Antigens Recognized by Monoclonal Antibody to Mouse Acrosomal ...

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