embedded in epoxy resin. They were then sectioned at 50 nm with a Reichert E ultramicrotome (Reichert Jung, Vienna, Austria) and stained with uranyl acetate and lead citrate. Observations were made with a JEOL JEM 1200 EX transmission electron microscope (JEOL, Tokyo, Japan). Identification of germ cell types was based on their characteristic morphology (37). Cells were identified as apoptotic by typical ultrastructural changes, including nuclear and/or cytoplasmic condensation and, in the late stage of apoptosis, by dense pycnotic bodies (38).
Statistical analysis
The experiments were repeated on at least three independent occasions. Quantitative data represent low molecular weight DNA (measured from x-ray films as optical density). The 0 h point was set at 1.0 (100%) to which the other settings were compared. Data obtained from the replicate experiments (mean ± SEM) were analyzed by one-way ANOVA followed by the Student-Newman-Keuls test for appropriate statistical comparisons. P < 0.05 was considered significant.
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Results |
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As detected by Southern blot analysis of DNA fragmentation,
incubation of seminiferous tubule segments without survival factors
(i.e. serum and hormones) for 4 h in 21% oxygen
induced clear apoptosis of the germ cells (Fig. 1
). As shown in Fig. 1B, the PCD
occurring in the seminiferous tubules was significantly inhibited by
lowering the oxygen level. After incubation for 4 h, DNA
fragmentation was suppressed by 61% at 0.5% oxygen (P
< 0.001), by 58% at 2% oxygen (P < 0.001), and by
51% at 5% oxygen (P < 0.01) compared with samples
cultured in 21% oxygen. Incubation at 10% oxygen did not
significantly inhibit apoptosis.
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(data not shown), thus indicating that PCD in
human male germ cells is regulated by oxygen regardless of the
patient’s age. Suppression of germ cell apoptosis by inhibitors of the mitochondrial permeability transition
Human testicular cell apoptosis was inhibited in a dose-dependent
manner by CsA, which binds to mitochondrial cyclophilin D and prevents
PT. After incubation for 4 h, DNA fragmentation was suppressed by
64% at a CsA concentration of 10 µmol/L (P <
0.001), by 50% at a concentration of 5 µmol/L (P <
0.05), and by 36% at a concentration of 0.5 µmol/L
(P < 0.05) compared with that in samples cultured
without CsA (in 21% oxygen; Fig. 2). The
possibility that this finding reflected the immunosuppressive effect of
CsA was excluded, because the nonimmunosuppressive CsA derivative
N-methyl-Val4-CsA also prevented germ
cell PCD (Fig. 2).
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The apoptotic cell types were identified by ISEL (Fig. 3A). Representative samples of cells from
human seminiferous epithelium were obtained by gently squashing
segments of seminiferous tubules under coverslips. In this technique,
cells from seminiferous epithelium migrate and produce a monolayer.
Incorporation of Dig-dd-UTP was found in spermatocytes and spermatids
(Fig. 3A). Not all apoptotic cells could be identified, however,
because of nuclear pycnosis in the late stages of apoptosis.
Consistent, with the results of Southern blot analysis, a
time-dependent increase in apoptotic DNA fragmentation in
situ and its inhibition by low levels of oxygen or by treatment
with CsA were observed in squash preparations (Fig. 3B). There was no
staining when the terminal transferase enzyme was substituted for the
same volume of distilled water (negative control).
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The apoptotic nature of the cell degeneration was further
confirmed by electron microscopy (Fig. 4). As in the ISEL analysis,
morphological signs of apoptosis were most frequently identified in
spermatocytes (Fig. 4) and spermatids (22), and apoptotic cells were
impossible to identify at a later stage. The mitochondria in apoptotic
germ cells appeared enlarged and swollen, and their membranes
discontinuous. In contrast, the mitochondria in the neighboring
nonapoptotic cells of the same preparation maintained their normal
structure (Fig. 4). The morphological alterations, including
mitochondrial changes, that were described after culture were also
found in apoptotic germ cells of the samples that were fixed
immediately after the operation i.e. before culturing
(Fig. 4, C and D).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In many cell types, oxidative and mitochondrial changes in conjunction with generation of ROS have been suggested to play key roles in the PCD process (14, 15, 20, 32). Apoptosis in response to withdrawal of growth factors has been suggested to involve modulation of antioxidant defenses, resulting in increased sensitivity to the ROS produced during normal metabolism (21). In this context, PCD has been demonstrated to be suppressed by antioxidants or by overexpression of antioxidative enzymes (16, 17, 18). However, there are also data indicating that ROS may not be obligatory mediators of all forms of PCD (14, 23), and it has been questioned whether the observed accumulation of ROS is a causal factor or only a side-effect of the other changes accompanying the apoptotic process (14).
In previous studies, the role of oxygen in PCD seems to have been enigmatic. Hypoxia and anoxia have been shown either to induce or to inhibit apoptosis. For example, hypoxic conditions have been shown to prevent PCD in neuronal cells (24) and thymocytes (13), but to enhance apoptosis in many other cell types, including cardiac myocytes and hepatoma and pheochromocytoma cells (25, 26, 27). In the present study, we demonstrate clear prevention of testicular apoptosis by lowering the oxygen tension. Thus, even though the direct effects of ROS were not investigated, oxygen, which is a necessary substrate for ROS production, appears to play an important role in male germ cell apoptosis. From the present results we infer that germ cell PCD in the human testis, which is induced by withdrawal of survival factors (i.e. serum and hormones), definitely involves oxygen-dependent steps during the apoptotic cascade.
However, the mechanism(s) underlying the association between oxidative changes and PCD is unclear. Recent data have suggested that mitochondria participate in the apoptotic cascade by loss of their energy-producing function and by enhanced production of ROS (20, 33). On the other hand, the mitochondrial PT (28, 29, 30, 32) and the release of apoptogenic factors through the outer mitochondrial membrane (28, 41) have particularly been implicated as key factors in PCD signaling. PT is associated with dissipation of the mitochondrial transmembrane potential due to opening of pores in the inner membranes of mitochondria (14, 15, 30, 31, 32, 42). The pore complex can be affected by several factors, including thiol-reactive agents such as NAC (32, 40) and cyclophilin D ligands, including CsA and its nonimmunosuppressive derivative N-methyl-Val4-CsA (14, 15, 31, 32, 34, 35).
The inhibition of PCD by CsA seems to depend both on cell type and on the inducer of apoptosis. Thus, CsA has been shown to block apoptosis in some cell systems, such as lymphocytes, hepatocytes, thymocytes, and liver endothelial cells (35, 41, 43, 44, 45). In some other systems, the same concentrations of CsA did not affect PCD. For example, in rat spermatocytes, CsA did not prevent apoptosis induced by methoxyacetic acid (46). Moreover, in leukemic and renal cells, CsA has been shown to induce PCD (47, 48). The present study demonstrates clear inhibition of apoptosis in human male germ cells by both CsA and N-Met-Val-CsA. Thus, in these cells the PCD cascade seems to include mitochondrial PT-related steps.
Recent evidence shows that mitochondria undergo structural changes during apoptosis (30, 36, 49, 50), although these changes are still poorly understood. Interestingly, the structural changes are suggested to vary with the cell type and with the inducer of PCD. For example, during aphidicolin-induced apoptosis of Chinese hamster ovary cells, the mitochondria were initially shown to be elongated, with an increased number of cristae, but later they appeared as condensed structures obscuring the cristae (36). In the same study, in contrast, the mitochondria of T cells undergoing dexamethasone-induced apoptosis were demonstrated to be swollen (36). In the present study, the mitochondria in the dying germ cells appeared enlarged and swollen, whereas in the neighboring nonapoptotic cells the mitochondrial structures remained normal. Thus, the morphological alterations observed in the apoptotic cells were due to PCD and not, for example, to nonspecific swelling or fixation artifacts. Furthermore, the morphological changes observed after culture in vitro were also seen in apoptotic cells of the samples that were fixed before culture. Thus, the mitochondrial alterations appear to occur during physiological apoptotic process also in vivo and not due to an artifact of the culture conditions in vitro.
In recent IVF techniques, successful fertilization has been achieved by injecting immature germ cells, namely round or elongated spermatids, into the cytoplasm of an oocyte (4, 7). However, much lower fertilization rates have been obtained with spermatid than with spermatozoa injections (4, 7). One of the reasons for this difference could be the harmful effects of oxygen, or ROS (5, 6, 18), as ex vivo the germ cells are normally manipulated in 21% oxygen. Actually, the current IVF systems could be argued to be far from physiological, as the oxygen tension in the reproductive tracts in vivo is considerably lower than 21%. Therefore, we suggest that in attempts to improve the success rates of various IVF techniques, the oxygen levels during germ cell manipulations should be lowered.
In conclusion, our study shows that PCD in human male germ cells is induced by incubating segments of seminiferous tubules without survival factors (i.e. serum or hormones) at 21% oxygen. This apoptosis of spermatocytes and spermatids is significantly inhibited at low levels of oxygen or by inhibitors of the mitochondrial PT. Mitochondria of the germ cells undergo morphological changes during the apoptotic process, which is consistent with the central role of PT in this process. Thus, oxidative mitochondrial functions are clearly involved in the regulation of human germ cell PCD.
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Acknowledgments |
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Footnotes |
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Received March 8, 1999.
Revised June 1, 1999.
Accepted June 18, 1999.
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References |
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-induced apoptosis in U937 cells: the role of mitochondria. Exp Cell Res. 220:232–240.[CrossRef][Medline]
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