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From the Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland.
| Correspondence to: Dr Barry R. Zirkin, Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205 (e-mail: brzirkin{at}jhsph.edu). |
| Received for publication June 29, 2007; accepted for publication January 23, 2008. |
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Abstract |
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Key words: Spermatogenesis, Sertoli cells, cell adhesion
We demonstrated previously that BMF is expressed in germ cells of the rat testis, specifically in the subacrosomal space of postmeiotic spermatids of steps 4 to 16 (Show et al, 2004). Experimental conditions in which normal testosterone concentrations within the testes are reduced substantially have been shown to result in the apoptotic death of meiotic spermatocytes and in the premature sloughing and subsequent degeneration of postmeiotic round spermatids (Russell and Clermont, 1977; O'Donnell et al, 1994, 1996). Under such conditions, BMF is expressed in spermatocytes and more mature spermatids and is redistributed throughout round spermatids rather than confined to its normal subacrosomal localization (Show et al, 2004).
We hypothesized that in response to the detachment of germ cells from their associated Sertoli cells, MAPK8 would become activated by phosphorylation and that its activation would be associated with the expression/redistribution of BMF in the detached germ cells and thus with the death of the detached cells. We demonstrate that phosphorylated (ie, active) MAPK8 (p-MAPK8) is detected in spermatocytes and elongated spermatids but not in round spermatids. The increased apoptosis of germ cells that occurs when they are cultured in the absence of Sertoli cells was found to be associated with increased p-MAPK8 expression and with the expression and/or redistribution of BMF within the p-MAPK8–expressing apoptotic germ cells. The results suggest that the activation of MAPK8 and the expression/redistribution of BMF may be involved in the mechanism by which specific germ cells undergo programmed cell death in response to loss of their attachment to Sertoli cells.
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Materials and Methods |
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Immunohistochemistry
Rats were anesthetized and whole-body perfused with phosphate-buffered
saline (PBS) to clear the testes of blood and then with neutral buffered
formalin for 1 hour at a rate of 7 mL/min. The testes were removed and
immersed in neutral buffered formalin overnight at 4°C. The tissue was
dehydrated in ice-cold (4°C) 70%, 90%, and 99% ethanol for 1 hour each and
then in absolute ethanol for 1 hour at room temperature. Tissue was
infiltrated with 50% polyester wax/50% ethanol for 2 hours at 42°C
followed by a 90% polyester wax/10% ethanol mixture for 1 hour at 42°C.
The tissue was then transferred into 90% wax/10% ethanol in plastic embedding
dishes and chilled on ice for 30 minutes or until the wax solidified. Sections
(5 µm) were cut and mounted on HiPure subbed glass slides. The slides were
dewaxed by immersion into 100%, 90%, and 70% ethanol baths for 10 minutes
each. Some slides were blocked in normal goat serum diluted in PBS (1:60;
Vector Laboratories Inc, Burlingame, California) and then incubated for 1 hour
at room temperature with a rabbit polyclonal antibody raised against the
N-terminus of the human BMF protein (1:200; Abcam Ltd, Cambridge, United
Kingdom). Bound primary antibodies were detected with a fluorescein
isothiocyanate (FITC)–conjugated goat anti-rabbit secondary antibody.
Other slides were blocked in PBS-diluted normal horse serum and then incubated
with a mouse monoclonal antibody raised against p-MAPK8 (1:100; Santa Cruz
Biotechnologies, Santa Cruz, California). In this case, bound primary
antibodies were detected with a FITC-conjugated horse anti-mouse secondary
antibody (1:100; Vector Laboratories). The p-MAPK8 antibody has been used to
localize p-MAPK8 by immunohistochemistry in several previous studies
(Shiraishi et al, 2002;
Kins et al, 2003;
Lysiak et al, 2003;
Ishii et al, 2004). Nuclei were
stained with 4,6 diamidoino-2-phenylindole (DAPI; Vector Laboratories) in
Vectashield anti-fade mounting medium. Antibody specificity was assessed by
incubating slides with mouse immunoglobulin G (IgG) followed by secondary
antibody application. BMF primary antibody specificity was demonstrated by
performing the above immunostaining procedure preceded by the preabsorption of
equal concentrations of the BMF primary antibody with the BMF N-terminal
peptide used for antibody production (Show
et al, 2004). Images were obtained with a Nikon Eclipse 800
microscope equipped with a Nikon planfluor x40 objective using a
Princeton Instruments 5-Mhz cooled CCd camera with custom CRI color filter and
IPLab digital image analysis software for Macintosh.
Seminiferous Tubule Microdissection
Seminiferous tubule segments were isolated from rat testes by
transillumination-assisted microdissection, as previously described
(Parvinen, 1982). For protein
analyses, approximately 15 to 20 cm of seminiferous tubules from stages I to
V, VII, VIII, and IX to XIV of the spermatogenic cycle were dissected from the
testes of control rats. Testes from 3 different rats were included as
independent samples for protein isolation.
Germ Cell Isolation and Culture
Each germ cell preparation included both testes of an adult rat. Testes
were decapsulated and incubated with 0.5 mg/mL collagenase (Sigma-Aldrich, St
Louis, Missouri) in F12 Dulbecco modified Eagle medium (DMEM; Invitrogen,
Carlsbad, California) supplemented with 1 mM sodium pyruvate (Sigma-Aldrich)
and 13 mM lactate (Sigma-Aldrich) in a shaking water bath for 12 minutes at
34°C. The free seminiferous tubules were washed 3 times with F12 DMEM
supplemented with lactate and pyruvate and allowed to settle between each
wash. The tubules were digested with 0.5 mg/mL trypsin in supplemented F12
DMEM for 8 minutes in a shaking water bath at 34°C. Following digestion by
trypsin, the remaining seminiferous tubule fragments were mechanically
dispersed by pipetting until the cellular suspension became homogeneous. The
crude germ cell suspension was then filtered through nylon mesh to remove any
undigested seminiferous tubule fragments. The filtrate was centrifuged for 5
minutes. The pellet was washed 3 times with supplemented F12 DMEM and
centrifuged as above. Finally, the pellet was resuspended and again filtered
through nylon mesh. The isolated germ cells were assessed for purity by
morphologic analysis and counted with a hemocytometer. The cells averaged
approximately 90% purity, with Sertoli and myoid cells the principal
contaminants. Following determination of purity, some germ cells were
immediately snap-frozen for later protein analysis, and others were labeled
with annexin V to determine apoptotic initiation or were cultured in F12 DMEM
supplemented with lactate and pyruvate for 4, 8, or 12 hours at 34°Cina5%
CO2 atmosphere so as to initiate apoptosis due to loss of Sertoli
cell-germ cell adhesion.
Annexin V Labeling of Apoptotic Germ Cells
Freshly isolated germ cells, as well as germ cells that had been cultured
in liquid media without Sertoli cells for 4, 8, or 12 hours, were assessed for
the initiation of apoptosis by the annexin V-FITC apoptosis detection kit
(Pharmingen, San Diego, California), according to the manufacturer's
specifications. Briefly, 1 x 106 cells were added to 0.5 mL
of Ca2+-binding buffer followed by the addition of 5 mL of annexin
VFITC. The cells were incubated for 10 minutes at room temperature, during
which they were protected from light. An aliquot of the annexin
V–labeled cells was then transferred to a microscope slide. Germ cells
labeled with annexin V-FITC were considered to be apoptotic. An apoptotic
index was calculated by averaging the total number of annexin
V-FITC–positive germ cells vs the total number of germ cells counted
using phase-contrast microscopy. Images were obtained with a Nikon Eclipse 800
microscope, as above, equipped with a Princeton Instruments 5-Mhz cooled CCd
camera.
Western Blot Analyses
Protein isolated from germ cells or from stage-specific seminiferous tubule
segments were homogenized in radioimmunoprecipitation assay buffer (1% Triton
X-100, 15 mM HEPES-NaOH [pH 7.5], 0.15 mM NaCl, 1% sodium deoxycholate, 0.1%
SDS, 1 mM sodium orthovanadate, 10 mM EDTA, and 0.5% protease inhibitor
cocktail [Sigma-Aldrich]), and stored at –80°C until analyzed.
Protein concentrations were determined by the bicinchoninic acid method
(Pierce, Rockford, Illinois), according to the manufacturer's specifications.
Protein samples were added to an equal volume of 2x loading buffer (100
mM Tris [pH 6.8], 4% sodium dodecyl sulfate [SDS], 0.2% bromophenol blue, 20%
glycerol). Samples were reduced with 0.1% β-mercaptoethanol, boiled for 2
minutes, and separated by 12% SDS-polyacrylamide gel electrophoresis. Protein
was transferred to Protran nitrocellulose membranes (Schleicher & Schuell,
Keene, New Hampshire) with a Trans-Blot SD semi-dry electrophoretic transfer
cell (Bio-Rad, Hercules, California), according to the manufacturer's
specifications.
Membranes were blocked for 1 hour with 5% nonfat dry milk in PBS (blocking solution) at room temperature, followed by anti–p-MAPK8 (1:300) overnight in blocking solution at 4°C. The membranes then were washed 3 times with PBS + 0.1% Tween-20 for 5 minutes and incubated for 30 minutes at room temperature with secondary anti-mouse horseradish peroxidase (HRP)–linked IgG (1:3000; Amersham Pharmacia, Piscataway, New Jersey) in PBS. Signals were detected using the SuperSignal WestPico chemiluminescent kit (Pierce), according to manufacturer's specifications. Protein membranes were stripped using Restore Western blot stripping solution (Pierce), according to the manufacturer's specifications. Membranes were then blocked for 1 hour and probed with anti-MAPK8/MAPK9 (JNK1/JNK2; 1:1000; Sigma-Aldrich) and anti–β-actin (1:1000; Sigma-Aldrich) for 1 hour in blocking solution, followed by washing and subsequent incubation for 30 minutes at room temperature with anti-rabbit HRP-linked IgG (1:3000; Amersham Pharmacia) for anti-MAPK8/MAPK9 or anti-mouse HRP-linked IgG (1:3000) for anti-β-actin. The relative ratios of p-MAPK8 to total MAPK8 normalized to the amount of β-actin in the samples were quantified by determining signal density using Scion Image 4.0.2 (Scion Corp, Frederick, Maryland).
Immunocytochemistry
Freshly isolated germ cells or germ cells cultured for 8 hours without
Sertoli cells were dried to microscope slides and fixed with neutral buffered
formalin immediately following determination of cell purity. Slides were
blocked in PBS-diluted normal horse serum (1:60) and then incubated with
anti-mouse p-MAPK8 (1:50) and anti-rabbit BMF (1:200) overnight at 4°C.
Slides were washed 3 times in PBS for 5 minutes each. Signal intensity for
p-MAPK8 was enhanced by incubating the slides with an anti-mouse biotin
secondary antibody (Vector Laboratories) in PBS for 1 hour at room
temperature. Bound primary antibodies were detected with a FITC-conjugated
anti-rabbit IgM secondary antibody (1:100) and Texas Red–conjugated
avidin (1:100; Vector Laboratories). Nuclei were stained with Vectashield
anti-fade mounting medium containing DAPI. Images were obtained as above using
a Nikon Eclipse 800 microscope and Princeton Instruments 5-Mhz cooled CCd
camera.
Statistical Analysis
Data are expressed as means ± SEM. Group mean differences were
determined by 1-way analysis of variance (ANOVA). If group differences were
revealed by ANOVA (<.05), differences between individual groups were
determined using Scheffe's least-significant-difference test. Means were
considered significantly different at P < .05.
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Results |
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Apoptosis and MAPK8 Activation in Cultured Germ Cells
Germ cells were isolated via enzymatic digestion, mechanical dispersion,
and filtration to a purity of approximately 90%. The isolated germ cells then
were cultured at 34°C, in the absence of Sertoli cell support, in media
supplemented with lactate and pyruvate. Apoptosis of freshly isolated or
cultured germ cells was measured by annexin V-FITC staining, which is an early
morphologic marker for cells undergoing programmed cell death
(van Engeland et al, 1996). In
germ cells cultured for 4 and 8 hours, increases of 2.5- and 3.1-fold,
respectively, were seen in the percentages of annexin V–positive cells
relative to total cells (Figure
4). Cells cultured for 12 hours were extremely fragile and also
exhibited a high percentage of annexin V–positive cells.
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Discussion |
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When rat germ cells were cultured in the absence of Sertoli cells, BMF staining became distributed throughout the round spermatids rather than restricted to the subacrosomal space; associated with this, there was increased p-MAPK8 staining in these cells. Increases in the intensity of p-MAPK8 and BMF staining also occurred in spermatocytes in response to cultured cells in the absence of Sertoli cells. The mechanisms that explain these increases remain unknown. Culturing the cells resulted in increased levels of apoptosis, as well as in increased levels of p-MAPK8 and redistribution of BMF. It was shown previously that the increased germ cell apoptosis (deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive cells) seen after vasectomy also was correlated with increases in p-MAPK8 levels in these cells (Shiraishi et al, 2001, 2002). From these results, it is tempting to speculate that the separation of germ cells from Sertoli cells might lead to the activation of MAPK8 and that this, in turn, might lead to the phosphorylation of BMF and to apoptosis of germ cells. It also is tempting to speculate that a similar sequence of events might account for germ cell apoptosis when germ cells slough from Sertoli cells in response to hypogonadism, testicular torsion, vasectomy, or other testicular insults. However, it is not clear that this temporal sequence occurs in response to any of these conditions. For example, it is possible that one or more of these conditions leads to biochemical changes that result in sloughing rather than the other way around. Clearly, cause-effect relationships are yet to be established.
Moreover, the sequence of events may be considerably more complex than described above. Thus, the MAPK superfamily is subdivided into 2 groups: the extracellular regulated kinases (Erk), which are activated by mitogens; and the stress-activated protein kinases/JNK, which are activated in response to various stimuli associated with the induction of apoptosis (Strniskova et al, 2002). Mixed-lineage kinase 2 (MLK2) is a MAPK kinase kinase that, when activated, can lead to the activation of MAPK8 (Dorow et al, 1995; Hirai et al, 1997). Phelan et al (1999) demonstrated that MLK2 is present in germ cells throughout all stages of spermatogenesis. It is possible that in response to culturing germ cells in the absence of Sertoli cells, the observed increase in MAPK8 phosphorylation seen in apoptotic germ cells might be due to activation of MLK2. Further studies will be needed to elucidate the role of MLK2 and MAPK8 in the initiation of germ cell apoptosis as well as to define the events that occur upstream of MAPK8 activation. Downstream events also are complex. BMF is a member of the "BH3-only" BCL2 family of proteins (Puthalakath and Strasser, 2002), each member of which is capable of detecting a specific apoptotic stimulus and transmitting this stimulus to downstream effectors to initiate programmed cell death. For example, in the case of some healthy cells, BMF is sequestered to myosin V motors by association with dynein light chain 2. Following certain damage signals, including loss of cell attachment, BMF translocates and binds prosurvival Bcl-2 proteins and thus causes cell death (Puthalakath and Strasser, 2002).
Clearly, BMF has an important, if not exclusive, role in ensuring that germ cells that lose attachment to the Sertoli cells will be eliminated by apoptosis and thus is involved in the quality control mechanism that ensure the completion of proper spermatogenesis. Further studies will be needed to determine the mechanism for increase in MAPK8 activation in relationship to the loss of adhesion, phosphorylation of BMF in relationship to the activation of MAPK8, and increased apoptosis in relationship to these events.
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Footnotes |
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* Present address: Department of Physiology, University of California, San
Francisco, San Francisco, CA 94143. 
Present address: Department of Gynecology and Obstetrics, Johns Hopkins
University School of Medicine, Baltimore, MD 21205.
Present address: Center for Reproductive Biology, School of Molecular
Biosciences, Washington State University, Pullman, WA 99164.
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