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Original Studies |
Hospital for Children and Adolescents, University of Helsinki (K.E., V.H., L.D.), FIN-00290 Helsinki; the Surgical Unit, Helsinki City Health Department (E.W.), FIN-00180 Helsinki; 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, FIN-00290 Helsinki, Finland. E-mail: krista.erkkila{at}helsinki.fi
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Abstract |
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We conclude that NAC, which is an antioxidant, plays an important role in germ cell survival in the human seminiferous tubules in vitro. We also suggest NAC as a possible new therapeutic factor for some men with idiopathic oligospermia.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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N-Acetyl-L-cysteine (NAC) is a well established thiol antioxidant that after uptake, deacetylation, and conversion to glutathione, functions as both a redox buffer and a reactive oxygen intermediate scavenger (8, 9, 10). In experimental studies, NAC has been reported to be an effective inhibitor of physiological cell death in several systems (9, 11, 12). In addition, it is a compound with which there is extensive clinical experience in the treatment of patients suffering from acetaminophen overdose, pulmonary disorders, or acquired immunodeficiency syndrome (8, 10, 13). Recently, NAC has also been suggested as a potential therapeutic factor for male patients with infertility, because by reducing reactive oxygen species (ROS) in semen, it was able to improve human sperm function in vitro (14).
In the rat testis, the expression of antioxidant enzymes has been demonstrated (15, 16), and ROS have been implicated in the testicular damage following torsion and reperfusion (17). Furthermore, human spermatozoa have been shown to be able to produce ROS (18, 19). Increased production of free radicals has been suggested to play a major role in defective sperm function (18, 20, 21). Protection of sperm from ROS damage by antioxidants (including NAC) as well as the preventive effects of oxygen radical scavengers on testicular function after acute experimental torsion (14, 17) suggest that antioxidants act as factors of survival, ensuring germ cell function.
During regular spermatogenesis, most of the testicular germ cells degenerate before reaching maturity, the mechanism being apoptosis (22, 23, 24). Programmed death in these cells has been shown to be sensitive to hormonal withdrawal, as lack of hormonal stimulation causes increased apoptosis in them (1, 23, 25). In keeping with this, we have recently shown the importance of testosterone as a survival factor for human testicular germ cells in vitro (1). However, the exact mechanisms by which hormonal signals are transduced within the cells to decide their future course, and whether other effectors of the apoptotic pathway take part in the control of human germ cell survival, are not known.
As the role of free radical formation in human testicular apoptosis has not been evaluated, the aim of the present study was to investigate in vitro whether apoptosis in human spermatogenic cells is regulated by antioxidant NAC. Germ cell death in the seminiferous tubules was induced by incubating segments of tubules under serum-free culture conditions. Apoptosis and apoptotic cells were detected by Southern blot analysis of DNA fragmentation, by DNA labeling in situ, and by morphological analysis under the electron microscope.
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Subjects and Methods |
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Tissue was obtained from 16 adult men undergoing orchidectomy as a hormonal treatment for prostate cancer. Three of the patients had signs of bone metastases, and the rest of them suffered from nonmetastasized cancer. The operations were performed between February and July 1997 at the Helsinki City Health Department, Surgical Unit (Helsinki, Finland). The age range of the patients was 57–81 yr. The patients had undergone neither hormonal nor chemotherapeutic medication, nor had they had radiotherapy before the operation; they had no endocrinological disease, and none of them had suffered from cryptorchidism.
Tissue preparation
The tissue was prepared as recently described (1). The testis
tissue was microdissected under a transillumination stereomicroscope in
a petri dish containing phosphate-buffered saline. Segments of
seminiferous tubules 1–2 mm in length and small tissue samples
(
1 x 2 x 2 mm) consisting of seminiferous tubules were
isolated and transferred in 10 µL phosphate-buffered saline to a
96-well culture plate. For squash preparations, segments of
seminiferous tubules were transferred in 10 µL medium onto a
microscope slide, squashed under a coverslip, and fixed as previously
described (1, 26). For Southern blot analysis of DNA fragmentation,
in situ detection of apoptotic DNA, and morphological
identification of apoptosis, the samples were processed as described
below.
Culture
The samples were incubated under serum-free conditions for different lengths of time (4, 24, and 48 h) in the absence or presence of NAC (Sigma Chemical Co., St. Louis, MO) or testosterone (Sigma). Each well contained 100 µL tissue culture medium (Ham’s F-10, Life Technologies Europe, Paisley, UK) supplemented with 0.1% human albumin (Sigma) and 10 µL/mL gentamicin (Life Technologies). The final concentrations of NAC were 125, 100, 50, and 25 mmol/L; those of testosterone were 10-6 and 10-7 mol/L. The incubations were performed at 34 C in a humidified atmosphere containing 5% CO2.
Southern blot analysis of apoptotic DNA fragmentation
Tissue samples (1 x 2 x 2 mm) were snap-frozen in
liquid nitrogen. Genomic DNA was extracted as previously described (27)
with modifications (1, 28). After isolation and quantitation, DNA
samples were 3'-end labeled with digoxigenin-dideoxy-UTP (Dig-dd-UTP;
Boehringer Mannheim, Mannheim, Germany) using the terminal transferase
(TdT; Boehringer Mannheim) reaction, fractionated through 2% agarose
gels, and blotted onto a nylon membrane. Dig-dd-UTP 3'-end-labeled DNA
from the nylon membrane was detected with the aid of the antibody
reaction (anti-DIG-AB, AFOS-conjugated; Boehringer Mannheim) as we have
recently described (1). The luminescence reaction was performed in CSPD
solution (Boehringer Mannheim) as previously described (1). The
membrane was then exposed to x-ray film. The information (optical
density) given by the x-ray films was digitized by a scanner (Microtec
ScanMaker, Microtec International, Inc., Taiwan), and the data in
pixels were analyzed with the NIH Image (1.61) analysis software (NIH,
Bethesda, MD). To identify low mol wt DNA fractions (<1.3 kb), a
labeled DNA marker was used. The pixel number of the low mol wt 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 low mol wt DNA
pixel number by that of the 0 h sample. Thus, the results were
expressed as relative to the 0 h value.
Nonradioactive in situ end labeling (ISEL)
Preparations were squashed and fixed as previously described (1, 26, 29). Small tissue samples (1 x 1 x 2 mm) consisting
of seminiferous tubules were fixed in glutaraldehyde, embedded in
paraffin, sectioned at 4 µm, and mounted on slides coated with
Vectabond (Vector Laboratories, Burlingame, CA). The ISEL of the squash
preparations and tissue samples was performed as previously described
(1, 29) but with a few modifications. After rehydration, the tissue
samples were incubated in 10 mmol/L citric acid (pH 6.0) in a microwave
oven for 5 min + 2 min and washed in distilled water. DNA 3'-end
labeling with Dig-dd-UTP (Boehringer Mannheim) by the TdT (Boehringer
Mannheim) reaction, the antibody reaction (antidigoxigenin antibody,
conjugated with alkaline phosphatase, 1:4000; Boehringer Mannheim), and
exposure to the substrates for alkaline phosphate were performed as
recently described (1). The slides were weakly counterstained with
hematoxylin. For the negative controls, the TdT enzyme was replaced by
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). The identification of germ cell types was based on their characteristic morphology (30). 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 (2).
Statistical analysis
The experiments were repeated on at least three independent occasions. Quantitative data represent low mol wt DNA (optical density from x-ray films). The 0 h point was set as 1.0 (100%), and the other settings were compared to it. Data obtained from the replicate experiments (mean ± SEM) were analyzed by one-way ANOVA followed by Student-Newman-Keuls test for appropriate statistical comparisons. P < 0.05 was considered significant.
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Results |
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Apoptosis was revealed by low mol wt DNA fragmentation (185
bp multiples) with the aid of 3'-end-labeled DNA (Fig. 1
). Incubation of segments of tubules
under serum-free culture conditions induced rapid germ cell death, as
incubations for 4 and 24 h without any treatments (i.e.
NAC or testosterone) increased apoptosis by 110% (P <
0.01, 0 vs. 4 h) and 156% (P < 0.01,
0 vs. 24 h), respectively, relative to that at 0
h. Consistent with our recent findings (1), after incubation for 4
h, testosterone significantly inhibited the deaths occurring in the
seminiferous tubules cultured under serum-free conditions (Fig. 1, A
and C). NAC was also able to suppress these programmed germ cell
deaths. Apoptosis was inhibited by NAC concentrations of 125, 100, 50,
and 25 mmol/L in a dose-dependent manner (Fig. 1, B and D). As
demonstrated in Fig. 1D, after incubation for 4 h, DNA
fragmentation was suppressed by 68% at a NAC concentration of 125
mmol/L (P < 0.001, 4 h 125 mmol/L NAC
vs. 4 h nontreated), by 63% at 100 mmol/L
(P < 0.001, 4 h 100 mmol/L NAC vs.
4 h nontreated), and by 54% at 50 mmol/L (P <
0.01, 4 h 50 mmol/L NAC vs. 4 h nontreated)
compared with samples cultured without NAC. NAC concentration of 25
mmol/L did not significantly inhibit germ cell death. The suppression
of apoptosis by antioxidant NAC was still effective after 24 h
(Fig. 1) and 48 h of incubation (data not shown).
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The apoptotic nature of cell degeneration was confirmed by ISEL
(Fig. 2). Consistent with the results of
Southern blot analysis, a time-dependent increase in the apoptotic DNA
fragmentation in situ and its inhibition by NAC were
observed in both histological sections and squash preparations (Fig. 2). Incorporation of Dig-dd-UTP was most often found in spermatocytes
and spermatids. Not all apoptotic cells could be identified, however,
because of nuclear pycnosis in the late stages of apoptosis. There was
no staining when TdT enzyme was substituted for the same volume of
distilled water (negative control).
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The apoptotic nature of cell degeneration was further confirmed by
electron microscopy (Figs. 3 and 4). As demonstrated by the ISEL analysis,
morphological signs of apoptosis were most frequently identified in
spermatocytes (Fig. 3) and early spermatids (Fig. 4). In addition,
different stages of apoptosis were observed in late spermatids by
electron microscopy (Fig. 4). As in the ISEL samples, late apoptotic
cells were impossible to identify.
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). Simultaneously, the synaptonemal complex, which is characteristic
of these cells, became unidentifiable (Fig. 3). During apoptosis of
early spermatids a ring-like condensation of chromatin was observed
(Fig. 4E). Some of the late spermatids showed abnormal condensation of
chromatin (Fig. 4D), which we suggest to be a sign of apoptosis in late
human spermatids. Clumping of chromatin and compact, nonidentifiable
organelles were also seen in apoptotic late spermatids (Fig. 4G). |
Discussion |
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An in vitro culture may naturally have some limitations as to approximation of germ cell physiology in vivo. However, we believe that the present tissue culture model provides a system sufficiently physiological to study the regulation of male germ cell physiology, firstly because in the present model the germ cells are allowed to stay in their natural surroundings (i.e. in the seminiferous tubules, which also include somatic sells, e.g. Sertoli cells), and secondly because cell death is blocked by a well established germ cell survival factor, testosterone.
Programmed cell death during regular spermatogenesis is a mechanism by which most of the germ cells in the testis degenerate before reaching maturity (22, 23, 24). Survival of these cells has been shown to be sensitive to hormonal withdrawal, as lack of hormonal stimulation causes increased apoptosis among them (1, 23, 25, 29). In this context, we have recently (1) and in this study shown the importance of testosterone as a survival factor in human testicular germ cells in vitro. Elucidation of how hormonal signals are transduced within different cell lines to direct their life or death has become a focus of interest in several research fields (31, 32, 33). Recent studies support the concept that there is a conserved pathway(s) of intracellular effectors responsible for preventing or bringing about cell death (34, 35).
Control of antioxidant defenses is likely to be one of the critical factors in the regulation of apoptosis (5, 35). Unstable, partially reduced forms of oxygen, i.e. ROS, are generated in cells through normal metabolic activities and hormone-mediated signaling events (5). During spermatogenesis, the production of ROS has been described in developing germ cells or during the phagocytic action of Sertoli cells (15). Furthermore, the expression of antioxidant enzymes has been reported in the rat testis (15, 16). Correspondingly, human spermatozoa are able to produce ROS (19, 21).
In many cell types, activation of an apoptotic cascade has been shown to occur as a consequence of uncontrolled generation of ROS (7). In addition, oxidative changes take place during apoptosis induced by nonoxidative stimuli (7). Programmed cell death can be delayed or inhibited by inclusion of antioxidants or by overexpression of antioxidative enzymes in different cell systems, including female germ cells (6, 7, 36). In the rat ovary, gonadotropins increase the expression of antioxidative enzymes while suppressing granulosa cell apoptosis and follicular atresia (36). Moreover, in follicles deprived of hormone support, granulosa cell apoptosis is prevented by inhibitors of oxidative stress, including NAC (36). In agreement with these previous results regarding regulation of apoptosis by antioxidants, in the present study antioxidant NAC was a significant suppressor of apoptosis in human testicular germ cells.
The mechanism underlying the association between apoptotic pathways and
oxidative changes is unclear. Apoptosis in response to withdrawal of
growth factors has been suggested to involve down-regulation of
antioxidant defenses, resulting in increased sensitivity to the ROS
produced during normal metabolism (37). In support of this, hormones
have been reported to be able to modulate the expression of genes
encoding key antioxidant enzymes (36) as well as the proposed functions
of the Bcl-2 family in regulating the intracellular reduction-oxidation
state (6). On the other hand, antioxidant-mediated protection of
apoptosis has been suggested to be due to a direct effect on
mitochondrial function (11, 35, 38), to cytochrome c action,
or to the regulative role of a transcriptional cascade after
hormone-receptor interaction (31). The mechanisms underlying the
preventive role of antioxidants on cell death have also been suggested
to be related to activation or inhibition of various factors, including
the apoptosis-inducing factor, nuclear factor-
B, Apaf 1–3,
caspases, Fas, tumor necrosis factor, p53, or the products of the
bcl-2 gene family (12, 33, 39, 40, 41, 42). In the present study,
testosterone as well as antioxidant NAC prevented testicular germ cell
apoptosis caused by hormone withdrawal. The inhibition of oxidative
stress by NAC was not directly demonstrated, because apoptosis was
induced by depletion of hormones and not by controlling oxidative
conditions. However, the present findings support the idea of an
association between oxidative stress and apoptosis, but leave the
targets of action on the intracellular level to be further
evaluated.
Different categories of testicular cell are suggested to display different degrees of susceptibility to oxidative stress. The antioxidant system of somatic Sertoli and peritubular cells differs from that of spermatocytes, spermatids, and spermatozoa (15, 16). In the present investigation, germ cells were the most sensitive to hormonal withdrawal in the late stages of differentiation, as the apoptotic cells were primarily spermatocytes and early spermatids. A finding that has not been reported previously is that different stages of apoptosis in late spermatids were observed under the electron microscope. It was the cells in the later phases of spermatogenesis that survived in the presence of antioxidant NAC or testosterone, indicating the sensitivity of these cells to regulation of apoptosis and survival. However, as in our previous studies, some of the cells in the late stage of apoptosis could not be identified. For this reason and because there were only a few spermatogonia compared to the number of other types of testicular cell, the possibility of spermatogonial apoptosis cannot be excluded.
Although controlled ROS production by spermatozoa has important beneficial functions (19), increased ROS production is known to impair sperm function (19, 20). As yet, the mechanisms that play a role in the overproduction of ROS and their deleterious effects on sperm function are not clear. The detection of increased ROS generation in the semen of infertile men has been suggested to reflect an imbalance between production and degradation of ROS in spermatozoa and seminal plasma (18, 43). However, little is known about whether antioxidants act as regulators of germ cell survival in seminiferous tubules ("upstream sperm"). In the rat, ROS have been implicated in the testicular damage after torsion and reperfusion (17), in cryptorchidism (44), or upon exposure to toxic chemicals (45, 46), thus indicating that antioxidative mechanisms are also of importance for the survival of germ cells in the seminiferous tubules during spermatogenesis. In agreement with this hypothesis, we found in the present study that NAC protected human testicular germ cells from the apoptosis induced by hormone withdrawal.
The use of antioxidants (including NAC) for preventing sperm from ROS damage and the protective effect of oxygen radical scavengers on testicular function after acute experimental torsion (14, 17) have been described in the rat. Reduction of ROS activity in semen by addition of a scavenging agent has been suggested as an approach to the treatment of male factor infertility, as NAC significantly decreases ROS activity in human semen in vitro (14). The results of the present study of human tissue indicate that NAC also improves the production of spermatozoa by inhibiting the death of their precursor germ cells. Thus, at least in a subgroup of men with idiopathic oligospermia, NAC may offer new therapeutic possibilities.
According to the free radical theory of aging (47), antioxidant mechanisms may change with age. In the present study, the testis tissue was obtained mostly from elderly men. However, we have performed similar experiments with testes derived from younger men or adolescents suffering from testicular cancer, using samples taken from an unaffected area. The findings in these experiments (data not shown) were similar to those in which tissue from elderly patients was used, thus indicating that apoptosis in the human seminiferous tubules is regulated by testosterone and antioxidants regardless of age.
In conclusion, in the present study, the antioxidant NAC and testosterone significantly inhibited apoptosis in human testicular germ cells in vitro, thus indicating the importance of antioxidative mechanisms for germ cell survival in the seminiferous tubules during spermatogenesis. NAC is also suggested as a possible new therapeutic factor in some men with idiopathic oligospermia.
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Acknowledgments |
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Footnotes |
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Received December 8, 1997.
Revised March 23, 1998.
Accepted April 3, 1998.
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References |
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