Departments of Physiology and Pediatrics (W.Y., J.T.)
University of Turku 20520, Turku, Finland
GERM-INSERM U
435 (M.S., B.J.) Université de Rennes I 35042, Bretagne,
France
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ABSTRACT |
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INTRODUCTION |
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Bcl-w is a prosurvival member of Bcl-2 family proteins, and its physiological significance has been highlighted by two studies generating bcl-w-deficient mice (5, 6). Male bcl-w-deficient mice display normal testicular development before puberty, whereas after puberty Sertoli cells and germ cells of all types are severely reduced in number, and numerous apoptotic cells and no mature sperm are present in the seminiferous tubules, indicating an essential role of Bcl-w in normal spermatogenesis. However, the molecular mechanisms accounting for the testicular phenotypes in the bcl-w-deficient mice are still largely unknown.
In the present study, we analyzed the expression of Bcl-w mRNA and protein in the immature and mature rat testes, the potential dimerization of Bcl-w with the proapoptosis factors of Bcl-2 family proteins Bax, Bak, and Bad, and the regulation of Bcl-w mRNA levels by FSH and testosterone (T), as well as the responses of Bcl-w to specific germ cell-apoptosis-inducing signals by using three animal models.
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RESULTS |
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Localization of Bcl-w mRNA and Protein
In the newborn rat testis, specific hybridization signals were
detected in both Sertoli cell and gonocytes (Fig.
2). At 10 and 20 days of age, the signals
were present in Sertoli cells, spermatogonia, and spermatocytes. In the
adult testis, the most intense signals were confined to the cytoplasm
of Sertoli cells and spermatogonia, whereas spermatocytes showed weaker
signals (Fig. 3). Stage-specific pattern
of signals were observed, with more intense signals at stages IVI and
IXVIX, and less intense signals at stages VIIVIII (Fig. 3, A
and A').
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The localization and stage-specific expression of Bcl-w mRNA and
protein is summarized in Fig. 5.
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ISEL staining revealed that most of the spermatocytes are still
ISEL-negative at 6 h after MAA treatment. Spermatocytes start to
undergo apoptosis at around 8 h, and apoptosis peaks at 12 h,
and upon 24 h after MAA treatment, most of apoptotic spermatocytes
at stages IVI have been depleted (data not shown). Bcl-w protein
levels were significantly reduced (Fig. 12, D and E; P
< 0.05, n = 3), whereas Bax and Bak levels were increased
significantly (Fig. 12
, D and E; P < 0.01, n = 3)
at 12 h after MAA treatment, when spermatocytes were undergoing
intensive apoptosis. Bcl-w levels were lower than those of the
controls at day 4, when seminiferous tubules at stages IVI were
devoid of spermatocytes. At days 18 and 30, Bcl-w levels were much
higher than those in the controls (Fig. 12
, D and E). Ratios of
Bax/Bcl-w and Bak/Bcl-w were significantly elevated at 12 h and
24 h after MAA treatment (Fig. 12F
; P < 0.01,
n = 3).
Responses of Bcl-w, Bax, and Bak to the Germ Cell Apoptosis Induced
by T Withdrawal after Ethylene Dimethane Sulfonate (EDS) Treatment
Germ cells, mainly spermatocytes and spermatids, undergo massive
apoptosis between days 5 and 15 after EDS treatment due to lack of T
caused by Leydig cell depletion (12, 13, 14). In the present study,
controls (3 rats/time point) displayed similar levels at every time
point (0 day, 14 days, 7 days, 10 days, 20 days, 40 days, data not
shown) studied, and therefore only the controls (n = 3) at 1 day
are shown in Fig. 12, GI. Bcl-w protein levels were significantly
reduced at days 415, as compared with the controls (Fig. 12
, G and H;
P < 0.01, n = 3). Two- to 5-fold increases of Bax
protein levels were found during the same period of time (Fig. 12
, G
and H; P < 0.01, n = 3). Bak protein levels were
significantly increased at day 10 after EDS treatment (Fig. 12
, G and
H; P < 0.05, n = 3). The ratios of Bax/Bcl-w and
Bak/Bcl-w were significantly elevated (Fig. 11I
; P <
0.01, n = 3) during days 415 after EDS treatment.
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DISCUSSION |
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By using the whole testis lysate, Bcl-w was found to be coimmunoprecipitated with Bax and Bak. However, it has been documented that in the presence of detergent, Bcl-2 family proteins can associate (22). Since the distribution of Bcl-w and Bax/Bak are mainly Sertoli cells and spermatocytes, respectively, it is reasonable to assume that interaction of these molecules can occur during processing of the tissue. Therefore, we prepared highly purified Sertoli cells, spermatogonia, spermatocytes, and spermatids and performed immunoprecipitation and cellular fractionation. Consistently, Bcl-w was found to be associated with Bax and Bak, but not with Bad in all three types of cells, indicating that the heterodimers of Bcl-w/Bax and Bcl-w/Bak do exist in each type of cell that expresses them in vivo. Bcl-w, Bax, and Bak were all detected in Sertoli cells, spermatogonia, and spermatocytes, but not in spermatids. The results further confirm that Sertoli cells, spermatogonia, and spermatocytes are the sites of the production of these three proteins. Bad was detected in Sertoli cells and spermatogonia by immunoprecipitation, which is consistent with the previous immunohistochemical data showing that Bad was localized to these cells (23).
Bcl-2 family proteins are believed to be a group of membrane proteins that regulate apoptosis through in vivo dimerizations. With few exceptions (Bad, Bid, A1, E1B19K), the Bcl-2 family proteins possess a C-terminal hydrophobic transmembrane domain of approximately 20 residues, which determines their insertion into different intracellular membranes including those of the endoplasmic reticulum, the nuclear envelope, and mitochondrial membranes (1). The intracellular distribution of the Bcl-2 family proteins can depend on the cell type and also on the family member (1). In murine thymocytes, Bax was found predominantly as a soluble protein, while Bcl-2 was not detected in the soluble fraction, but was present in both high-speed membrane and nuclear fractions (24). In human leukemia cells, Bcl-2 was mainly located to mitochondria and was not present in the cytosol and Bcl-XL distributed in both mitochondria as a membrane-bound protein and the cytosol as a soluble protein. Bcl-XS, Bax, and Bad, on the other hand, were mainly located to the cytosol (25). In the present study, we demonstrate that Bcl-w, a homolog of Bcl-2, is predominantly an integral membrane protein, whereas Bax and Bak are mainly cytosolic in the healthy Sertoli cells, spermatogonia, and spermatocytes. The observation that Bax, Bak, and a significant portion of Bcl-w in Sertoli cells are cytosolic suggests that their C-terminal hydrophobic domains may be hidden either within the interior of the proteins or may be involved in binding other cytosolic factors. Interestingly, recent evidence suggests that the subcellular localization of at least certain members of the Bcl-2 family changes during apoptosis (24, 25). For example, Bax and Bcl-xL, but not Bcl-2, have been found to redistribute from the cytosol to intracellular membranes upon induction of thymocyte apoptosis, and Bax, Bad, and Bcl-Xs can redistribute from cytosol to mitochondria in two human leukemia cell lines. Therefore, it would be very interesting to determine, in a future study, how these Bcl-2 family proteins potentially translocate in response to the induction of germ cell apoptosis.
FSH has a prosurvival effect on germ cells in vitro (26). Since in the in vitro seminiferous tubule culture system most of the apoptotic cells are germ cells and Sertoli cells are more resistant to apoptosis than germ cells, it is plausible to ascribe the changes of mRNA levels in response to FSH stimulation to germ cells. Thus, up-regulation of Bcl-w mRNA levels by FSH might account for its prosurvival effect on germ cells in vitro. Since FSH receptor is mainly located on Sertoli cells, the prosurvival effect of FSH must be mediated through Sertoli cells (27). In fact, FSH prosurvival effect has been found to be partially mediated through SCF/c-kit interaction (28), and SCF from Sertoli cells could up-regulate Bcl-w protein levels in germ cells during 48 h culture in vitro (15).
Prosurvival members of Bcl-2 family exert prosurvival function by forming complexes with antisurvival members of Bcl-2 family and therefore suppressing their death-inducing effects (4). In the adult rat testis, Bcl-w forms complexes with Bax and Bak, but not with Bad. Consistently, Bax and Bak have similar expression sites to Bcl-w. Higher levels of Bax and Bak and lower levels of Bcl-w in spermatogonia and spermatocytes might imply that these types of cells are more susceptible to apoptosis than Sertoli cells. Neither Bcl-w, Bax, nor Bak is expressed in spermatids, suggesting that spermatid apoptosis might be regulated by other members of the Bcl-2 family. Indeed, Bcl-xL (19) and Diva (29) appear to be expressed only in spermatids, but little is known about these two factors in spermatid apoptosis. A recent study shows that Bcl-w forms a complex with Bax, but not with Bak, in the mammary gland (30). This might reflect the tissue-specific action of Bcl-2 family members since Bcl-2 family members, both the proapoptotic and antiapoptotic, are differentially expressed in different tissues and cells, and therefore the dimerization status could be different as well (1, 2, 3).
The changes of Bcl-w levels during apoptosis of specific germ cell types induced by different methods strongly support that Bcl-w is a prosurvival factor of spermatogonia and spermatocytes in vivo. It has been well characterized that the proliferating spermatogonia can be depleted when SCF/c-kit interaction is blocked by a function-blocking antibody, ACK-2, in the testis (7, 8). In the present study, spermatogonial apoptosis was induced. After 4 days of treatment with ACK-2, the number of apoptotic spermatogonia increased dramatically. DNA laddering analysis indicated that the ISEL-positive cells were apoptotic rather than necrotic. The time-dependent reduction of Bcl-w and elevation of Bax and Bak correlate with the time-dependent spermatogonial apoptosis. Three specific findings from this study are of interest: 1) depletion of proliferating spermatogonia by blockade of SCF/c-kit interaction is mediated via apoptosis, implicating the involvement of SCF/c-kit system in the regulation of spermatogonial apoptosis; 2) increased ratios of Bax/Bcl-w and Bak/Bcl-w correlate with spermatogonial death; 3) blockade of SCF/c-kit interaction differentially affects Bcl-w, Bax, and Bak expression, implying the involvement of other downstream factors or transcription factors in the regulation of gene expression of these three regulators of apoptosis.
MAA has been found to be able to selectively deplete spermatocytes through apoptosis within 24 h after oral administration (9, 10, 11). In the present study, we used this model to specifically induce spermatocyte apoptosis and monitored the changes of Bcl-w, Bax, and Bak levels every 6 h during the first 24 h. Morphological observation and ISEL staining indicate that MAA depletes spermatocytes at stages I-VI within 24 h after administration, and spermatocyte apoptosis peaked at 12 h after treatment (data not shown). There are three findings from this model that deserve further comment: 1) selective induction of apoptosis of spermatocytes at stages IVI by MAA correlates with increased ratios of Bax/Bcl-w and Bak/Bcl-w; 2) reduction of Bcl-w levels at day 4 after MAA treatment is apparently due to the depletion of spermatocytes, which express Bcl-w. Elevations of Bcl-w levels at day 18 and day 30 result from the enrichment of Bcl-w-expressing cells in the testis after depletion of round spermatids and elongating spermatids. These results further validate our localization data, showing that Sertoli cells, spermatogonia, and spermatocytes are the expression sites of Bcl-w; 3) since Bcl-w, Bax, and Bak are commonly expressed in spermatocytes at all stages in the epithelial cycle, the selective apoptosis of spermatocytes suggests that there might be some switching points that are responsible for turning on the apoptosis machinery only at stages I-VI.
It is well known that EDS can selectively deplete Leydig cells within 48 h after administration, and T declines abruptly to an undetectable level at day 3 and remains there until day 15 (12, 13, 31, 32). From day 5 to day 15, germ cells, mainly spermatocytes and spermatids, undergo a massive wave of apoptosis as a consequence of T withdrawal. In the present study, the reduced levels of Bcl-w and elevated levels of Bax and Bak correlate with the wave of apoptosis. The reversed ratios at day 20 correlate with the reduced number of apoptotic germ cells since at this time point the testosterone level has already increased and the number of apoptotic germ cells has decreased dramatically (Refs. 12, 32 and W. Yan, J. Kero, I. Huhtaniemi, and J. Toppari, paper submitted). Since Bcl-w and its partners, Bax and Bak, are expressed in spermatogonia and spermatocytes, the changes should result from the apoptotic events taking place in these cells. Thus, the increased ratios of Bax/Bcl-w and Bak/Bcl-w might produce more homodimers, such as Bax/Bax, Bax/Bak, or Bak/Bak, which promote apoptosis. Several studies (Refs. 12, 32 and W. Yan, J. Kero, I. Huhtaniemi, and J. Toppari, paper submitted) have shown that after EDS treatment, not only does the testosterone level decrease, and the LH level goes up as a feedback, but the FSH level increases as well. Therefore, the changes of these three Bcl-2 family members could not be simply ascribed to the effect of T withdrawal. However, as FSH could up-regulate Bcl-w levels and in the EDS-treated rats Bcl-w levels were down-regulated when FSH levels were severalfold higher than the controls and testosterone levels were undetectable from day 2 to day 10 (Ref. 32 and W. Yan, J. Kero, I. Huhtaniemi, and J. Toppari, paper submitted), it is very likely that down-regulation of Bcl-w and up-regulation of Bax and Bak were due to lack of testosterone.
Both Bcl-2 and Bcl-xL levels were measured by Western blotting for all three models used. We could not detect Bcl-2 in the adult rat testis, while the positive control (prostate protein) gave a clear Bcl-2 band in Western analysis (data not shown). This is consistent with several previous reports showing that in the mature testis Bcl-2 is absent (16, 18). Bcl-xL levels are relatively low in the adult rat testis. We could not detect significant changes of Bcl-xL levels in the ACK-2-treated rats and MAA-treated rats (data not shown). However, in the EDS model, we did find changes of Bcl-xL levels (data not shown). Since it is localized to round spermatids (19), which express neither Bcl-w nor Bax nor Bak, it is likely that Bcl-xL might be involved in regulation of spermatid apoptosis.
Immunohistochemical detection of Bcl-w, Bax, and Bak in the Bcl-w deficient mice enabled us, first, to test the specificity of the antibodies used in the present study, and second, to explain possibly the reason why both Sertoli cells and germ cells start to undergo apoptosis after puberty in the absence of bcl-w. There is no discernable abnormality during testicular development in the bcl-w-deficient mice before puberty (6). However, this does not mean that Bcl-w has no role or the proapoptotic members are not present yet by this stage. In fact, in the immature testis, the antiapoptotic members, including Bcl-xL, Bcl-w, and Mcl-1, are all highly expressed by almost all cell types including Sertoli cells and germ cells in the seminiferous epithelium (15). Similarly, the proapoptotic members of Bcl-2 family, including Bax, Bak, and Bad, are also expressed in the immature testis, and the expression sites are quite similar to those of the antiapoptotic members (15). It appears that the differential localization of Bcl-2 family proteins to different cell types in the seminiferous epithelium takes place around puberty. Thus, it is very likely that the absence of Bcl-w could be compensated by other antiapoptotic members of Bcl-w family, e.g. Bcl-xL, during prepubertal development. After puberty the failed spermatogenesis in bcl-w-deficient mice might result mainly from cell-extrinsic effects rather than cell-intrinsic effects (6), given the fact that the Bcl-w is much more abundant in Sertoli cells than in spermatocytes and spermatogonia. It is very likely that Sertoli cells are first implicated due to the absence of Bcl-w and undergo apoptosis via Bax- and/or Bak-mediated cell death. Death of Sertoli cells is devastating for germ cells, particularly for elongating spermatids and round spermatids, which is consistent with the findings that these two types of germ cells suffer from the most severe reduction in number in both lines of bcl-w-deficient mice (5, 6). However, depletion of spermatogonia and spermatocytes may be either mediated via Bax and Bak, due to lack of Bcl-w in a cell-intrinsic way, or due to Sertoli cell death in a cell-extrinsic manner.
Taken together, the present study suggests that Bcl-w is an important prosurvival factor of Sertoli cells, spermatogonia, and spermatocytes and participates in the regulation of apoptosis by binding proapoptotic factors Bax and Bak. The ratios of Bax/Bcl-w and Bak/Bcl-w might be decisive for the fates of Sertoli cells, spermatogonia, and spermatocytes.
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MATERIALS AND METHODS |
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All animal experiments were approved by the Turku University Committee on Ethics of Animal Experimentation.
To induce apoptosis of proliferating spermatogonia by blocking SCF/c-kit interaction (7, 8), a function-blocking anti-c-kit antibody, ACK-2 (kindly provided by Dr. T. Kunisada, Department of Immunology, Faculty of Medicine, Tottori University, Japan) was injected i.v. at 3.5 mg/kg body weight in physiological saline. The injection was given twice, once every 48 h. Rats that received ACK-2 injection were killed at 24 h, 48 h, 72 h, and 96 h, respectively. One testis was snap frozen in the liquid nitrogen and then stored at -70 C for isolation of RNA; the other was fixed overnight at 4 C in 4% paraformaldehyde followed by dehydration and embedding onto paraffin for detection of spermatogonial apoptosis by ISEL.
For induction of spermatocyte apoptosis, MAA (Aldrich Chemie, Steinheim, Germany) was diluted in physiological saline and administered orally with a single dose of 650 mg/kg BW. The control rats received physiological saline. The rats were killed at 6 h, 12 h, 24 h, 4 days, 18 days, and 30 days, respectively. One testis was snap frozen in liquid nitrogen, and the other was fixed in 4% paraformaldehyde for preparation of paraffin block for checking apoptosis by ISEL staining.
To study the response of Bcl-w to germ cell death induced by testosterone withdrawal after EDS treatment, the rats were injected i.p. with a single dose of EDS (75 mg/kg BW). EDS was synthesized as previously described (33) and dissolved in dimethylsulfoxide (DMSO)-water (1:3, vol/vol). Control animals (3 rats/time point) for every time point [1 day (1d), 2d, 3d, 4d, 7d, 10d, 20d, 40d] received injection of vehicle. Rats (n = 3/group) were killed by cervical dislocation under CO2 anesthesia at day 1, 2, 3, 4, 7, 10, 15, 20, 30, and 40 after administration of EDS. The testes were snap frozen in the liquid nitrogen and then stored at -70 C for isolation of RNA.
Riboprobe Preparation
The bcl-w cDNA was kindly provided by Dr. Jerry M.
Adams (The Walter and Eliza Hall Institute of Medical Research,
Victoria, Australia). The fragment, corresponding to nucleotide (nt)
1131-nt 1943 of the bcl-w cDNA, was subcloned into
pBluescript II KS- (Stratagene, La
Jolla, CA). The plasmid was linearized with EcoRI or
XhoI for preparation of antisense or sense probe using
T3 or T7 RNA polymerase
(Promega Corp., Madison, WI), respectively. For Northern
hybridization, 32P-UTP was used for labeling the
antisense riboprobe: for in situ hybridization,
35S-UTP was used for labeling both antisense and
sense probes.
Northern Hybridization
RNA preparation, gel fractionation, and Northern blotting, as
well as hybridization, were performed as described previously (34).
In Situ Hybridization
Five-micrometer thick sections were cut from
paraffin-embedded testis samples and mounted onto SuperForst Plus
glass slides (Menzel-Gläser, Steinheim, Germany). The slides were
then incubated at 37 C overnight and then stored at 4 C before use.
In situ hybridization was performed as described previously
(35).
Preparation of Purified Sertoli Cells, Germ Cells, and Leydig
Cells
Sertoli cells were isolated from 20-day-old Sprague Dawley rats
as described previously (36, 37). Spermatogonia were prepared from
9-day-old Sprague Dawley rats using a protocol described by
Bellvé (38) with minor modifications introduced by Dym et
al. (39). This procedure involved the use of enzymatic
dissociation followed by filtration through 80- and 40-µm nylon mesh.
Cells from the dissociated seminiferous tubules were separated by
sedimentation velocity at unit gravity at 4 C using a 24% BSA
gradient in Hams F-12/DMEM. The cell suspension were bottom loaded
into an SP-120 chamber in 30 ml of Hams F12/DMEM containing 0.5%
BSA, and a gradient was simultaneously generated using 275 ml each of
medium supplemented with 2 and 4% BSA, respectively. The cells were
allowed to sediment for a standard period of 2.5 h, and 300 ml
were then collected from the bottom of the gradient and centrifuged at
100 x g for 10 min. Pellet cells were then resuspended
in Hams F12/DMEM supplemented with gentamycin (50 µg/ml) and 10%
FCS and incubated at a density of 2.5 x
106/ml in a humidified atmosphere of 5%
CO2-95% air. After 14 h of culture,
contaminant cells were plated, and nonadherent spermatogonia were used.
Postmitotic germ cells were obtained from 90-day-old rat testes by
mechanical dissociation (38). These cells were separated by centrifugal
elutriation into two populations: primary spermatocytes and early
spermatids. Flow rate and/or rotor speed were changed progressively, as
described by Pineau et al. (40). Cell viability was
evaluated by the trypan blue exclusion test and was found to be at
least 95%. Pachytene spermatocytes and early spermatid fractions were
found to be about 90% pure. Leydig cells were isolated and purified
from rat testis as described previously (41), and the purity was
85%.
Preparation of Subcellular Fractions
Soluble, crude membrane and nuclear fractions of the purified
Sertoli cells, spermatogonia, and spermatocytes were prepared as
described by Hsu et al. (24). Briefly, cells were suspended
at a cell density of 5 x 107 cells per ml
in 1 ml of buffer A containing 10 mM HEPES, pH
7.4, 38 mM NaCl, phenylmethylsulfonyl fluoride
(25 µg/ml), aprotinin (1 µg/ml), and leupeptin (10 µg/ml). The
cell suspension was homogenized in a glass Dounce homogenizer and then
centrifuged at 900 x g to pellet the nuclei in a
Sorvall SA-600 rotor. The postnuclear supernatant was further
centrifuged at 130,000 x g in a Ti 80 rotor
(Beckman Coulter, Inc., Palo Alto, CA) to pellet
the membranes. The crude membranes were solublized in a volume of the
lysis buffer (buffer A supplemented with 0.5% NP-40) equal to that of
the supernatant. The lysates were subjected to Western blotting.
Immunoprecipitation
Whole testis, purified Sertoli cells, or germ cells were lysed
in a buffer containing 25 mM Tris-Hcl, 120 mM
NaCl, 0.5% NP-40, 4 mM NaF, 100 µM
Na3VO4, 100 KIU/ml
aprotinin, 1 mM phenylmethylsulfonylfluoride , and 10
µg/ml leupeptin at 4 C for 30 min with vigorous shaking. Cell lysates
were centrifuged for 20 min at 13,000 rpm, and then the supernatants
were transferred to new tubes for measuring concentration of protein as
described previously (42).
An aliquot of 300 µg of protein was incubated with 1 µg of polyclonal rabbit antirat Bax (PharMingen, San Diego, CA), Bak (G-23, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or Bad (K-17, Santa Cruz Biotechnology, Inc.) for 2 h at 4 C with gentle mixing. An aliquot of 20 µl of Protein A-agarose (Pharmacia Biotech, Uppsala, Sweden) was added, and the incubation proceeded for another 2 h at 4 C with gentle shaking. The mixture was centrifuged at 2500 rpm for 5 min at 4 C, and the pellet was washed four times with the buffer containing 20 mM Tris-Hcl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 100 mM NaF, 200 µM Na3VO4, 100 IU/ml aprotinin, 1 mM phenylmethylsulfonylfluoride and 10 µg/ml leupeptin and once with TBS. The pellet was resuspended in the sample buffer (400 mM Tris-HCl, 40% glycerol, 8% SDS, 0.4 M dithiothreitol, and 0.1% bromophenol blue) and boiled for 2.5 min. The suspension was centrifuged at 4 C, and the supernatant was loaded onto a 12.5% SDS-polyacrylamide gel using Mini Protean II system (Bio-Rad Laboratories, Inc. Hercules, CA). After electrophoresis, the protein was electrophoretically transferred onto a nitrocellulose membrane (Hybond, Amersham Pharmacia Biotech, Aylesbury, UK). Immunoprecipitation experiments were repeated three times using cells from different rats.
Immunoblotting
The membrane was incubated in a blocking buffer (10
mM Tris-HCl, pH 8.0, 0.1% Tween 20, and 5% non-fat milk
powder) at room temperature for at least 1 h, followed by
incubation in the blocking buffer containing polyclonal rabbit
anti-bcl-w antibody (StressGen Biotechnologies Corp., Victoria, British
Columbia, Canada) at 1:1001:200 dilution for 1 h. After three
times of wash in a washing buffer (10 mM Tris-HCl, pH 8.0,
0.1% Tween 20), the membrane was incubated in the blocking buffer
containing horseradish peroxidase-conjugated donkey antirabbit antibody
(Amersham Pharmacia Biotech) at 1:200 dilution for 1
h. After three times of wash, the membrane was subjected to
chemiluminescent detection using an ECL Western Blotting Detection Kit
(Amersham Pharmacia Biotech) and, finally, the membrane
was exposed for 110 min to x-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan). The membranes were stripped using the
method provided by the manufacture of the membrane (Hybond,
Amersham Pharmacia Biotech) and reprobed using a mouse
anti-Actin monoclonal antibody (ICN Biomedicals, Inc.,
Aurora, OH) for normalization of the loading.
Tissue Culture and Hormone Stimulation
Seminiferous tubule segments were isolated in DMEM/F12 (1:1)
(Life Technologies, Inc., Paisley, Scotland, UK)
supplemented with 15 mM HEPES, 1.25 g/liter sodium
bicarbonate, 10 mg/liter gentamycin sulfate, 60 mg/liter G-penicillin,
1 g/liter BSA and 0.1 mM 3-isobutyl-1-methylxanthin (MIX)
(Aldrich Chemie) under a stereomicroscope by transillumination-assisted
microdissection technique as described previously (43).
Twenty pieces of 5-mm seminiferous tubule segments from stages IIVI, VIIVIII, IXXII, or XIIII were incubated in 1 ml of above mentioned culture medium in the presence and absence of FSH (10 ng/ml), testosterone (T) (10- 6 M), FSH+T for 8 h and 30 h. After incubation, RNA was isolated as described previously (34), and Bcl-w mRNA levels were detected by Northern blot hybridization.
Immunohistochemistry
Two 5-µm-thick consecutive sections were cut from each sample
and mounted onto polylysine-coated slides. One section was used for
immunohistochemical staining of Bcl-w, Bax, and Bak, and the other was
used for periodic acid-Schiff-hematoxylin staining for accurate
determination of stages.
After rehydration, the slides were washed twice in TBS buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl,) for 5 min each followed by microwave antigen retrieval at 700 W for 15 min in 10 mM sodium citrate solution, pH 6.0. After two washes with TBS, an aliquot of 50 µl of blocking solution (TBS containing 1% BSA, 3% FCS, and 3% normal horse serum) was applied to each section and incubated for 1 h at room temperature. After blocking, an aliquot of 50 µl primary antibody (1:200 diluted in TBS containing 1% BSA) was applied to each section and incubated at 4 C overnight. Incubation with secondary antibody and visualization of positive cells were performed using Vectastain Elite-kit (Vector Laboratories, Inc., Burlingame CA) according to the manufacturers instructions.
As a control experiment, the testis sections from bcl-w-/- (line 043 and line 044) and bcl-w+/+ (line 042) mice, which were kindly provided by Dr. Suzanne Cory (The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia), were employed to perform immunohistochemical staining using anti-Bax, Bak, and Bcl-w antibodies. Preabsorbed antibodies by corresponding peptides used for immunization and rabbit IgG were also used as control antibodies.
A semiquantitative analysis was conducted for estimating the relative abundance of Bcl-w, Bax, and Bak proteins in Sertoli cells, spermatogonia, and spermatocytes in the rat. Immunoreactivity in different types of cells and in different stages was semiquantified according to the following criteria: +, weak; ++, strong; +++, very strong immunoreaction.
ISEL Staining of Apoptotic Spermatogonia
Two consecutive sections (5-µm thick) were cut from each
paraffin block; one for ISEL staining and the other for periodic
acid-Schiff-hematoxylin staining. ISEL staining was performed as
described (28). Briefly, after rehydration, the sections were incubated
in 2 x SSC at 80 C for 20 min followed by washing twice with
water and once with Proteinase K buffer (20 mM Tris-HCl, pH
7.4, 2 mM CaCl2) for 5 min each. The
slides were then treated with proteinase K (10 µg/ml, Roche Molecular Biochemicals, Indianapolis, IN) in proteinase K buffer
at 37 C for 30 min. An aliquot of 20 µl of 3'-end labeling reaction
mixture containing 4 µl 5xTdT buffer (Promega Corp.),
0.1 µl Dig-11-ddUTP (10 nmol/µl, Roche Molecular Biochemicals), 0.2 µl dd-ATP (5 mM, Promega Corp.), 1 µl terminal deoxynucleotidyl transferase
(Roche Molecular Biochemicals) and 14.7 µl nuclease-free
water (Promega Corp.) was applied to one cross-section.
The slides were kept in a humidified box and incubated at 37 C for
1 h and then washed three times with TBST buffer (10
mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.1%
Tween-20) for 10 min each. An anti-Dig-horseradish peroxidase
monoclonal antibody (DAKO Corp., Glostrup, Denmark, 1:200
dilution in TBST containing 1% BSA,) was applied, and the slides were
incubated in a humidified box at room temperature for 1 h and then
washed three times with TBST for 5 min each time. Finally, the labeled
cells were visualized by 3,3'-diaminobenzidine tetrahydrochloride
(Sigma, St. Louis, MO) for 0.52 min.
Nonradioactive DNA Laddering
DNA was isolated from five pieces of 2 mm-long tubule segments
by phenol/chloroform extraction after digestion in TES buffer (10
mM Tris-HCl, pH 7.6, 10 mM EDTA, 100
mM NaCl) containing 1% SDS, proteinase K (100 µg/ml,
Roche Molecular Biochemicals), and RNase A (10 µg/µl,
Roche Molecular Biochemicals) at 55 C for 30 min. The
3'-end labeling was performed in a 20 µl reaction volume containing
100 ng DNA, 4 µl 1x Terminal transferase buffer (Promega Corp.), 0.5 µl terminal deoxynucleotidyl transferase (20
U/µl, Promega Corp.), 0.1 µl DIG-11-ddUTP (10
nmol/µl, Roche Molecular Biochemicals). The reaction
mixture was incubated for 30 min at 37 C and then 1 µl 0.5
M EDTA, pH 8.0, was added to terminate the reaction. The
reaction mixture was size fractionated in a 1.6% agarose gel followed
by blotting onto a nylon membrane overnight with 10xSSC. The DNA was
fixed by baking in an oven at 80 C for 1 h followed by UV
cross-linking. The membrane was incubated in 20 ml 1x blocking buffer
(Roche Molecular Biochemicals) containing 1 µl
anti-DIG-AP (Roche Molecular Biochemicals) at room
temperature for 1 h followed by three times of washing with TBST
buffer for 15 min each. One milliliter chemiluminescent substrate CSPD
(Roche Molecular Biochemicals) was applied onto the blot.
The blot was incubated at room temperature for 10 min and then sealed
in a plastic bag. The blot was exposed to x-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan) for 510 min.
Quantitative Analysis of Northern Hybridization and Western
Blotting Results
The x-ray films of Northern hybridization and Western blotting
results were first scanned by a UMAX scanner (UMAX Inc., Fremont, CA)
and a Photoperfect software package (Binuscan Inc., New York, NY). The
images were saved as TIFF-type files (*.tif, Microsoft Corp. Co. and Aldus Co., New York, NY) and then quantified by
TINA 2.0 densitometric analytical system (Raytest Isotopenmeß gerate
GmbH, Straubenhardt, Germany) according to the manufacturers
instructions. For Northern blotting results, after normalization to 28S
rRNA, the densitometric value of a control was designated as 100%, and
values of other controls and treated samples were expressed as the
percentages of the control. For Western blotting results, after
normalization to actin, similarly, the densitometric value of a control
was designated as 100% and values of other controls and treated
samples were expressed as the percentages of the control. The values of
the controls that were designated as 100% were excluded from
statistical analysis.
Statistical Analysis
The values from three independent experiments were pooled for
the calculation of the SEMs and for one-way ANOVAs and
Duncans new multiple range test to determine the significant
differences between different experimental groups by using StatView
4.51 statistic program (Abacus Concepts Inc., Berkeley, CA).
P < 0.05 was considered statistically significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This research was supported by grants from the Academy of Finland, the Finnish Research Program on Environmental Health, and the Turku University Central Hospital.
Received for publication September 15, 1999. Revision received January 24, 2000. Accepted for publication February 15, 2000.
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REFERENCES |
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