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Experimental Studies |
Children’s Hospital (K.E., V.H., L.D.) and the Department of Urology (S.R., J.S.), University of Helsinki, SF-00290 Helsinki; and the Department of Anatomy, University of Turku (K.H., M.P.),SF-20520 Turku, Finland
Address all correspondence and requests for reprints to: Dr. Leo Dunkel, Children’s Hospital, University of Helsinki, SF-00290 Helsinki, Finland. E-mail: leo.dunkel{at}sci.fi
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Abstract |
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We conclude that apoptosis is induced in human seminiferous tubules under serum-free conditions in vitro. That this apoptosis is suppressed by testosterone indicates that testosterone in the human male is a critical germ cell survival factor. The model created in the present study provides a valuable tool for further investigation of hormonal and gene regulation of human germ cell death and survival.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Most of the germ cells degenerate before reaching maturity (4, 5, 6, 7). In rodent testis, the demise of spermatogonia (8), spermatocytes, and spermatids (9, 10, 11) is shown to occur through an apoptotic mechanism regulated by gonadotropins and androgens. In rat testis, hypophysectomy or treatment with a GnRH antagonist (12) as well as immunoneutralization of FSH (13) or gonadotropin ablation (14) induces germ-cell apoptosis. This effect is partially inhibited with testosterone, FSH agonist, or hCG (12). A decrease in the testosterone concentration induces a significant increase in the number of apoptotic germ cells in most stages of the cycle of the seminiferous epithelium (11). These data suggest that androgens (stimulated by LH) are indispensable for the maintenance of spermatogenesis.
As present knowledge of apoptosis in testis is mostly based on rodent models, the first aim of the present study was to create an in vitro model for evaluation of the role of apoptosis in human adult testes. Germ cell death in seminiferous tubules was induced by incubating segments of tubules under serum-free culture conditions. As androgens are indispensable for the maintenance of spermatogenesis, and they are reported to be important factors regulating apoptosis in rodent testis, the second objective of our study was to examine in human testicular apoptosis the possible role of testosterone. The detection of apoptosis and apoptotic cells was performed by Southern blot analysis of DNA fragmentation, DNA labeling in situ, and morphological analysis under light and electron microscopy.
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Subjects and Methods |
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Tissue was obtained from 10 adult men undergoing orchidectomy for prostate or testicular cancer; samples were dissected out from an unaffected area. The operations were performed between September 1995 and August 1996 at the Department of Urology, University of Helsinki (Helsinki, Finland). The age range of the patients was 28–87 yr. The patients had undergone neither hormonal nor chemotherapeutic medication, nor had they had radiotherapy before the operation. The patients had no endocrinological disease, and none of them had suffered from cryptorchidism. The pathologic anatomical diagnosis (PAD) of the patients with testicular cancer were seminomas, and their hCG levels were normal.
Tissue preparation
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
sections (
1 x 1 x 2 mm) consisting of seminiferous
tubules were isolated and transferred in 10 µl phosphate-buffered
saline onto a 96-well culture plate for the culture. For the squash
preparations, segments of seminiferous tubules were transferred in 10
µL medium onto a microscopic slide, squashed under the coverslip, and
fixed as previously described (11, 15). For Southern blot analysis of
DNA fragmentation, in situ detection of apoptotic DNA
fragmentation, and morphological identification of apoptosis, the
samples were processed as described below.
Culture
The samples were transferred onto 96-well culture plates as described above. They were then incubated under serum-free conditions for increasing lengths of time (4, 8, 24, and 48 h) in the absence or presence of testosterone (Sigma Chemical Co., St. Louis, MO). Each well contained 100 µL tissue culture medium (Ham’s F-10, Life Technologies Europe, Paisley, UK) supplemented with 0.1% human albumin (Sigma), 10 µL/mL gentamicin (Life Technologies), and 50 ng/mL vitamin A (retinoic acid, Sigma). The final concentrations of testosterone were 10-6 and 10-7 mol/L. The incubation was performed at 34 C in a humidified atmosphere containing 5% CO2.
Southern blot analysis of apoptotic DNA fragmentation
Small tissue sections (1 x 1 x 2 mm) were
snap-frozen in liquid nitrogen and stored at -70 C for further DNA
isolation. Genomic DNA was extracted as previously described (16) with
modifications (17). After isolation and quantitation, DNA samples were
3'-end labeled with digoxigenin-dideoxy (dd)-UTP (Boehringer Mannheim,
Mannheim Germany) by use of the terminal transferase (Boehringer
Mannheim) reaction, fractionated through 2% agarose gels, and blotted
onto a nylon membrane overnight. The next day the nylon membrane was
autocross-linked and washed for 5 min with maleic acid buffer (0.1
mol/L maleic acid and 0.15 mol/L NaCl, pH 7.5). It was then blocked
with blocking solution \[1% blocking reagent (Boehringer Mannheim) in
maleic buffer\] for 30 min. The membrane was incubated for 30 min in
antibody solution (anti-DIG-AB, Boehringer Mannheim; 1:10,000 in
blocking solution), after which it was washed twice for 15 min with
washing buffer (maleic acid buffer with 0.3% Tween-20). The membrane
was equilibrated for 5 min in detection buffer (0.1 mol/L Tris-HCl, 0.1
mol/L NaCl, and 50 mmol/L MgCl2, pH 9.5). For the
luminescence reaction, incubation was performed in CSPD solution
(Boehringer Mannheim; 1:100 in detection buffer) for 5 min. The
membrane was incubated in a hybridization bag for 15 min at 37 C to
enhance the luminescent reaction. It was then exposed to x-ray film.
The information (optical density) obtained from x-ray films was
transformed to pixels by scanning and use of the MCID Image Analyzing
System (Imaging Research, Canada). To analyze low mol wt DNA fractions
[<1.3 kilobases (kb)], a DNA marker, which was also labeled, was
used. On each Southern blot, the 0 h sample was used as a control.
The number of pixels in each lane was divided by the number of pixels
in the 0 h lane to eliminate the role of background. All reactions
were performed at room temperature if not otherwise stated.
Biochemical detection of apoptotic cells using nonradioactive in situ end labeling (ISEL)
Preparations were squashed and fixed as previously described
(11, 15, 18). Small tissue sections (1 x 1 x 2 mm)
consisting of seminiferous tubules were fixed in Bouin’s solution,
paraffin-embedded, sectioned at 4 µm, mounted on slides coated with
Vectabond (Vector Laboratories, Burlingame, CA), and deparaffinized.
ISEL of the squash preparations and histological samples was performed
as described previously (11, 19, 20), with some modifications. The
samples were hydrated. The histological sections were incubated in 10
mmol/L citric acid (pH 6.0) in a microwave oven 5 min 3 min (21) and
washed twice for 5 min each time in distilled water. DNA 3'-end
labeling with digoxigenin-dd-UTP by terminal transferase reaction as
well as detection of digoxigenin-dd-UTP by antidigoxigenin antibody
conjugated to alkaline phosphatase and exposure to the substrates for
alkaline phosphate were performed as previously described (11). Slight
counterstaining was performed with hematoxylin. For the negative
controls, the terminal transferase enzyme was substituted for the same
volume of distilled water.
Morphological analysis of apoptotic cells under light and 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 1.5% K-ferrocyanide, embedded in epoxy resin (Glycidether 100, Merck, Darmstadt, Germany), and stained with toluinide blue for light microscopy. For the electron microscopy, the samples were sectioned at 70 nm with a Reichert E Ultramicrotome (Reichert Jung, Vienna, Austria) and stained with uranyl acetate and lead citrate. Observations were made with a JEOL 100 SX electron microscope (JEOL, Tokyo, Japan). The identification of germ cell types was based on their characteristic morphology and staining affinity as well as their localization in the seminiferous tubules. The germ cells were identified as apoptotic by nuclear and/or cytoplasmic condensation and in the late stage of apoptosis by dense pycnotic bodies.
Statistical analysis
All experiments were repeated on at least three independent occasions. Quantitative data obtained from the replicate experiments (mean \ SEM) were analyzed by nonparametric Wilcoxon’s 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) by use of 3'-end-labeled DNA (Fig. 1
). DNA
samples prepared from the tissues immediately after the operation (0 h)
showed no apoptotic ladder pattern (Fig. 1). A rapid increase in germ
cell apoptosis was induced by serum-free conditions, because 4-h
incubation resulted in a 15-fold increase in apoptotic DNA laddering
(P < 0.02; Fig. 1) relative to levels measured at
0 h. The Southern blot analysis revealed a time-dependent increase
in intensity in apoptotic fragmentation. After 24 h of incubation,
the extent of low mol wt DNA reached a 20-fold intensity relative to
the 0 h value (P < 0.02; Fig. 1).
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Apoptosis in the seminiferous tubules cultured without hormones
was suppressed by testosterone concentrations of 10-6 and
10-7 mol/L (Fig. 2A). As demonstrated in
Fig. 2B, after 4 h of incubation, DNA fragmentation was suppressed
by 71% at a testosterone concentration of 10-7 mol/L
(P < 0.05) and by 43% at 10-6 mol/L
(P < 0.05) compared with that in samples cultured
without testosterone. After 24 h of incubation, no suppressive
effect of testosterone was observed.
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To verify the specific cell types involved in DNA
fragmentation, ISEL of DNA was performed (Fig. 3, panel
B). Incorporation of digoxigenin-dd-UTP was found most
often in spermatocytes, and occasionally, DNA labeling was also
localized in spermatids. Due to nuclei pycnosis in the late stages of
apoptosis, however, not all apoptotic cells could be identified. There
was no staining when terminal transferase enzyme was substituted for
the same volume of distilled water (negative control).
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, panel A; A compared
with C).
The apoptotic nature of cell degeneration was further confirmed
by electron microscopy. Small clumps of heterochromatin could be
distinguished in the nuclei of the cells in early stages of apoptosis;
in later stages of apoptosis, the heterochromatic areas became larger.
Condensed chromatin and degenerating cytoplasmic organelles were
visible in the late stages of apoptosis. As demonstrated in the ISEL
and light microscopy analyses, morphological signs of apoptosis were
most frequently identified in spermatocytes (Fig. 4).
Occasionally, some spermatids (Fg. 5) showed signs of
apoptosis, with some of the apoptotic spermatids showing a ring-like
condensation of chromatin (Fig. 5). As in the ISEL and light microscopy
samples, late apoptotic cells were impossible to identify.
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Discussion |
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Before reaching maturity, most of the testicular germ cells degenerate (4, 5, 6, 7); in rat testes, this occurs through an apoptotic mechanism (9, 12, 22). There is evidence that the germ cells in the three main phases of spermatogenesis, i.e. spermatogonial multiplication, meiosis, and spermiogenesis, do, in fact, undergo programmed cell death (8, 9, 10, 11, 22). Morphometric and morphological analyses have indicated that the spontaneous loss of germ cells is greatest during the mitoses of spermatogonia and the first meiotic division (6, 23, 24, 25). On the other hand, in situ analysis of adult rat testes demonstrates that mainly spermatocytes and occasionally some spermatids are affected in response to a lack of hormonal stimulation (9, 10, 11, 14, 22). In agreement with this, the present study shows that germ cells in the later phases of spermatogenesis seem to be most sensitive to hormonal withdrawal.
Germ cell apoptosis in the rodent testes has been reported to be stage specific (9, 10, 11, 14, 26). In the human, the cellular associations follow each other in spiral-like fashion, and a cross-section through human seminiferous tubule reveals several stages (27, 28) instead of the one seen in the rat. Therefore, because in the present study each squash preparation as well as each histological section for light and electron microscopy contained several stages, possible stage specificity could not be demonstrated.
Hormonal withdrawal has a significant and rapid effect on human germ cells. In vitro and in vivo studies in the rodent have indicated changes in apoptosis after at least 24-h exposure to hormonal manipulation (11, 10, 22, 26). In human testicular cells in the present study, serum-free conditions resulted in a rapid increase in apoptosis; after merely a 4-h incubation in vitro, a 15-fold apoptotic laddering occurred relative to that at 0 h, indicating that human adult testicular cells are sensitive to hormonal deprivation.
The testis tissue was obtained from a heterogeneous group of patients. Even though some of the patients were quite old, the responses of their germ cells to hormonal withdrawal and to testosterone substitution were similar to those of cells from the younger men, thus indicating that the possible age-related hormonal and vascular changes within their testes as well as their concomitant medications did not significantly affect the regulation of apoptosis in the seminiferous tubules in vitro.
LH or hCG stimulates Leydig cell production of androgens, which is essential for seminiferous tubule differentiation (27, 29, 30). The absence of androgens caused by hypophysectomy in adult rats resulted in degeneration of primary spermatocytes and spermatids (31). This demise was shown to be apoptotic in hypophysectomized and GnRH-antagonist-treated rats (9, 10, 12). The role of gonadotropins and sex steroids as survival factors in testis is further confirmed by the preventive effect of FSH, hCG, or testosterone on germ cell death (12).
In the rat, a decrease in both serum and intratesticular testosterone concentrations mediated by destruction of Leydig cells with ethane dimethane sulfonate induced apoptotic germ cell death in nearly all stages of the cycle (11, 22). This is in agreement with an experiment in which a reduced serum testosterone concentration and an increased concentration of FSH (even though neither significantly) mediated by methoxyacetic acid resulted in an increase in germ cell apoptosis (14). The apoptotic cells were primarily spermatocytes and spermatids, although some spermatogonia were also affected (11, 14, 22). One interesting exception concerned the testosterone-dependent regulation of testicular apoptosis, in which testosterone seemed to be a positive regulator of apoptotic germ cell death at one of the stages (stage XII) (11).
In the present study, the suppressive role of a testosterone concentration of 10-7 mol/L on testicular apoptosis was more effective than that of a testosterone concentration of 10-6 mol/L. As the stage specificity of testosterone regulation and its possible role as a positive regulator of apoptosis at some of the stages could not be demonstrated, the mechanism of that unusual dose-response remains unclear. Anyhow, the concentration of 10-7 mol/L is closer to the physiological concentration than that of 10-6 mol/L, and it seems reasonable that it is the more effective germ cell survival concentration than 10-6 mol/L.
In agreement with findings in the rat, apoptotic germ cells in the human testis in the present study were mostly spermatocytes and occasionally spermatids, suggesting that the cells of later differentiation are the most sensitive to hormonal control. Some of the cells in the late stage of apoptosis could not, however, be identified. Due to this and to the fact that there are only a few spermatogonia compared to the number of spermatocytes and spermatids, the possibility of spermatogonial apoptosis cannot be totally ruled out. In our in vitro model the suppressive effect of androgens on germ cell apoptosis was demonstrated only in short term incubations. That this effect was not observed in the 24-h incubation indicates the importance of some unknown factors in the maintenance of spermatogenesis and the survival of germ cells.
The microdissected seminiferous tubules contained some peritubular and interstitial cells. We assume that they could not have any significant effect on the results because 1) their relative proportion was very low; 2) those cells were present at all time points and in both the serum-free as well as the testosterone-treated conditions, so the suppressive effect of testosterone on germ cell apoptosis had to be mediated by exogenous testosterone; and 3) we obtained consistent results in each experiment in the nonhormone-treated controls.
In conclusion, data regarding apoptosis mainly involving the rodent testis appear to be consistent with our findings in the human. In the present study, by means of an in vitro model, we demonstrate that apoptosis is a normal, hormonally controlled phenomenon in the adult human testis. Apoptosis in germ cells was induced under serum-free conditions. That testosterone suppressed this apoptosis in seminiferous tubules indicates that in the human testis, testosterone is a critical germ cell survival factor. Our model thus provides a valuable tool for further investigation of hormonal and gene regulation of germ cell death and survival.
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Acknowledgments |
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Footnotes |
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Received November 20, 1996.
Revised February 25, 1997.
Accepted March 19, 1997.
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