and Gynecology. University of Pennsylvania .... vern, PA). Animals. Male retired breeder guinea pigs. (Hartley strain) were obtained from .... to O'Farrell. (1975).
BIOLOGY
OF REPRODUCTION
39, 43 1-441
(1988)
Antigens Recognized by Monoclonal in Guinea GEORGE
Antibody
Pig Spermatogenic
L. GERTON,2’3
DEBORAH
Division Department University
A. O’BRIEN,4
National
Insitute
of Medicine
19104-6080
and Biology
Section4
and
Developmental
of Environmental
National Research
Institutes
Triangle
Park,
E. M. EDDY4
Gynecology
School
Pennsylvania
Gamete
and
Biology3
and
of Pennsylvania
of Reproductive
Differ
Cells and Sperm’
of Reproductive of Obstetrics
Philadelphia,
Laboratory
to Mouse Acrosomal Components
Toxicology
Health
Sciences
of Health North
Carolina
27709
of the
developing
ABSTRACT Monoclonal until the spermatozoa,
antibody terminal it
antigens
recognized
in mouse
spermatids.
180,000, and weights were immunoblots, The molecules with periodic
1D4
in extracts
Three
>200,000 released
spermatocytes. mal granule. sperm
a marker
with
a glycoconjugate
of guinea
major
pig
bands
antigen
Although region
1D4 does not label the of guinea pig epididymal
spermatogenic
of reactivity
sperm
Ji
that 1D4 reacted with antigens having and C6. immunofluorescent labeling of developing spermatids and sperm,
in general, immunoblots demonstrated
of the
staining was of extracts that
developing
the
acrosome
associated of guinea
1985).
sperm
are
molecular
spermatid
different
weights
antigens for
change
studies
acrosome,
an
exocytotic
Morphological
features
during
germ
of the synthesis,
vesicle
of
similar molecular of guinea pig germ and occasionally
with the periphery pig spermarocytes,
from (M7)
cell
and
(Leblond
Little
is known
translational ents, although
Accepted April 4, 1988. Received December 7, 1987. 1 This study was partially G. L. Gerton and was presented the Society for the Study of Reprod 34 (Suppl. 1):82. 2 Reprint requests.
in the acrosome (Clermont supported
by
NIH
in part at the 19th Reproduction, July
grant Annual 14-17,
HD-20736 Meeting 1986,
Thus,
assembly
detected
pig sperm extracts. not react strongly of immunoblots of
Clermont,
concerning modification the Golgi
1D4
of the sperm
berg, 1963; Holt, 1981; 1981). However, the panying these structural
acrosome
do
and not with condensing
and development described in and
those
of97,000-145,000,
weights to glycoconjugates reccells showed that 1D4 reacted with the juxtanuclear region of
differentiation.
Structure,
acrosome
with the same molecular 187. On two-dimensional
of the acrosome round spermatids,
formation have been
over-
lying the anterior aspect of the nucleus, is required for mammalian fertilization. It contains a variety of hydrolytic enzymes in addition to an uncharacterized matrix substance (Bellv#{233}and O’Brien, 1983; Huang et al.,
and
were detected in extracts of guinea pig sperm. Soluble antigens after the acrosome reaction was induced with ionopbore A23
INTRODUCTION The
cells
with apparent
mouse
acrosome of mouse epididymal sperm. Here we report that the
1D4 recognized a microheterogeneous population of molecules in guinea recognized by this antibody are not major Concanavalin A receptors and acid-Schiff’s Stain or periodic acid-dansyihydrazine. However, comparisons
sperm extracts indicate ognized by antibodies only wish the acrosomes
and
reacts
steps of spermiogenesis. does bind to the acrosomal
detail
the acrosospermatids,
can
be
during for
1952;
used
as
organelle.
spermiogenesis several species
Fawcett
and
Hollen-
Holstein and Roosen-Runge, biochemical events accomchanges are not well defined. the of apparatus
delivery of glycosylated during the early steps and Tang, 1985.)
synthesis acrosomal clearly
and
post-
constituplays a role
molecules to of spermiogenesis
the
Recently, we described a monoclonal antibody, termed 1D4, that reacts with the acrosomes of developing spermatids, but not with those of late spermatids or mature spermatozoa in the mouse
to of Biol
431
GERTON
432 (O’Brien
et al.,
1988).
On
immunoblots,
this
immuno-
Cell
globulin M (1gM) antibody reacted with multiple antigens having molecular weights in excess of 200,000 and a minor 85,000 molecular weight constituent. The antigens are glycoconjugates that can be labeled biosynthetically with [3H1 glucosamine and immunoprecipitated
from
cell
extracts.
These
that the epitope recognized during the terminal steps ation,
possibly
side-chains
due
of the
Although
did
of
alterations not
in the
bind
guinea
suggested
to
carbohydrate
pig
the
acrosome
in
and
rabbit
in the
sperm.
To
compare the reactive antigens further and explore the possibility of species variations in the post-translational modifications of this epitope, we have examined 1D4 reactivity in spermatogenic the guinea pig. The antigens
cells and sperm in guinea pig cells
different
than
molecular
weights
those
from have
detected
in
mouse spermatid extracts and show stage-specific patterns of expression. In addition, these antigens were compared by immunoblotting to antigens recognized by three monoclonal antibodies that react with well-characterized carbohydrate determinants.
MATERIALS
AND
METHODS
Electrophoresis-grade from Bio-Rad Laboratories Research
Laboratories
chemicals (Richmond, (Gaithersburg,
(Westbury, NY). Trypsin, soybean trypsin inhibitor, were from Sigma Chemical Horseradish immunoglobulins
peroxidase-conjugated, (1gM, IgG,
isothiocyanate(F immunoglobulins vern,
were purchased CA), Bethesda MD),
or
Serva
deoxyribonuclease I, and protease inhibitors Co. (St. Louis, MO), and
ITC )-conjugated were obtained
goat IgA) and goat from
anti-mouse fluorescein
anti-mouse Cappel (Mal-
PA).
Male
retired
breeder
were obtained (Wilmington, intracardial pentobarbital NE) prior
guinea
pigs
(Hartley
strain)
from Charles River Laboratories MA). The animals were killed or
intraperitoneal
(Beuthanasia to the removal
injection D, Burns-Biotec, of tissue.
of
by
with 6 mM sodium 2 mM glutamine,
pg/ml
streptomycin).
lactate, 100 pg/mI The
maintained
flask
1 mM sodium penicillin, and was
then
seminiferous treatment decanting
tubules released by the collagenase were recovered and washed three times by the GC-MEM after the tubules had settled
at unit gravity. final concentrations respectively, and
mm
Trypsin
inhibitor mg/ml.
from
and DNAse of 0.12 mg/ml flask was shaken
the
at 3 3#{176}C. After
removed
at 33#{176}C.After
shaken
bath
the
this
time,
water
the
bath
15 mm,
the
were added to and 1 i.tg/ml, for another 15
cell and
suspension soybean
was trypsin
was added to a final concentration The cell suspension was briefly pipetted
of
0.5 with
a large-bore plastic pipette prior to being filtered through an 80-pm mesh nylon screen. After centrifugation at 450 X g for 10 mm, the resulting single cell suspension was washed twice by centrifugation with 0.5% bovine serum albumin, 0.25 mg/ml soybean trypsin counted
inhibitor, in a
7.5-8.0
X i0 cells were applied per Scientific, Toronto, Ontario)
0.5 pg/ml DNAse hematocytometer.
in GC-MEM Approximately
and
SP-180 chamber containing
a
1 100-ml gradient of 2-4% bovine serum albumin in GC-MEM. Fraction collecting was initiated 1.5 h after the loading of the cells to the gradient. Cell fractions were assayed by Nomarski differential interference contrast microscopy, taking into account the morphological characteristics of guinea pig spermatogenic cells (Clermont, 1960). Guinea pig sperm were prepared from the vasa deferentia and caudae epididymides by the procedure of Primakoff et al. (1980). For the preparation of the soluble acrosomal contents of sperm, the acrosome reaction was induced with the Ca-ionophore A2 3187, and
Animals
Extraction
in a water
(Johns
Chemicals
and
Using a modification of the procedures of Romrell et al. (1976), spermatogenic cells were purified from guinea pig testes (Joshi and Gerton, 1987). Briefly, testes were removed and decapsulated and then added to a flask containing 0.5 mg/ml collagenase in GCMEM (Eagle’s minimal essential medium supple-
100
or vas deferens did react with
Isolation
mented pyruvate,
antigens.
the cauda epididymidis rat, or hamster, it of mature
results
1D4 was modified acrosomal differenti-
glycoconjugate
1D4
sperm from the mouse, acrosome
to
by
ET AL.
the
supernatant
was
recovered
tion to remove sperm and released (Primakoff et al., 1980). For the preparation of detergent pig spermatogenic cells and sperm,
sodium
resuspended
Omaha,
sodium (PBS)
at
0-4#{176}C in
deoxycholate containing
10
1.0%
after
centrifuga-
membrane
vesicles
extracts of guinea cell pellets were Triton
in phosphate-buffered pg/ml leupeptin,
X-100,
1.0%
10
saline pg/mI
MONOCLONAL
aprotinin,
1 mM
ANTIBODIES
p-aminobenzamidine. buffer,
After
homog-
enization
in this
at 15,000 resulting
rpm (20,000 X g) for 15 mm, and supernatant from this extraction was
lected.
Extracts
method
of Schaffner
the suspension
AND
were
assayed and
The
was centrifuged
for
Weissman
protein
by
PIG SPERMATOGENESIS
GUINEA
blots
were
antibodies
washed twice were incubated
the
gated second two washes
(1973).
Analytical gel
sodium
dodecyl
electrophoresis
according lar weights
to the were
method estimated
was
carried
of Laemmli (1970). after SDS-PAGE
and
produced
a gradient
from
pH 4.4
the ly
these
1gM
class
produced
studies, were from
membranes
four used.
Antibody
a BALB/c
prepared
(O’Brien munizing syngeneic
monoclonal
to 7.9.
from
mouse
was
of
previous-
lar cells (Fenderson
and
boosting them with et al., 1983). Antibody
ly
demonstrated
D-glucosamine residues Antibody C6 recognizes structures, lactosamine irrespective
bind
to
washed
with
standard transfer,
nitrocellulose
protocols (Towbin et al., the nitrocellulose sheets were
1979). washed
After for 10
mm with tris(hydroxymethyl)aminomethane (Tris)buffered saline (TBS:20 mM Tris-HC1, 500 mM NaC1, pH 7.5) and then blocked with blocking buffer. For 1D4, Ji, and C6, the blocking buffer and antibody diluent was 5% Carnation non-fat dry milk in TBS (Johnson et al., 1984). For A5, 3% bovine serum albumin studies
was substituted indicated milk
for inhibited
milk since preliminary A5 binding to blots.
2-5 times
ml
TBS,
60
1:3) and (1:50).
incubation
for
20
p130%
coverslips Next,
PBS
5-15
anti-mouse incubation
terminated
by
coverslips
were
mounted
9.0.
Photographs
glycerol a Zeiss
in PBS,
pH
photomicroscope
at an ASA
using
incubated supernatant immunoperiod with washes
of
on slides
with
were Kodak
5
taken Tri-X
of 400.
Immunofluorescen Antigens in Guinea
t
LTS
Localization Pig Sperm
1 demonstrates
of
that
the
by 1D4 were present in the guinea pig sperm, with the in the apical the antigens
permeabilization of that the antigens
portion could sperm are
or distributed
antigens
acrosomal greatest
recognized region intensity
of of
of the acrosome. Furtheronly be detected after with methanol, intracellular
However, at the resolution of the was impossible to tell whether the brane-associated
PBS
III
RESU
staining more,
with
FITC-goat Each 1-h
The
metha-
treatment
(culture
was
cold
were perwere
antibody
three
in PBS
coverslips
successively
mm
each.
mm
into
before
were
at were
coverslips cells were
the
as by
applied
coverslips
The the
and
mm. in
and
After the
the
coverslips
monoclonal
film
by
fixed
placing
The
Figure to
and
for
three
50%
A5 recognizes N-acetylGaIj3l-”4GlcNAcJ3l -+3R) status (Fenderson et al.,
transferred
for 2 h. After the blots were
antibody
on
Immunoblotting were
blots
cells/ml
mm,
by
(-20#{176}C)
diluted glubulins
N-acetyl-
PBS
5-15
meabiized
with
cells
for
3.7% formaldehyde. in PBS again
antibodies.
1986).
Proteins
Next, the peroxidase-conju-
methanol,
of 106 coverslips.
containing then rinsed
washed
(Symington et al., 1984). branched N-acetyllactosamine
while antibody (type 2 chain, of the branching
temeprature
cells
terminal
then
(100 in
primary and
(diluted 1:1000) each) with TBS,
4-chloronaphthol
the 1:3)
pig spermatogenic cells were prepared above. Sperm were washed as described et al. (1980). Cell pellets were resuspended
room
seminiferous
Nuili-SCC1 EC Ji hassubsequent-
TBS.
with horseradish
a final concentration polylysine-coated
with
A5 were obtained by hyperimmumice with dissociated rat testicu-
to
to to
immunized
et al., 1988). J1 was obtained by hyperima male mouse with mechanically dissociated mouse cells (Fenderson et al., 1984). C6 and BALB/c
mm
10 with
4-chloronaphthol
Guinea described Primakoff
nol
mouse
Antibodies nizing male
been
antibodies 1D4
h with diluted
Immunofluorescence
Molecuby using
Antibodies For
2-4
out
the method of Weber and Osborn (1969). Twodimensional gel electorphoresis was performed according to O’Farrell (1975). For the first dimension of isoelectric focusing, pH 3.5-10 ampholines were used
with
ml 0.3% H2 02).
sulfate-polyacrylamide
(SDS-PAGE)
for
for
supernatant
antibody (10 mm
developed Electrophoresis
incubated
(culture
the col-
433
indicating components.
light microscope, it antigens were mem-
uniformly
throughout
the sperm acrosome. Indeed, although it is probable that the antibody reacts with components on the inside of the acrosome, it is possible that the antigens
GERTON
434
FIG. 1. Localization of antigens in the text. Antibody 1D4 labeling Sperm that had lost their acrosomes
are located membranes.
on
the Until
ET AL.
recognized by 1D4 in guinea pig sperm. lmmunofluorescence was confined to the acrosomal region of these sperm with as a result of the procedure (arrow) showed very little, if any,
cytoplasmic face the localization
was performed on guinea pig sperm no detectable binding to other areas staining. Bar indicates 10 tim.
as described of th cells.
of the acrosomal of the antigens
recognized by antibody 1D4 are defined by immunoelectronmicroscopy, the use of the terms “acrosome” or “acrosomal region” reflect the possibilities that the
2OO
antigens
I-
the
recognized
acrosomal
cytoplasm
the
Immunoblot and Guinea To
lumen,
antibody
1D4
its limiting
immediately
enclosing
mouse sperm,
by
to
Analysis Pig Sperm
compare spermatids proteins
weights
bands
the
in
or the membranes
of Mouse Antigens
Spermatid
the
antigen(s) with those were extracted
in excess
recognized by 1D4 in detected in guinea pig from these cells with
of
of 200,000
weight the other
reactivity
43
and Figure antigens and
a minor
anti-
of 85,000. Guinea pig hand, produced three
with
molecular
the soluble released
acrosomal after the
acrosome
reaction
components ionophore
(Primakoff
weights
of
that
the
of guinea pig sperm A23 187-induced et al.,
molecular
1980). weights
26
transferred 2, mouse with mo-
97,000-145,000, 180,000 and >200,000. To determine whether there were detectable alterations to the antigens following the acrosome reaction, we examined
d demonstrates
97
acrosome.
gen with a molecular sperm extracts, on major
located
membranes,
subjacent
detergents, separated by SDS-PAGE, to nitrocellulose. As shown in round spermatids contained major lecular
are
Figure
2c,
of the
an-
FIG.
2.
lmmunoblot
reactivity
of
mouse
spermatids,
guinea
pig
sperm, and the soluble acrosomal components of guinea pig sperm. Extracted protein (50 g) from guinea pig sperm (a) and mouse spermatids (b) were separated by SDS-PAGE (10% polyacrylamide gel) and blotted to nitrocellulose. After treatment with 1D4 and horseradish peroxidaseconjugated second antibody, it can be seen that the antibody recognized components of different molecular weights in these cells from two different species. The major antigens in guinea pig sperm had molecular weights of 97,000-145,000, 180,000, and >200,000. The mouse spermatids had molecular weights >>200,000 and 85,000 (arrow). Proteins (50 g each) from a detergent extract (c) and the soluble acrosomal components (d) of guinea pig sperm were also analyzed by immunoblotting. No changes in the molecular weights of the 1 D4 antigens were apparent after the acrosome reaction. Numbers on the sides of the figure refer to the molecular weights (X 1O) Protein standards;
MONOCLONAL
tigens
detected
rosome peptin fect
with
reaction.
1D4
in the
acrosome
the
immunoblotting
of
did
Inclusion
reaction
medium
anti-guinea to lower
pig testicular proacrosin molecular weight forms
leupeptin
(data
these
contrast,
not
after of
pattern
1D4.
ial for
change
or omission
antibody
products from not examined
In
not
ANTIBODIES
shown).
had reactive
were in the
the
ac-
5 pM
obtained
antigens
AND
leu-
no
ef-
the
analysis
would
be produced
Antigens
Localization
in Spermatogenic
Figures antigens togenic
3 and
4
soluble
under
Cells demonstrate
the
This
early
spermatids
where
the
localization
cytoplasm mouse
are
of guinea pig round paired phase-contrast
guinea
acrosomal
vesicles
Immunoblot
the
more
intense.
a region
spermatocytes
pig.
Golgi needs
locali-
Analysis
Spermatogenic
those
Immunofluorescence the text. Shown
reactive,
in the
de-
condensing spermatids (Fig. 4a-c). This region membrane and corresmatrix material of the
of pachytene and with
suggesting
in
less
spermatids
was
to photograph,
associated
acrosome,
3.
the
granule detected by electron microscopy and Hollenberg, 1963). Occasionally, minor was observed in the juxtanuclear region of
the
The molecular
FIG. described
in
condensing
Whether apparatus
from this
both
reactivity
or is due
is
to pro-
to be determined.
of
pig spermathe antigens
membrane
both
in late,
immunofluorescence
the
readily apparent immediately after the of meiosis. In spermatids at various differentiation, the staining pattern apbe restricted to the boundaries of the acrosomal
evident and
difficult
acrosomal (Fawcell reactivity
were first completion stages of peared to
an
was
veloping acrosome of early bound little or no 1D4 antibody was on the inner acrosomal ponded to the electron-dense
of
in a mixed population of guinea cells. As was found for the mouse,
zation.
435
with
conditions.
Immuno/luorescent
SPERMATOGENESIS
Although
spontaneous acrosome reactions were because insufficient amounts of mater-
biochemical
PIG
with
converted absence of
In addition,
GUINEA
Cell
of Extracts
antigens from guinea weights than those
However,
it was
contained
possible
antigens from
spermatids. A mixed population of spermatogenic (a, c) and fluorescence (b, d) micrographs
that
similar
mouse
spermatids,
of
pig sperm of mouse guinea in
pig
molecular but
that
had lower spermatids. spermatids weight they
were
cells was treated for immunofluorescence the same cells. Note the lack of staining
pachytene spermatocyte (PS) in the top panel, while an early round spermatid (RS) is stained around the periphery In the lower panel, a step 4-5 round spermatid illustrates the beginning of the cap phase of acrosomal development plane of focus, the periphery of the acrosome is clearly visible in the cap region adjacent to the nucleus. Bar indicates
of the acrosomal vesicle during spermiogenesis. 4 sm.
to
as the
(arrow). In this
GERTON
436
ET
AL.
FIG. 4. Immunofluorescence of condensing guinea pig spermatids. Shown are examples of phase-contrast micrographs (b, c, e, g) of condensing spermatids stained with 1D4. The top panel is a step 9-10 spermatid the acrosome. Also shown in b is a focal plane illustrating that the labeling is absent in the region where The middle panel demonstrates a side view of a later spermatid (approximately step 11-12). The bottom as the primary antibody. There was no detectable immunofluorescence in this case. Bar indicates 4 m.
modified molecular
during weight
spermiogenesis forms found
examine this possibility, enriched togenic cell populations were a modification of the unit gravity cedure used to isolate mouse (Romrell et al., 1976; Joshi and these experiments, pachytene round
spermatid
tion isolated 40%), residual tids
purities contained bodies
exceeded condensing (35%-40%),
to produce in mature
the lower sperm. To
guinea pig spermaprepared following sedimentation prospermatogenic cells Gerton, 1987). For spermatocyte and 77%.
The
spermatids and round
third
frac-
(35%sperma-
(25-30%). The enriched spermatogenic cells were extracted with detergents and analyzed with immunoblotting procedures. Pachytene spermatocytes from either
mice (Fig.
micrographs (a, d, f) and fluorescence again showing the peripheral labeling of the acrosomal granule is located (arrow). panel is a control where 1 D4 was omitted
or guinea pigs reacted 5 a and e). In the guinea
a molecular weight greater Round (early) spermatids produced
two
molecular and greater
weights than
spermatids (Fig. 5g).
major,
than
broad
ranging 200,000
very pig,
weakly a single
with band
200,000 was detected; from the guinea pig
bands
of reactivity
from 170,000 to (Fig. Sf). Condensing
yielded four major bands of Two of these bands had molecular
over 200,000 lar weights 155,000-195,000.
in
while the
the other ranges of No
1D4 with
two bands had 120,000-140,000
antigens
guinea pig spermatogenic cells with near the 85,000 molecular weight round spermatids (Fig. 5b).
were
with 195,000 (late)
reactivity weights molecuand
detected
molecular antigen
weights of mouse
in
MONOCLONAL
a
b
cd
ANTIBODIES
AND GUINEA
PIG SPERMATOGENESIS
showed
efghi
with
no
obvious
antibody
1D4
Reaction Ji,
correlation (data
Three
A5
Guinea
sperm
FIG. 5. Immunoblot analysis of mouse and guinea pig spermatogenic cell extracts. Spermatogenic cells were purified by sedimentation velocity at unit gravity and extracted with detergent as described in the text. Fify micrograms of protein from each extract were subjected to immunoblot analysis with 1D4 (10% polyacrylamide gel). Lanes a-d are from mouse cells; Lanes e-i are from guinea pig cells: pachytene spermatocytes (a, a), round spermatids (b, f), condensing spermstids/residual bodies (c, g), cytoplasts (h), sperm (d, i). It is clear from this experiment that mouse sperm do iot retain the 1D4 epitope. Note the different molecular weights of the antigens detected in different spermatogenic cells and between species. Arrow indicates the 85,000 molecular weight antigen detected in mouse spermatids. Numbers on left side of the figure refer to the molecular weights of protein standards (X i0’).
in
broad
bands
immunoblots
of
reactivity
that
of spermatogenic
cell and
resulted sperm
extracts
suggested that the antibody recognized a heterogenous set of proteins. This was further confirmed by an examination of the immunoblot patterns generated by two-dimensional gel electrophoresis of guinea pig sperm extracts and the soluble acrosomal components of guinea pig sperm (Fig. 6). Several isoelectric point variants (pI range 6.4-6.8) of each of the three bands seen by one-dimensional gel electrophoresis were recognized guinea
by pig
separated
1D4. In other spermatogenic
by
SDS-PAGE,
experiments, cells and and
the
extracts of sperm were
gels
were
for carbohydrate with periodic transferred to nitrocellulose and
acid-Schiff’s examined
ity
A (Con
with
to the the
the
lectin
procedure patterns
Concanavalin of
generated
Hawkes
(1982). from
these
stained
stain or for reactiv-
7,
detected
with
Analysis
of Spermatogenic Ji,
that the
C6 and
experiments
and
1984).
To
determine
if
with the
J
1D4,
J1,
1 and
not
C6,
C6
to
and
AS.
immunoblot
identical, reactivity
to the was
shown).
A5 of guinea pig sperm C6, and 1D4, it was
Ji,
extracts apparent
there might be similar patterns of expression reactive antigens during spermatogenesis.
examine
this
possibility,
spermatogenic and those
we tested
cells
by
A5 to determine with 1D4 would
almost
negligible
additional
extracts
whether be obtained.
with
cells
(Fig.
to was
8a-e);
band of reactivity with in elongating spermatids
C6, the pattern seen with 1D4 with
1, C6,
J
patterns similar Ji reactivity
in spermatogenic
band
for To
of enriched
immunoblotting
the exception was a minor molecular weight of 120,000
of reactivity except that
a molecular
was there
weight
a
very was
in excess
of 200,000 (Fig. 8f-j). The highest molecular weight band recognized by C6 persisted throughout spermiogenesis but was not present in sperm extracts (Fig. 8j). AS reacted weakly by immunoblotting with extracts of guinea pig spermatogenic cells. By immunofluorescence, J1, C6, and A5 all reacted with the acrosomes
of permeabilized
spermatids ji did not
and sperm. significantly
round
less
and
but
not
intact
However, unlike react with the
mature
condensing
guinea
pig
C6 and acrosomes
A5, of
spermatids
(not
shown). The molecular weight ranges of the components recognized by J1, C6, and 1D4 in guinea pig spermatogenic cells are summarized in Table 1. DISCUSSION
A) according Comparison
C6,
very high molecular spermatogenic cells
similar, but not with 1D4. Minor
AS (data
When immunoblots were probed with
an
from
probed
were very obtained
Ji,
with guinea pig sperm, extracted by SDS-PAGE, transferred
Figure
patterns pattern
with
with of mouse
et al.,
and
shown
(Fig. 8c). With similar to that The
react
react separated
nitrocellulose,
Cells
antibodies,
(Fenderson
these antibodies proteins were As
Pig Sperm
monoclonal
AS, are known to weight glycoconjugates and
immunoblots
shown).
Antibodies
with
other
with
not
of Monoclonal
C6, and
437
Monoclonal
of of
the
developing
antibody acrosome
1D4
recognized in both
constituents the
mouse
and
GERTON
438
ET AL.
pH
:1..
L97 I,.
68
FIG. 6. Two-dimensional A23 187 and the soluble 1980). The proteins were each diffuse band detected
immunoblot analysis of sperm antigens. The acrosomal components collected after centrifugation separated by two-dimensional gel electrophoresis by one-dimensional immunoblotting contains
extracts. during
the
guinea
pig.
Because
epididymal
the
guinea
pig
retained
acrosomal
and sperm from to characterize
Numbers isoelectric
on the right side of the focusing are represented
obtained for sperm pH gradient generated
spermatozoa
figure refer at the top
acrosome reaction in guinea pig sperm was to remove sperm and released membrane and blotted to nitrocellulose. Treatment several components of different isoelectric to the molecular of the figure.
weights
(X
10’)
of protein
induced with the ionophore vesicles (Primakoff et al., with 1D4 demonstrates that points. Similar results were standards.
The
values
of the
from
immunoreactivity
the mouse did not, we have used the reactive antigens in these
1D4 two
species during spermatogenesis. In extracts of guinea pig sperm, three antigens with diffuse electrophoretic mobility were identified on immunoblots. These antigens had lower molecular weights than antigens previously detected in mouse spermatids (O’Brien et
200”-
97ai-1
al., 1988). Furthermore, immunoblot comparisons of extracts from round and condensing spermatids demonstrated that 1D4 recognizes antigens of different molecular weights in these species throughout spermatid differentiation. These differences do not rule out the possibility that the molecules are closely related in the two species. For example, acrosins with multiple forms and distinct molecular weights have been reported for several mammalian species (Polakoski and Siegel, 1986). Alternatively, the epitope recognized molecules sis is that
by 1D4 may be present on unrelated in the mouse and guinea pig. One hypothedistinct acrosomal constituents might be
43Ip-
FIG. 7. Identification of sperm antigens reactive with monoclonal antibodies J 1 and CO. Extracted proteins (50 tig) from guinea pig sperm were separated by SDS-PAGE on 7.5% polyacrylamide gels, transferred to nitrocellulose, and probed with antibodies Ji (a) CO (b), and 1D4 (c). Components with similar molecular weights were recognized by all three antibodies. Numbers on the left side of the figure refer to the molecular weights (X i0’) of protein standards.
MONOCLONAL
ANTIBODIES
AND
GUINEA
PIG
processed
abcde
by
genically Several
similar
mechanisms
related end experiments
nature
of
studies
the
demonstrated
products. have
antigens
et al.,
to
produce
examined
recognized that
cells were large bolically labeled precipiated from (O’Brien
439
SPERMATOGENESIS
the
the by
molecular
1D4.
antigens
Previous
in mouse
glycoconjugates that could with [3H]glucosamine and cell extracts with the 1D4 1988).
Two-dimensional
of extracts from guinea pig sperm band detected on one-dimensional several
components
Such weight
heterogeneity is consistent
with
different
in both with the
anti-
germ
be metaimmunoantibody
immunoblots indicated blots
that each comprised
isoelectric
charge and preliminary
points. molecular classifica-
tion of these antigens as glycoconjugates (Dunbar, 1987). Attempts to correlate 1D4 reactivity on blots with Con A binding or staining with periodic acid-
FIG.
8. Immunoblot analysis of guinea pig spermatogenic cell extracts with antibodies J 1 and C6. Results with sperm suggested that 104 may be reacting with a set of antigens similar to those recognized by Ji and C6. To further examine these relationships, extracts (50 12g) of guinea pig pachytene spermatocytes (a, I), round spermatids (b, g), condensing spermatids (c, h), cytoplasts (d, i), and sperm (e, j) were separated by SDS-PAGE in 7.5% polyacrylamide gels, trasnferred to nitrocellulose, and antigens were detected with J 1 (a-e) and C6 (f-i). In spermatogenic cells, ii faintly reacted with only one component of condensing spermatids (arrow). The pattern seen with CO was very similar to that obtained with 1D4. However, reaction with a very high molecular weight component (top of running gel) occurred in all spermatogenic cell populations analyzed. Numbers on the left side of the figure refer to the molecular weights (X 10’) of protein standards.
TABLE
1. Molecular
weights
of guinea
pig
spermatogenic
cell
components
Schiff’s or periodic acid-dansylhydrazine tive, suggesting that the antigens
are
high-mannose-type
or
with acid with again sialic
oligosaccharides
sugar
exposed vicinal hydroxyl groups (such residues). Treatment of mouse testicular neuraminidase did not alter 1D4 suggesting that acid (unpublished
the epitope observations).
The antigens recognized by 1D4 cells were compared to carbohydrate
recognized
were neganot rich in
by
three
monoclonal
does
not
in guinea antigens
residues as sialic sections binding, contain pig germ reactive
antibodies.
Antibody Cell
population
Pachytene
Ji
104
C6
>>200,000 >200,000
spermatocyte >200,000
Round
spermatid >200,000 170,000-195,000
Condensing
Cytoplasts
Sperm
spermatid
>>200,000 >200,000 153,000-195,000 120,000-140,000
>200,000
>>200,000
120,000
>>200,000 >200,000 108,000-185,000
>200,000 180,000
97,000-145,000
>>200,000 >200,000 165,000-185,000 130,000
>200,000 145,000-180,000 120,000-1 30,000 >>200,000 >200,000 155,000-180,000 130,000
>200,000 180,000
95,000-135,000
>200,000 175,000 115,000-150,000
GERTON
440 with
monoclonal
three
antibodies
on the cell spermatogenic more,
the
and extensively
C6,
et
al.,
antibodies bind surface cells
Ji,
to large
AS
are distinct (Symington
1986).
extracts,
On
the
acrosome et al., 1984).
epitopes
recognized
and have et al.,
immunoblots
antigens
reactive
A5.
These
with
by
of
mouse Furtherby
J 1,
been characterized 1984; Fenderson of
guinea
1D4,
Ji,
pig and
obtained
with
marked recognize
similarities, a determinant
these
antibodies,
which
sperm C6
had
there
on
To test this hypothesis, it will be necessary terize the determinant further. The observation that antigens recognized change during the haploid differentiation tids
indicates
acrosomal noblots
that
there
constituents of guinea
are
suggest that 1D4 polylactosaminoglycans.
are alterations
during pig germ
may
weight) sperm.
not the
is unknown, but one possibility is that hydrolytic enzymes present in the acrosome might modify acrosomal constituents, thereby exposing or deleting epitopes during development.
dense inner
acrosomal acrosomal
nuclear
granule membrane
membrane
comprises of guinea
and
the remainder pig spermatogenic
that a
less
dense
where with region
the the that
of the lumen. Examination cells by immunofluores-
cence has demonstrated that the intra-acrosomal localization of molecules recognized by 1D4 is different from that found for the protease zymogen proacrosin. It has been shown recently that guinea pig proacrosin is present throughout the developing acrosome (Arboleda binding the the
and is prominent and Gerton, appeared
acrosome acrosomal
to periphery granule
in the acrosomal 1988). In contrast,
be localized and region.
granule 1D4
preferentially appears
to be
is released
along absent
antigens
recognized
membrane
Although lie near much
the the of
in a soluble
needs
to
be
epitope-bearing periphery of the the 1D4-binding
form
from
guinea
pig
to
the
acrosome
nisms need
reaction.
of release to be examined
Results
The
of molecules in this
Formation process
report
demonstrate
macromolecules spermatids and
of the mammalian and may involve
carbohydrate of different cells and
studies of the acrosome such as 1D4, Ji, C6, helpful in understanding of this
and by
mecha1D4
will
further.
presented
1D4 recognizes multiple acrosomes of developing
assembly
location recognized
unique
structures molecular between
sperm
that in the of the
acrosome the synthesis
is
that are shared weights within species. Further
using immunological tools and anti-acrosin should be the synthesis, structure, and organelle.
ACKNOWLEDGMENTS We thank Elana Braz for excellent technical assistance. We also express our appreciation to Drs. Christina Teng and Michael Dresser for their comments on the manuscript. The use of guinea pigs for this study was approved by the University of Pennsylvania Institutional Animal Care and Use Committee.
REFERENCES
guinea pig spermatids based upon morphollevel: the electron-
is anchored makes contact
of
acrosomal
sperm after the induction of the acrosome reaction with ionophore A23 187. This could occur by hydrolysis of anchoring sites on the acrosomal membrane or release from acrosomal binding proteins in response
of complex by molecules spermatogenic
On immucondensing
association the
further. appear to acrosome,
by 1D4 of sperma-
was not detected in extracts of the The nature of these modifications
The acrosome of developing can be divided into two regions ogy at the electron microscope
investigated molecules developing
guinea pig. a complicated
spermatid fraction has two additional bands detected in round spermatids. Furthermore, largest antigen in condensing spermatids (>>200,000 molecular epididymal
with
to charac-
to pre-existing
this period. cells, the
presumed
1D4
material
similar molecular weights. The immunoblot patterns detected with 1D4 on earlier spermatogenic cells also were very similar to those obtained with C6, but not Ji. Although there are some differences in the patterns
AL.
The
polylactosaminoglycans
and in (Fenderson
carbohydrate
and
C6,
El
in
Arboleda CE, Gerton GL, 1988. Proacrosin/acrosin during guinea pig spermatogenesis. Dcv Biol 125:277-87 Bellv#{233}AR, O’Brien DA, 1983. The mammalian spermatozoon: structural and temporal assembly. In: Hartmann JR (ed), Mechanisms and Control of Fertilization. New York: Academic Press, pp. 55 -13 7 Clermont Y, 1960. Cycle of the seminiferous epithelium of the guinea pig. A method for the identification of the stages. Fertil Steril 6:563-73 Clermont Y, Tang XM, 1985. Glycoprotein synthesis in the Golgi apparatus of spermatids during spermiogenesis of the rat. Anat Rec 213:33-43 Dunbar BS, 1987. Basic principals of posttranslational modification of proteins and their analysis using high-resolution two-dimensional polyacrylamide gel electrophoresis. In: Two-Dimensional Electrophoresis and Immunological Techniques. New York: Plenum Press, pp. 77-102 Fawcett DW, Hollenberg RD, 1963. Changes in the acrosome of guinea pig spermatozoa during passage through the epididymis. Z Zellforsch Mikrosk Anat 60:276-92 Fenderson BA, Hahnel AC, Eddy EM, 1983. Immunohistochemical localization of two monoclonal antibody-defined carbohydrate antigens during early murine embryogenesis. Dcv Biol 100: 3 18-27 Fenderson BA, Nichols EJ, Clausen H, Hakomori 5, 1986. A mono-
MONOCLONAL clonal antibody lactosooctasylceramide probe normal
for
23:747-54 Fenderson BA,
defining
determining and transformed O’Brien
ANTIBODIES
a binary N-acetylactosaminyl (1V6 Gal1 ->4GlcNAcnLc6): differential human
glycosylation fibroblasts.
AND
structure in a useful patterns Molec
between Immunol
DA, Millette CF, Eddy EM, 1984. Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies. Dcv Biol 103:117-28 Hawkes R, 1982. Identification of Concancavalin A-binding proteins after sodium dodecyl sulfate-gel electrophoresis and protein blotting. Anal Biochem 123:143-46 Holstein AF, Roosen-Runge EC, 1981. Atlas of Human Spermatogenesis. Berlin: Gross-Verlag Holt WV, 1981. Development and maturation of the mammalian acrosome. J Ultrastruct Res 68:58-71 Huang TTF Jr. Hardy D, Yanagimachi H, Teuscher C, lung K, Wild C, Yangimachi R, 1985. pH and protease control of acrosomal content stasis and release during guinea pig acrosome reaction. Biol Reprod 32:451-62 Johnson DA, Gautsch JW, Sportsman JR, Elder JH, 1984. Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Tech 1 :3-8 Joshi DA, Bautsch JW, Sportman JR. Elder JH, 1984. Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Tech 1: 3-8 Joshi MS, Gerton GL, 1987. Isolation and short-term culture of guinea pig spermatogenic cells. J Cell Biol 105:83a Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage 14. Nature (Lond) 227:680-85
GUINEA Leblond
441
PIG SPERMATOGENESIS CP,
Clermont
Y,
1952.
Spermiogenesis
and guinea pig as revealed by the “periodic technique. Am J Anat 90:167-216 O’Brien DA, Gerton CL, Eddy EM, 1988. identified with a monoclonal antibody spermiogenesis in the mouse. Biol Reprod O’Farrell PH, proteins.
1975. High resolution, J I3iol Chem 250:4007-21
two-dimensional
of rat,
mouse,
acid-fuchsin Acrosomal are modified 38:955-67
hamster sulfurous”
constituents during
electrophoresis
late of
Polakoski KL, Siegel MS, 1986. The proacrosin-acrosin system. In: Paulson JO, Negro-Villar A, Lucena E (eds.), Andrology:MaIe Fertility and Sterility. New York: Academic Press, pp. 3 59-75 Primakoff P. Myles DC, Bellv#{233}AR, 1980. Biochemical analysis of the released products of the mammalian acrosome reaction. Dcv Biol 80: 324-3 1 U, Beliv#{233}AR, Fawcett OW, 1976. Separation of mouse spermatogenic cells by sedimentation velocity. Dcv Biol 49: 119-3 1 Schaffner W, Weissman C, 1973. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 56:502-04 Symington FW, Fenderson BA, Hakomori 5, 1984. Fine specificity of a monoclonal antitesticular cell antibody for glycolipids with terminal N-acetyl-D-glucosamine structure. Molec Immunol 21:877-82 Towbin H, Staehelin 1, Gordon J, 1979. Electrophoretic transfer of proteins from polyacrylamide to nitrocellulose sheets: procedure and some applications. Proc Nail Acad Sci USA 76:43 50-54 Weber K, Osborn M, 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406-12 Romrell