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INTRODUCTION |
Most physiological cell death is caused by apoptosis, and
apoptotic cells are believed to immediately undergo heterophagic elimination by surrounding phagocytic cells, such as macrophages (reviewed in Refs. 1-3). However, the molecular basis underlying the
phagocytosis of apoptotic cells remains to be clarified.
Apoptosis and subsequent phagocytosis also occur in areas where
macrophages do not infiltrate, such as the brain and the testis. In the
testis, more than half of the differentiating spermatogenic cells die,
probably by apoptosis, before they mature into spermatozoa (reviewed in
Refs. 4-8), although the mechanism and meaning of this phenomenon are
unknown. The occurrence of spermatogenic cell apoptosis at various
stages of differentiation has been reported (Refs. 9-13 and reviewed
in Refs. 7 and 8). Only a limited number of apoptotic spermatogenic
cells, however, are detectable when testis sections are histochemically
examined. This may be due to the elimination of degenerating
spermatogenic cells by testicular phagocytes at the early stage of
apoptosis. Electron microscopic studies with rodent testis sections
have shown that degenerating spermatogenic cells are engulfed by
Sertoli cells, a type of testicular somatic cell (14-17). Some murine
Sertoli cell lines show phagocytic activity against latex beads
(18-20). Sertoli cells are thus likely to be responsible for
eliminating apoptotic spermatogenic cells in the testis (21). However,
little is known about the regulation of this Sertoli cell function.
We previously reported that primary cultured rat spermatogenic cells of
20-day-old rats, which mostly consisted of spermatocytes, underwent
apoptosis and were phagocytosed by Sertoli cells (22). In the apoptotic
spermatogenic cells, phosphatidylserine
(PS),1 which is otherwise
confined to the inner leaflet of the membrane bilayer, was translocated
to the outer leaflet, and phagocytosis was inhibited in the presence of
liposomes containing PS (23). In the present study, we examined whether
spermatogenic cells at various stages of differentiation are
phagocytosed by Sertoli cells in a manner similar to spermatocyte
phagocytosis and tried to identify the PS receptor presumed to be
present on the surface of Sertoli cells and to be responsible for
recognition and subsequent phagocytosis of apoptotic
spermatogenic cells.
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MATERIALS AND METHODS |
Testicular Cell Preparation--
Dispersed testicular cells were
prepared from testes dissected from either 20- or 45-day-old Donryu
rats as described previously (24, 25). The dispersed spermatogenic
cells were separated through a 2-4% (v/v) density gradient formed on
a 20% cushion of Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden)
using a Celsep apparatus (Brinkmann Instruments) (26) according to the
protocol supplied by the manufacturer. From six 20-day-old rats,
2-3 × 106 4n-rich cells and 6-10 × 105 2n-rich cells were obtained; and from
45-day-old rats, 1-2 × 107 1n-rich cells
(first elution), 6-10 × 105 2n-rich
cells, and 6-10 × 105 1n-rich cells
(second elution) were obtained. Sertoli cells were isolated as
described (23).
Phagocytosis Assay--
Phagocytosis of spermatogenic cells by
Sertoli cells was performed as described previously (23) with a few
modifications. In brief, spermatogenic cells that had been cultured
without Sertoli cells for 14-15 h were labeled with biotin
(NHS-LS-Biotin, Pierce) and added to Sertoli cell cultures maintained
either in 96-well plates (Corning, Cambridge, MA) for phagocytosis of
fractionated spermatogenic cells or in Lab-Tek Chamber Slides (Nalge
Nunc, Naperville, IL) for phagocytosis of unfractionated spermatogenic cells of 20-day-old rats at a ratio of 10:1 spermatogenic cells/Sertoli cells. The culture was kept at 32.5 °C for 2 h, and unreacted spermatogenic cells were removed first by pipetting with
phosphate-buffered saline and then by trypsin (0.5 mg/ml) treatment for
3 min at room temperature. The remaining cells were fixed, supplemented with fluorescein isothiocyanate (FITC)-labeled avidin
(fluorescein-avidin D, Vector Labs, Inc., Burlingame, CA), and examined
under a fluorescence/phase-contrast microscope (IX70, Olympus, Tokyo,
Japan). The ratio of the number of Sertoli cells having fluorescent
signals to total Sertoli cells was determined in each microscopic
field. Six to eight fields from different culture wells were examined
in each experiment, and the results were analyzed statistically. The
means ± S.D. of a typical example from at least two independent
experiments were taken as the phagocytic index.
Since Sertoli cell-derived cell lines only weakly attached to the
culture container, the phagocytosis assay with these cells was slightly
modified. The phagocytic cells and cultured spermatogenic cells were
mixed and maintained at 32.5 °C for 2-3 h, and the cells were all
detached from the culture container by treatment with 0.1% (w/v)
trypsin and 0.02% (w/v) EDTA, placed on
poly-D-lysine-coated glass slides, and further treated as
described above.
Phospholipid Externalization Assay--
Cell-surface PS was
detected using FITC-conjugated annexin V (Bender MedSystems, Vienna,
Austria) as described previously (27). Spermatogenic cells were
simultaneously treated with propidium iodide and FITC-conjugated
annexin V and analyzed with a flow cytometer (EPICS XL, Coulter Corp.,
Hialeah, FL). The cells negative for propidium iodide staining, which
were considered to retain integrity of the plasma membrane, were gated
and analyzed for binding of the FITC-labeled probe.
Liposome and High Density Lipoprotein (HDL)
Preparation--
Liposomes were prepared as described previously (28).
PS-containing liposomes were composed of phosphatidylcholine (PC) and
PS at a molar ratio of 7:3. Fluorescence-labeled liposomes were
prepared with N-(lissamine rhodamine B
sulfonyl)-L-
-phosphatidylethanolamine (Avanti Polar
Lipids) at 1% of total phospholipids. Human HDL was prepared in the
density range 1.063-1.21 g/ml from plasma by ultracentrifugation
according to standard procedures (29).
Northern Blots--
Total RNA was prepared using acid
guanidinium thiocyanate (30), and poly(A)-containing RNA was enriched
by affinity chromatography (oligo(dT)-cellulose type 2, Collaborative
Biomedical Products, Bedford, MA). The RNA was separated on a
formaldehyde-containing 1.2% (w/v) agarose gel and blotted onto a
nitrocellulose membrane (BA85, Schleicher & Schuell, Dassel, Germany).
The membrane was probed with a DNA fragment derived from the human
-actin pseudogene (31) or a cDNA of the hamster class B
scavenger receptor type I (SR-BI) (32). The hybridization signals were
visualized by autoradiography using x-ray film (X-AR, Eastman Kodak
Co.). Electrophoresis, transfer, blotting, and hybridization were
conducted according to standard procedures (33). The probes were
labeled with 32P by random priming (33) using a commercial
kit (Takara Shuzo, Otsu, Shiga, Japan).
Cloning of Rat Sertoli SR-BI cDNA--
Poly(A)-containing
RNA (~6 µg) was prepared from Sertoli cells (1.8 × 108) of 20-day-old rats and used to make a cDNA library
using a commercial kit (Great Lengths cDNA synthesis kit,
CLONTECH). The cDNA was ligated with the
ZAPII vector (Stratagene, La Jolla, CA) and packaged using
GigapackIII Gold (Stratagene). The library, containing 1 × 105 independent clones, was screened by hybridization with
a probe of the hamster SR-BI cDNA. Final positive clones were
sequenced using an automated DNA sequencer (ABI Prism 377, Perkin-Elmer).
Transfection of Sertoli Cell-derived Cell Lines with Rat Sertoli
SR-BI cDNA--
Rat SR-BI cDNA was recloned into the pRc/CMV
vector (Invitrogen, NV Leek, The Netherlands) and introduced into
Sertoli cell-derived cultured cell lines 15P-1 (a gift from F. Cuzin)
(19) and TM4 (obtained from the American Type Culture Collection,
Rockville, MA) (34) by the calcium phosphate method using a commercial kit (Invitrogen), and the cells were selected by maintenance in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and G418 (0.5 mg/ml) (Geneticin, Life Technologies, Inc.) at 32.5 °C
for 7-14 days. The selected cells were cloned and used for further analyses.
Liposome Incorporation Assay--
Fluorescence-labeled liposomes
were added to cultured cells at 0.2 mM, and the mixture was
incubated for 1 h at 32.5 °C. The cells were washed with
phosphate-buffered saline, and the extent of liposome incorporation was
determined using either a flow cytometer or a
fluorescence/phase-contrast microscope.
Latex Bead Incorporation Assay--
Sertoli cell-derived cell
lines were mixed with fluorescence-labeled latex beads (Polybead
Microparticles (ø = 0.75 µm), Polysciences, Warrington, PA) for
1 h at 37 °C, and incorporation of the beads was examined using
either flow cytometry or microscopy.
Anti-SR-BI Antibody Preparation--
Peptides corresponding to
amino acid residues 76-95 and 110-132 of hamster SR-BI (32) with an
extra Cys residue at the carboxyl terminus were synthesized. The
peptides were coupled to keyhole limpet hemocyanin, emulsified with
Freund's adjuvant, and injected into the backs of New Zealand White
rabbits. Anti-SR-BI antibodies were affinity-purified from the rabbit
sera as described previously (35). Anti-SR-BI-76 and anti-SR-BI-110
stand for antibodies raised against peptides corresponding to amino
acid residues 76-95 and 110-132, respectively. Anti-SR-BI-76 was used
throughout the study, except that immunohistochemical analysis was done
with anti-SR-BI-110.
Western Blotting--
Membrane fractions were prepared from
liver and primary cultured Sertoli cells and spermatogenic cells of
20-day-old rats as described previously (36). The fractions were
denatured under reducing conditions and separated on an 8%
SDS-polyacrylamide gel. The proteins were electrophoretically
transferred onto a polyvinylidene difluoride membrane (Millipore Corp.,
Bedford, MA), and the membrane was blocked with 5% dry skim milk. The
membrane was incubated with an anti-SR-BI antibody in a buffer
consisting of 10 mM Tris-HCl (pH 8.0), 0.15 M
NaCl, and 0.5% Tween 20; washed; reacted with an alkaline
phosphatase-conjugated anti-rabbit IgG antibody (Bio-Rad); and
subjected to a chemiluminescence reaction using the Immun-Star system
(Bio-Rad).
Immunohistochemistry--
Cultured cell lines or Sertoli cells
of 20-day-old rats were fixed with 4%
paraformaldehyde/phosphate-buffered saline for 20 min at room
temperature and blocked with 3% bovine serum albumin for 1 h at
room temperature. The fixed cells were then mixed with an anti-SR-BI
antibody and left at room temperature for 1 h. To detect the
transcription factor Ad4-binding protein (Ad4BP) (37), the fixed
Sertoli cells were further treated with 0.1% Triton X-100/phosphate-buffered saline, then with methanol for
permeabilization of the plasma membrane, and finally with an anti-Ad4BP
antibody (37) for 1 h at room temperature. The cells were
supplemented with an FITC-conjugated anti-rabbit IgG antibody
(Immunotech, Marseilles, France) for 30 min at room temperature and
examined under a fluorescence/phase-contrast microscope.
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RESULTS |
Preparation of Spermatogenic Cell Populations with Distinct
Ploidy--
The cells present in seminiferous tubules of either 20- or
45-day-old rats were dispersed by successive treatment with trypsin and
collagenase and subjected to a Percoll density gradient. The morphology
of the separated cells was examined by microscopy, and cells with
similar morphology were combined. When the combined cells were
subjected to DNA flow cytometry, cell populations with distinct ploidy
were found at 60-90% purity (Fig.
1A). The cells were eluted
from the gradient in the order of 4n-, 1n-,
2n-, and 1n-rich populations; 1n-rich
cells were recovered in two different populations. 2n- and
4n-rich cell populations were obtained from 20-day-old rats,
and 1n- and 2n-rich cell populations from
45-day-old rats. The separated cell populations were distinctive in
appearance, and morphological examination allowed us to identify
particular spermatogenic cell types, i.e. 1n-rich
cells (first elution) consisted of round spermatids, 1n-rich
cells (second elution) contained many elongated spermatids, most
2n-rich cells were spermatogonia, and 4n-rich
cells were spermatocytes (Fig. 1B). Testicular somatic cells, including Sertoli cells, were not recovered as a major population in any fraction, and they probably remained attached to the
chamber wall of the separation apparatus.

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Fig. 1.
Characterization of fractionated
spermatogenic cells. A, DNA flow cytometry. Several
fractions eluted from the Percoll gradient were combined based on their
morphology, stained with propidium iodide, and analyzed in a flow
cytometer. Arrows indicate the positions of 1n-,
2n- and 4n-cells. Numbers are the
percentages of cells present in corresponding peaks. B,
microscopic analysis. The cells were stained with Hoechst 33342 and
examined under a fluorescence/phase-contrast microscope. Scale
bar = 10 µm.
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Phagocytosis of Fractionated Spermatogenic Cells by Sertoli
Cells--
We first determined how efficiently the fractionated
spermatogenic cells were phagocytosed by Sertoli cells. The cells were cultured without Sertoli cells for 14-15 h to remove residual contamination of somatic cells that adhered to the culture container. The cultured spermatogenic cells, the viability of which was 80-90% as determined by trypan blue exclusion, were subjected to a
phagocytosis assay with isolated rat Sertoli cells. Sertoli cells
prepared from 20-day-old rats phagocytosed 2n- and
4n-rich cells of 20-day-old rats as efficiently as they did
unfractionated spermatogenic cells (Fig.
2A). Similarly, 1n-
and 2n-rich cells prepared from 45-day-old rats were almost
equally phagocytosed by Sertoli cells from 20-day-old rats (Fig.
2B). These results showed that spermatogenic cells at
various stages of differentiation were phagocytosed by Sertoli cells
with similar efficiencies.

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Fig. 2.
Phagocytosis of fractionated spermatogenic
cells by Sertoli cells. Unfractionated and fractionated
spermatogenic cells prepared from 20-day-old (A) and
45-day-old (B) rats were subjected to a phagocytosis assay
with Sertoli cells of 20-day-old rats. The differences among the
phagocytosis efficiencies of the various cell populations were
statistically assessed by analysis of variance.
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Involvement of PS in Phagocytosis of Fractionated Spermatogenic
Cells--
We previously showed that PS was translocated from the
inner to the outer leaflet of the membrane bilayer of unfractionated spermatogenic cells of 20-day-old rats during culture without Sertoli
cells and that liposomes containing PS inhibited phagocytosis of the
spermatogenic cells by Sertoli cells (23). We thus examined whether PS
externalization occurs in fractionated spermatogenic cells.
Translocation of PS to the surface of the spermatogenic cells was
determined using flow cytometry with FITC-labeled annexin V, which
specifically binds to PS (38). All spermatogenic cell populations
examined showed the presence of annexin V-bound cells, although the
content differed among the populations (Fig.
3A), indicating that PS
externalization occurs in apoptotic spermatogenic cells at all stages
of differentiation.

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Fig. 3.
PS-mediated phagocytosis of fractionated
spermatogenic cells by Sertoli cells. A, exposure of PS
on the cell surface. Spermatogenic cell populations were analyzed by
flow cytometry for the binding of FITC-labeled annexin V. Numbers are the percentages of annexin V-bound cells,
indicated by horizontal bars. B, effect of
PS-containing liposomes on phagocytosis. Phagocytosis assays were
performed in the presence of liposomes composed of either PC only
(PC) or a mixture of PC and PS (PS). Phagocytic
activity was measured relative to that in a reaction with no added
liposomes, which was taken as 100. Significance was determined using
Student's t test. *, p < 0.002; **,
p < 0.001.
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We then examined the effect of PS-containing liposomes on phagocytosis
of fractionated spermatogenic cells by Sertoli cells. As shown in Fig.
3B, the addition of PS-containing liposomes significantly reduced phagocytosis of 1n- and 2n-rich cells of
45-day-old rats as well as of 4n-rich cells of 20-day-old
rats, whereas liposomes composed of PC alone had little effect. The
inhibition by PS-containing liposomes of phagocytosis of
2n-rich cells from 20-day-old rats was not significant, but
a decrease in the phagocytic index in the presence of the liposomes was
reproducibly observed. These results indicated the involvement of PS in
the phagocytosis of all apoptotic spermatogenic cells by Sertoli cells.
Identification of Sertoli Cell PS Receptor--
The above results
suggested the presence of a molecule(s) that recognizes PS and induces
phagocytosis on the surface of Sertoli cells. SR-BI (reviewed in Ref.
39) is a strong candidate for such a PS receptor since it recognizes PS
(40, 41) and is present in the testis (32, 42-44). We first examined
whether Sertoli cells contain SR-BI mRNA. Oligo(dT)-selected RNA
from Sertoli cells and spermatogenic cells of 20-day-old rats was
blot-hybridized with a hamster SR-BI cDNA probe. The RNA from both
cell types showed a discrete signal whose size roughly corresponded to
that of the rat ovary SR-BI mRNA (45) (Fig.
4). This indicated that rat Sertoli cells
express a gene coding for SR-BI. We then isolated an SR-BI cDNA by
screening a library prepared from Sertoli cell mRNA using the
hamster SR-BI cDNA as a hybridization probe. Three positive clones
were obtained, and sequence analyses of two clones revealed one to be a
part of the other. The longer clone included the entire coding region,
and the primary sequence of rat Sertoli SR-BI2 was found to
be identical to that of rat ovary SR-BI
(45).

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Fig. 4.
Presence of SR-BI mRNA in Sertoli
cells. Total RNA (lanes 1 and 4) and
poly(A)-containing RNA (lanes 2, 3, 5,
and 6) (2.5 µg each) from Sertoli (lanes 1,
2, 4, and 5) and spermatogenic
(lanes 3 and 6) cells of 20-day-old rats were
analyzed on Northern blots with probes of the hamster SR-BI cDNA
(lanes 1-3) and human -actin pseudogene DNA (lanes
4-6). The positions of 28 S and 18 S rRNAs are shown with
arrowheads.
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To determine the function of Sertoli cell SR-BI, the cDNA was
introduced into Sertoli cell-derived cultured cell lines 15P-1 and TM4.
From the cells that remained alive in G418-containing medium, 14 clones
of 15P-1 and 30 clones of TM4 cells were isolated. They were first
examined for the ability to incorporate fluorescence-labeled PS-containing liposomes by flow cytometry. The five 15P-1 clones and
the 10 TM4 clones tested incorporated PS-containing liposomes more
efficiently than the corresponding parental cells (Fig.
5A). The SR-BI mRNA was
detectable in both parental cell lines (Fig. 5B). When
parental cells and transfectants were immunohistochemically examined
for the presence of SR-BI with anti-SR-BI-110, extranuclear localization of the protein was observed in all of them, and all the
transfectants examined were found to contain more SR-BI than the
corresponding parental cells (Fig. 5C). These cell clones were then subjected to a phagocytosis assay with apoptotic
spermatogenic cells. Both parental cell lines possessed activity for
phagocytosing spermatogenic cells in a PS-dependent manner
(Fig. 5D). When the selected transfectants were tested, all
showed higher activity levels of PS-mediated phagocytosis than the
parental cells; the extent of the increase in the activity caused by
SR-BI expression was not large, but was significant (Fig.
5D). The levels of activity of engulfing latex beads were
comparable among parental cells and transfectants (data not shown). The
above results indicated that overexpression of SR-BI confers the
PS-mediated phagocytic activity of apoptotic spermatogenic cells on
Sertoli cell-derived cell lines.

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Fig. 5.
Function of SR-BI in Sertoli cell-derived
cell lines. The rat Sertoli SR-BI cDNA was introduced into
Sertoli cell-derived cell lines TM4 and 15P-1. The activities of
incorporating PS-containing liposomes and phagocytosing apoptotic
spermatogenic cells and the presence of SR-BI in cloned transfectants
were determined. A, incorporation of fluorescence-labeled
PS-containing liposomes. Solid lines, parental cells;
broken lines, cell clones derived from TM4 (left
panels) and 15P-1 (right panels). B,
presence of SR-BI mRNA in parental cells. Poly(A)-containing RNA
(2.5 µg) of 15P-1 (lane 1) and TM4 (lane 2)
cells was analyzed on Northern blots with the hamster SR-BI cDNA as
a probe. C, presence of SR-BI protein in transfectants. The
cells were treated with an anti-SR-BI antibody and an FITC-labeled
secondary antibody, and the signals were visualized under a
fluorescence microscope. Scale bar = 10 µm.
D, phagocytosis of spermatogenic cells. Phagocytic indices
and effects of PS- and PC-containing liposomes (1 mM) were
determined for each cell clone. Significance was determined using
Student's t test. *, p < 0.05; **,
p < 0.02; ***, p < 0.002; ****,
p < 0.001.
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We next studied whether SR-BI is actually involved in spermatogenic
cell phagocytosis by Sertoli cells. Sertoli cells were first examined
for the presence of SR-BI protein. Membrane fractions prepared from
primary cultured Sertoli cells and spermatogenic cells of 20-day-old
rats were analyzed by Western blotting with anti-SR-BI-76 (Fig.
6A). A discrete signal with a
molecular mass of ~70 kDa was detected in Sertoli cell proteins, and
it disappeared in the presence of the antigen peptide, but not a
peptide corresponding to another region of SR-BI. Moreover, the
migration of the signal was almost the same as that of a signal
detectable with rat liver proteins. SR-BI protein seemed much less
abundant in spermatogenic cells than in Sertoli cells. When Sertoli
cells were immunohistochemically examined with anti-SR-BI-110, many of
the cells showed extranuclear signals, but at differing intensities
(Fig. 6B). On the other hand, the nuclei of Sertoli cells
appeared to be uniformly stained with an antibody specific to the
transcription factor Ad4BP, which exists in Sertoli and Leydig cells of
the testis (37). Treatment with control normal rabbit IgG did not
produce signals (data not shown). These results indicated that SR-BI
exists in rat Sertoli cells. The antibodies used here do not
distinguish SR-BII (46) from SR-BI. Most of the signal was, however,
likely to be derived from SR-BI since SR-BII is much less abundant than
SR-BI in the testis (46). We then determined the effect of anti-SR-BI
antibodies on phagocytosis of spermatogenic cells by Sertoli cells
prepared from 20-day-old rats. The addition of anti-SR-BI-76 inhibited the phagocytosis reaction, whereas control normal rabbit IgG had a
minimal effect (Fig. 6C). Anti-SR-BI-110 showed a similar
inhibitory effect (data not shown). Significant levels of the
phagocytic activity always remained in the presence of maximal amounts
of the antibody, and the residual activity was reduced by the addition of PS-containing liposomes (Fig. 6D), indicating that some
part of PS-mediated phagocytosis by Sertoli cells is resistant to
anti-SR-BI antibodies. We next examined the effect of HDL, a specific
ligand for SR-BI (39). HDL inhibited both phagocytosis of spermatogenic cells (Fig. 7A) and
incorporation of PS-containing liposomes (Fig. 7B) by
Sertoli cells of 20-day-old rats in a dose-dependent
manner. All of the above results indicated that SR-BI acts as a
phagocytosis-inducing PS receptor of Sertoli cells, but that a small
portion of PS-mediated phagocytosis seems to be executed by a
molecule(s) other than SR-BI.

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Fig. 6.
Effect of anti-SR-BI antibody on phagocytosis
by Sertoli cells. A, Western blotting of SR-BI protein.
Membrane fractions from liver (50 µg of protein) (lanes 1,
4, and 6), primary cultured Sertoli cells (10 µg of protein) (lanes 2, 5, and 7),
and spermatogenic cells (10 µg of protein) (lane 3) of
20-day-old rats were analyzed by Western blotting with anti-SR-BI-76.
Peptides (7 pmol) corresponding to amino acid residues 76-95
(lanes 4 and 5) or amino acid residues 110-132
(lanes 6 and 7) of SR-BI were added to the
reaction simultaneously with the primary antibody. Shown on the left
are the positions of size markers. B, immunohistochemical
analysis of Sertoli cells. Primary cultured Sertoli cells of 20-day-old
rats were immunohistochemically examined with anti-SR-BI-110 or an
anti-Ad4BP antibody. Fluorescence and phase-contrast microscopic views
are shown. Scale bar = 10 µm. C, effect of
an anti-SR-BI antibody and PS-containing liposomes on phagocytic
activity of Sertoli cells. Left panel,
phagocytosis of apoptotic spermatogenic cells by Sertoli cells of
20-day-old rats was performed in the presence of anti-SR-BI-76 or
normal rabbit IgG. The extent of phagocytosis was measured and is shown
relative to that in a reaction with no added antibodies, which was
taken as 100. Right panel, anti-SR-BI-76 (3 µg) and
PS-containing liposomes (0.5 mM) were added to the
phagocytosis reaction either by themselves or simultaneously.
Significance was calculated using Student's t test. *,
p < 0.001.
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Fig. 7.
Effect of HDL on phagocytosis and liposome
incorporation by Sertoli cells. A, effect of HDL on
phagocytic activity of Sertoli cells. The phagocytosis reaction of
apoptotic spermatogenic cells by Sertoli cells of 20-day-old rats was
conducted in the presence of the indicated concentrations of HDL, and
the extent of the reaction is shown relative to that in a control
reaction with no added HDL, taken as 100. Vertical bars
indicate the standard error. B, effect of HDL on
incorporation of PS-containing liposomes by Sertoli cells.
Incorporation of fluorescence-labeled PS-containing liposomes by
Sertoli cells of 20-day-old rats was examined in the presence of HDL or
unlabeled PS-containing liposomes (PS) (1 mM).
Left panel, fluorescence microscopic views of
liposome-incorporating Sertoli cells are shown. HDL was added at 800 µg/ml. Scale bar = 10 µm. Right panel,
the level of the fluorescence signal was determined using a cooled
charge coupled device camera and is shown relative to a control
reaction, taken as 100.
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DISCUSSION |
As has been suggested from histochemical examination,
apoptotic spermatogenic cells at all stages of differentiation were phagocytosed by Sertoli cells in vitro. This indicated that
Sertoli cells are capable of phagocytosing all types of spermatogenic cells undergoing apoptosis during spermatogenesis. Moreover, the present results showed that all the apoptotic spermatogenic cells examined were recognized by Sertoli cells via PS exposed on the surface
of the dying cells. This suggests that the apoptotic pathway and mode
of subsequent recognition by Sertoli cells are common to all
degenerating spermatogenic cells regardless of their state of differentiation.
Membrane phospholipids of normal cells are localized asymmetrically on
the membrane bilayer, i.e. PC and sphingomyelin are mostly
present in the outer leaflet, whereas other phospholipids, including PS
and phosphatidylethanolamine, are restricted to the inner leaflet
(reviewed in Refs. 47 and 48). If such asymmetry is lost upon induction
of apoptosis, phospholipids that normally exist on the cytoplasmic side
of the plasma membrane would appear on the cell surface, serving as a
marker of apoptotic cells (49, 50). Translocation of PS from the inner
to the outer membrane leaflet has been reported in a variety of
apoptotic cells such as thymocytes (51), vascular smooth muscle cells
(52), neutrophils (53), spermatocytes (23), and some cultured cell
lines (38, 50, 54-56); and in cytoplasts treated with an
apoptosis-inducing reagent (57). We showed here that PS externalization
occurs in apoptotic spermatogenic cells at all stages of
differentiation. It is thus possible that a loss of membrane
phospholipid asymmetry, at least with regard to PS, is a general
feature of apoptotic cells.
It is still unclear how phagocytes discriminate between apoptotic
and normal cells. Several approaches have been taken to resolve this
issue, and it is presumed that certain molecules present on the surface
of phagocytic cells are responsible for the recognition of apoptotic
cells (reviewed in Refs. 3, 58, and 59). The externalization of PS is
one of the earliest changes in apoptosing cells (reviewed in Refs. 48
and 60), and a role for externalized PS in the recognition of apoptotic
cells by phagocytes has been demonstrated (reviewed in Refs. 3, 48, 58,
and 59). The presence of a phagocyte receptor(s) that recognizes PS
exposed on the surface of apoptotic cells has thus been postulated, and
several molecules have been identified as candidates (Ref. 61 and
reviewed in Ref. 60); among them are class B scavenger receptors. We
have provided evidence here that SR-BI, a member of the class B
scavenger receptor family, is a Sertoli cell PS receptor responsible
for phagocytosis of apoptotic spermatogenic cells. SR-BI has been
shown to function as the PS-recognizing phagocytosis receptor in some
cultured cell lines such as Chinese hamster ovary cells (41) and a
human monocyte-derived cell line, THP-1 (62). It is thus likely that
this particular member of the scavenger receptor family is the
phagocytosis-inducing PS receptor common to the non-macrophage-type
phagocytic cells. Since antibody inhibition of phagocytic activity of
Sertoli cells was only partial and the residual activity was
inhibitable by PS-containing liposomes, the presence of another
phagocytosis-inducing PS receptor is presumed. We previously showed
that integrin is not involved in the phagocytosis reaction by Sertoli
cells (23). Lectins are not likely to participate in the recognition
between Sertoli cells and spermatogenic cells either since the addition
of GlcNAc, GlcN, GalN, or Glc at 20 mM did not affect the
phagocytosis reaction (data not shown). Other approaches should be
taken to identify this additional phagocytosis receptor of Sertoli
cells. The level of SR-BI expression did not appear to be uniform among
Sertoli cells of 20-day-old rats. This suggests that their phagocytic activity varies at different spermatogenic stages.