BIOLOGY

OF REPRODUCTION

Alterations

21, 1295-1307

(1979)

in Lectin Binding to Guinea Pig Spermatozoa Accompanying in vitro Capacitation and the Acrosome Reaction MARTIN

A.

SCHWARZ

and

Department

JAMES

of Biological

University

K.

KOEHLER

Structure,

of Washington,

School of Medicine, Seattle, Washington 98195 ABSTRACT The

distribution of surface carbohydrates during in vitro capacitation of guinea pig (Cavia porcella) spermatozoa was probed by labeling membrane polysaccharides with fluorescent conjugates of the plant lectins. concanavalin A (Con A), wheat germ agglutinin (WGA) and soybean agglutinin (SBA). Lectin distributions over the surface of cauda epididymal cells were characteristic of each lectin studied. Labeling of live and prefixed cells during various temperature regimens indicated that no significant redistribution of lectin binding sites oocurs either to lectin-induced or intrinsic membrane mobility changes. Spermatozoa prescribed intervals during capacitating incubations revealed few alterations labeling patterns; however, an increase in SBA binding over the surface served. Some cells exhibited a diminished fluorescence over the equatorial

over

the sperm head due exposed to lectins at

in

Con A and/or

of the

flagellum region with

WGA

was obCon A or

WGA.

After Ca stimulation of the acrosome reaction, fluorescence previously observed with Con A and WGA (but not SBA) was no longer present over the plasma membrane surrounding the equatorial segment and medial region of the acrosome. In reacted cells, the putative inner acmsomal membrane displayed some labeling with all 3 lectins, whereas the postacrosomal region bound only Con A and WGA. These results further demonstrate the high degree of structural regionalization and mosaicism present within the surface membrane of mammalian spermatozoa. A well defined sequence of changes in lectin binding appears to accompany both in vitro capacitation and the acrosome reaction. These changes are consistent with the notion of membrane fusion requiring a prior clearance of nonlipid components from the presumptive fusion sites.

INTRODUCTION As

to

a preliminary

spermatozoa change

must termed

Barros,

is

dence

in

been

achieved

the

successfully

Such

mimicked

ciated

the

events

of

sperm-egg

acrosome

to

proceed

1973,

1974;

Gwatkin

1969).

Although

for

the

unclear,

it

is

tation

involves

incubation

surface

(Bedford,

et

a

species.

appears

to

surface

al.,

see

al.,

that of and

as

intrinsic

or

and

Oliphant,

1295

a means

for

al.

1977;

in

proceed

both

in

et

capacitative reaction a

random

vivo

and

recognize

reviews

Lis,

1974).

characterizing

mobili-

during

cellular

al.,

do

maturation

1978).

changes and the not occur syn-

sperm fashion

in vitro.

monitoring

(receptor and

Poo

mammalian in

have

(for

and

differentiation et

chronously hours

and

fluidity)

Unfortunately, acrosome

but

Sharon

properties

ensuing

Accept September 12, 1979. Received June 7, 1979.

which

glycocomponents

(Nicolson

surface

use of carbohydrate

lectins,

membrane

utilized we

oligosaccharides

provide

development,

sperm

been

approach

makes

membrane

and

and

sperm

The

1974;

probes

has

probes

mammalian

proteins

and

Oliphant

1976).

study

mono-

membrane

capaci-

the

this

compo-

Kinsey

1975; 1979;

surface

Nicolson,

ty

Oliphant,

the

in

Such

yet

and

capacitation.

specific

Yanagiis

during

redistri-

membrane

1973;Oliphant,

binding

and

specific

Koehler,

examine

spermatozoal

modification,

of

of

the

in

include

loss

variety

taken

mechanism

believed Brackett

a

be

et

1972;

to

alterations

1978;

A

asso-

reaction (Barros

restructuring 1970;

in

some

transformation

generally

Brackett,

has

a molecular

capacitative

phe-

and

and

the

machi,

1951;

Koehler,

tract

in

membrane

fusion

(Brackett

resi-

(capacitation)

for

nents

of

by

medium

incubation

1951;

a period

reproductive

defined

necessary

Chang,

could or

preconditioning during

female

chemically

1970;

This

Initial

bution

physiological

(Austin,

Bedford,

1977).

nomenon

mammalian

a

capacitation

1974;

Gwatkin,

fertilization,

undergo

1975). surface

populations, over several This lack of a

SCHWARZ

1296

well

defined

surface A

population

changes system

which

sampling

problem

matozoa

cultured

Yanagimachi allows

one

obtain

40-50%

Three

the

agglutinins,

specific agglutinin amine

residues)

specific

for

panying

The

and

to on

in

method

of

technique

the

can 20-3

cell

spermatozoa addition

of

be elicited

in

0 mm.

A

(Con

A,

agglutinin and/or

(SBA,

N-acetyl-D-

residues), coupled to fluorescein were utilized as surface probes relative

components

of

vitro

purpose

of

surface

carbohydrate

monitor

this

work

variations

guinea

pig

capacitation

and

was

the

to

distriin

spermatozoa

KOEHLER the acrosome cultures motility

reaction by the addition of CaCl2. These were also examined for viability, pattern of and occurrence of the acrosome reaction.

sper-

synchronous

soybean

D-galactose

study.

butions

the This

concanavalin

and

galactosarnine isothiocyanate this

this pig

mannose-like residues), wheat germ (WGA, specific for N-acetylglucos-

for

establish

to

Following

within

of

problem.

minimize

(1974).

reaction

cells

analysis

guinea

partially

acrosome of

of

preincubation

medium.

the

in

Usui

by

Ca-free

to

that

according

to

the difficult

appears is

and

populations in

makes

a statistically

AND

these accom-

acrosome

of Lectins and Preparation of Fluoresceinated Lectin Conjugates

Purification

Concanavalin A was purchased as a 30%, twice crystallized solution in saturated NaCI (Miles Yeda, Rhehovot, Israel). Wheat germ agglutinin (WGA) and soybean agglutinin (SBA) were purified by affinity chromatography on derivatized Sepharose 6B (Vretblad, 1976). Con A was coupled to fluorescein isothiocyanate (designated as CAF) or tetramethylrhodasnine isothiocyanate (Baltimore Biological) by modification of

methods of Aketa (1975) and Veron et al. (1977). Fluoresceinated SBA and WGA (designated SBAF and WGAF, respectively, were synthesized by conventional techniques originally utilized for fluorescent antibody conjugations (Klugeman, 1966; Goldman, 1968). In all cases, the appropriate hapten inhibitors were included in the conjugation reaction mixtures to protect the saccharide binding sites of each lectin. After conjugation, the active fluorescent conjugates were repurifled on their respective affinity matrices and stored in small aliquots at a concentration of 1.0-2.0 mg/mI at -20#{176}C.

reaction.

Labeling MATERIALS Cell Culture

AND

METHODS

Conditions

Spermatozoa released from transected tubules of the cauda epididymides of adult male guinea pigs (Cavia porcella) were suspended in “5 ml modified Tyrodes medium, pH 7.2-7.4, (Caand glucose-free) and preincubated at 37#{176}C(Vanagimachi and Usui, 1974). Spermatozoa were washed by 2 different procedures: 1) repeated centrifugation (600 X g for 4-6 mm.) and resuspension for the resulting sperm pellet in fresh medium at a concentration of 0.5-1.0 X 10’ cells/mI; or 2) passing the sperm suspension over a small column (5-10 ml disposable syringe with 18.5 gauge hypodermic syringe needle) of Sephadex G-25 (coarse, Pharmacia Fine Chemicals) equilibrated with Caand glucose-free Tyrodes solution (Gabel et al.,

1979). Cell cultures were prepared in sterile organ culture dishes (35 mm, Falcon Plastics). Each preparation consisted of 0.5-1.0 X 10’ cells/mI in a total volume of 1-2 ml. Cultures were incubated for up to 24 h at 37#{176}Cin an air atmosphere (5% CO2 :95% #{176}2) and periodically examined microscopically and assessed for viability, pattern of movement and occurrence of the acrosome reaction. Following preincubation for various periods, a quasisynchronous acrosome reaction was initiated by addition of 0.4-0.8 ml 10 mM CaCI2 to each dish. After further incubation for 5-30 mm, cultures were either labeled directly with fluorescent lectin conjugates or fixed in 1.0-2.0% buffered paraformaldehyde (see cultures were divided aliquots prior to Ca lectin labeling or fixation,

labeling procedures). Other into approximately equal addition, one for immediate the other for stimulation of

Procedures

Lectin labeling was performed aldehyde-fixed cells. Preliminary exclusively on live cells; however,

on

both

live

studies were the cytotoxicity

and

done of

lectins towards spermatozoa induced a high variability in the labeling pattern which was not observed in prefixed cells. Live cell suspenaions (0.1-0.2 ml; 1 X 101 cells/mI) were incubated with 0.1-0.2 ml of the lectin conjugates (300-400 g/ml) in a 12 X 75 mm disposable plastic test tube (Falcon Plastics) for 5-30 mm at 10#{176}Cor 37#{176}C,depending upon the nature of the experiment. Unbound lectin was removed by centrifugation (600 X g for 5-10 mm) through a one-step BSA gradient [2% bovine serum albumin (Sigma Chemical Co)l in Tyrodes medium or phosphate buffered saline (PBS) (O’Rand, 1977). Cell pellets were resuspended in Tyrodes, fixed with 2% paraformaldehyde for 5-15 mm, washed with PBS and observed through a Zeiss epi-illumination fluorescence microscope. Samples were photographed with Kodak Tri-X Pan film (ASA 400) (Eastman Kodak) using 30-60 sec exposures. Prefixed spermatozoa were labeled according to the following protocol. At prescribed intervals during the capacitation incbuation period (0, 2, 4, 8, 16 or 24 h) and after the addition of CaCl2 (5, 10, 20, 30 mm), spermatozoa were fixed by dropwise addition of 1-2% buffered paraformaldehyde directly to the cultre dish suspensions. Fixation was terminated after 5-15 mm by addition of an equal volume of cold PBS containing 0.2 M glycine to block unreacted aldehyde groups (Nicolson and Vanagimachi, 1974). The glycine incubation was continued for at least I h with several changes of solution. Cells were then centrifuged (600 X g, 3-5 mm), resuspended in PBS (without glycmne) at a concentration of I X 101 cells/mI and exposed to fluorescent lectin conjugates as previously described.

BINDING

LECTIN

Some

samples

TO GUINEA

of cells were pretreated with trypsin prior to the labeling procedure.

neurarninidase

To assess the relative effect lectin binding, both epidid1mal

or

more

compressed

thin

of temperature and

PIG SPERMATOZOA

on

incubated

sper-

matozoa were labeled at 10 C for 15-30 mm, washed in a “chilled” gradient and processed as outlined earlier. A second set of specimens was rewarmed to 37#{176}C after initial exposure to fluorescent conjugates at 10#{176}Cto investigate the possibility of ligand-induced redistribution on the sperm surface. All 3 lectins were utilized in these “temperature-shift” studies. Lectin-saccharide binding specificities were confirmed by preincubation of 100-200 jig of each lectin with 1 ml of a 0.1 M solution of the respective hapten inhibitors (alpha methyl mannoside (for Con A), ovomucoid (for WGA) and N-acetyl-D-galactosamine (for SBA)l for 30 mm at room temperature prior to labeling cells. Cell suspension (0.1-0.2 ml) was added to the inhibitor-lectin mixture and incubation continued for 20-30 mm. Following centrifugation through the BSA gradient, samples were fixed as described previously. In these controls, appropriate saccharide inhibitors were included in the gradient wash buffer (for Con A, the gradient was comprised of 2% BSA in either Tyrods or PBS plus 0.1 M alpha methyl-mannoside).

Labeling

the

of in vitro

The

that

of

to

acrosome

reaction

described

earlier

CAF.

less

(1974); served.

however,

optimal 50%

the

portion

extended

Tyrods,

usually

pletion

of

required

20-30

longer

16-24

high

of

the

h.

Initiation

This

result

3-6

mm

interval

“capacitated,”

to

reaction an

com-

in

the

In

1974).

demonstrating

responsiveness

since

was not

Usui, Ca+

a

aggregation.

10#{176}C(lower at 37#{176}Cwere

temperatures

Similar

results

SBA

labeled

vitro

Lectin

fertilization

Lectins cells.

Acrosome

several

bation

The may

morphologically

shown

in

The

sperm

Fig.

crescent-shaped

also consists

1 (see

head

anterior

surface be

of the

guinea

differentiated

into

distinct

regions

Friend

al., 1977). a characteristic

of

acrosomal

to

any

signifi-

sperm

surface

either

or caused cells labeled

Live were

with

harmful)

cells

reincubated

initially

mm)

(20

at

such a sequence of It is clear that there in

obtained

overall distrimodifications.

for

Con

A

and

during and

Capacitation

Reaction

distributions

of

glycocomponents

spermatozoon

performed

cells.

Distribution

in vitro

of

Epididymal

substantially

differences temperature

were

was

sheath,

exhibited

compared

significant after such

piece

postnuclear

phenomena

lectin-induced

are no bution

intensity

stimu-

employed.

Fluoresceinated

middle

the

mobility

10#{176}C and

anterior

The

were

at or

at

most

exhibited

by

The

pig

on

In

binding

lectins.

lectins

intrinsic

acrosome.

4).

experiments

the

redistribution to

the Distribution

of if

the

confined

(S BAF)

the

piece both

from

considered

were in

in

number

the

region

was

the

37#{176}C.Figures 5-7 show cells labeled with WGAF.

routinely

noted

and

with

labeled

is significantly

spermatozoa

acrosome

and

reaction

mm.

study,

(40-

occurred Ca””'-free

in

principal

due

ob-

addition

cells)

(Vanagimachi

degree

assay

conditions,

Ca”'”

acrosome

the

work

present

lating

were

culture

preincubation

the

than

original

to

variations

the

the

the

over

seen

labeling

of

(Fig.

over

that

label

to

acrosome

fluorescence to

of

obvious

of

agglutinin

restricted the

at

pg/mI distribution

not

soybean of

cant

Usui

3 0-40

fluoresced less intensely in Fig. 3, the binding of (WGAF) was similar to

anterior

determine

results

and

acrosome-reacted

after

the

present

responsiveness viable

only

several

Under

the

Yanagimachi

of

Although

totally

A

stimulated

approximated

epididymal

intense CAF labeling pattern, and equatorial segment and

intensity

the

was

while

by

the

concanavalin

nonuniform

a

a greater

comparison,

and

Ca’”

describing

cauda

at a concentration

figure,

of

the

in lectins.

washed,

postnuclear sheath (Fig. 2). As shown wheat germ agglutinin

Reaction

of

useful

a

shown).

(not

fluoresceinated

displayed a very while the medial

Acrosome

course

time

live,

with

into

piece

bound

a

postacrosomal

of binding sites over the various regions sperm head. The anterior acrosomal

Capacitation

Cat”' -Stimulated

be

37#{176}C demonstrated

similar

Kinetics

end

surface

of

spermatozoa

portion

RESULTS

will of

segment,

a

differentiated

is and

divisions

distribution

(CAF)

and

flagellum

principal

These

acrosomal

segment

The

middle,

A

medial

equatorial

region.

1297

as

guinea

conditions

and

stimulation

of

contrast

comparing

a

extracted

pig

sperm

following

8),

Ca”'”'

By phase no

obvious

were

detectable

with

those

cells the

incu-

capacitating

reaction.

(Fig.

incubated

associated during

under

acrosome

differences

from

surface

examined

immediately

the

microscopy

morphological

et

region,

of

were

epididymis.

Con

in freshly A

and

1298

AND

SCI-IWARZ

KOEHLER

FIG. 1. Phase contrast photomicrograph of epididymal guinea pig spermatozoon. The sperm head is demarcated into an anterior acrosomal region (as), a thin equatorial segment (es) and a postacrosomal region (pa). The flagellum is comprised of the middle (mp) and principal piece (pp) separated by the anulus (a). X 2000. FIG. 2. Epididymal guinea pig spermatozoon labeled with fluoresceinated concanavalin A (CAF) demonstrates a nonuniform distribution of bound other regions of the sperm head. X 2000. FIG. 3. Epididymal guinea pig sperm distribution is similar to that of CAF. A anterior acrosomal region and a decrease in FIG. 4. Epididymal guinea pig sperm are segregated over region are unlabeled.

WGA

distributions

majority

of

distinct

frequently

ment.

Aside

bution

of

from

(Fig.

that

A

variation, WGA

of epididymal

was

cells.

anterior

acrosomal

segment

in

the

the

lectin-free

equatorial the indistinguish-

intensely

labeled

than

fluoresceinated wheat germ agglutinin (WGAF). Lectin high concentration of binding sites is associated with the is noted over the surface of the flagellum. X 2000. with fluoresceinated soybean agglutinin (SBAF). Receptors

revealed to

is more

with

of the acrosome. The remainder of fluorescence is seen over the flagellum.

anterior

the

this and

labeled relatively binding labeled

cells

These

9).

included from

Con

some regions

region

The

unchanged

however,

fluorescence-free

areas

portion a faint

remained

cells;

postacrosomal

able

the anterior Occasionally,

lectin.

segdistri-

In

vitro

intense region segment, epididymal whereas

the

incubation

acrosome

and

postacrosomal

X 2000.

did

not

postacrosomal cells are spermatozoa

(capacitated lectin binding

cells) over

region

and

unlabeled

by

incubated demonstrated the flagellar

the

diminish

staining of the anterior with SBAF. The medial and

acrosomal equatorial flagellum this

for

of

lectin,

16-24

appreciable surface (Figs.

h

LECTIN

BINDING

TO GUINEA

PIG SPERMATOZOA

10,

11).

In

pieces

of

much

more

most

cases,

the

incubated

of

the

helical

middle

spots

11, top

or

was

clusters

and

piece

than

binding

flagellum

along

CaCl2

staining

were

labeling.

occasional

It

cells

not

(but a loss

could

region

of

mm

poststimulation

the

(Figs.

head.

anterior

which

to

described

earlier,

spermatozoa tions

a

20-30

mm of

process

have

between

postacrosomal

A

or

to

45%

was

WGA

staining

stimulation

and

a

new

membrane

(Bedford,

al.,

1977).

Acrosomal

caps,

the

process

of

and

stained

(Fig.

are 17)

and

vesiculated

outer

complex, brane binding

quite WGA.

the can sites

18-21,

both

binding

sites)

(WGA

putative

all

for

to

by

binding

in (Fig.

both

Con of

memsaccharide

As shown residues

in Figs. (Con

N-acetyl-D-glucosamine sites)

comprise

on

N-acetyl-D-galactosamine

the

membrane acrosomal

3 lectins.

et

fixed

possess

mannosyl-like

and

Friend

dissipation

inner

shown

inner

observed

intensely After

carbohydrate complement inner acrosomal membrane, and/or

1970; be

con-

the

acrosomal-plasma

be

mem-

acrosomal

presumably can

this

fusions

surface,

acrosomal

16)

CaCI2. of

acrosomal of

reacting,

reac-

multiple

outer

exocytosis

exposes

motile

within

analysis that

plasma

the

with

structural

to

of

acrosome fashion

indicated

leads and

viable

after fine

the

branes

tose

10

routinely

fluorescence

semisynchronous

Results

tents

up

underwent

in

dues

the

equa-

labeled

12-15).

As

A

Cells

in

Con

after

showing and

Ca

a decrease

apparent

now

sperm

that

incubated

medial

with

regions in

in

the

to

earlier

populations in

intro-

subjected

encountered

stimulated)

torial

region

seen

intervals

mentioned

fluorescence

revealed

a

be

after

similarly

was

were

Ca”'

of

the

cell).

of

lectin

did often

occasionally

Sperm cultures fixed at specified (5, 10, 20 or 30 mm) immediately duction

principal

SBAF

SBA

as discrete

extent

and

fluoresced

with

cells.

expressed

middle

spermatozoa

intensely

uncapacitated

(Fig.

1299

A resi-

a significant

the presumptive whereas D’galacresidues

FIGS. 5, 6, 7. Initially unfixed epididymal sperm labeled with fluoresceinated wheat germ agglutinin (WGAF) under various temperature regimens. Fig. 5. A cell treated at 10#{176}C. The distribution of fluorescence is similar to that of fixed cells (Fig. 3). Fig. 6. A cell treated with lectin at 37#{176}C. Fig. 7. ResuIts of initial labeling at 10#{176}Cfollowed by reincubation at 37#{176}C for 20 mm. No net changes in the distribution of lectin are seen among these cells. X 2000.

SCI-IWARZ

1300

KOEHLER

AND

FIG. 8. Phase contrast micrograph of guinea pig spermatozoa following incubation under capacitating conditions (16-24 h in Cat”- and glucose-free Tyrodes medium). The morphology appears identical to that of nonincubated epididymal cells. X 2000. FIG. 9. The distribution of CAF over the surface of incubated spermatozoa remains essentially unchanged from

that

observed

somal region

(SBA

binding

ponents

of

The

ing

sites) this

21).

some

the

With

the

width

part

or

all

of

the

lectin

or

(Figs. band

reveal

fluorescence-free

a to

bovine

of

ever,

This

serum

did

ing

region

17, 18, 20). may include

not

tional were

ever,

the

noted

over

tated)

cells

the

flagellae

persisted

acrosomal contents Lectin-saccharide confirmed using appropriate

of

cells, over

of

incubated

sites of

pg/mI,

Con

A and were

0.1

were the

SBA,

at

completely

incubation

prior

the

of the M alpha-methyl

lectins in manno-

with

plete

fluorescence

labeled

be

sperm

by

a

faint apex

fluorescence of

the

acrosome

could

be

regardless

and are not compartments

The the

studies

were

the

from

incubation

Con

the in

a-methyl

the

with

together

A

surface

gaining

of the

entrance sperm.

to

cytoplasmic

DISCUSSION

detected of

3 lectins

observations of the highly restrictive localization of SBA (epididymal cells) to the most anterior region of the acrosome strongly indicate that the lectins are surface associated

0.1 M N-acetyl-D-galactosamine, (micrographs not shown). In some

the

a com-

in

all

that

eluted

data

for

binding

lectin

with

showed

could

with pg/mI

“competition” which

Addiwhich

resulted

labeling

These

mannoside.

interaction surface.

of

lectins

Finally,

out

suggest-

(300-400

saturation

of

how-

WGA,

preincubation

unmarked

inhibition

carried of

(capaci-

exocytosis

21).

sugars. 100

containing

side and respectively

binding

binding specificities inhibition controls utilizing

hapten by

SBA

after (Fig.

concentrations inhibited medium

of

sites

in

by 2-5 rich in solution

spermatozoa

following

This

acro-

mg/mI), of

sperm

lectins

mm).

5-15

the

included

labeled

studied.

array

binding,

(2-5

binding at

controls

seen

punctate

WGA

lectin-protein

involved

ever prior

either how-

albumin

inhibit

a nonspecific

that

was

not

nonfluoresceinated

region reaction;

inhibitor.

medial

bind-

segment.

over the postacrosomal or after the acrosome

hapten

of

in the

turn, was inhibited almost completely mg/ml ovomucoid, a glycoprotein polymeric N-acetyl-D-glucosamine. A

depletion or clearance of carbohydrate moieties was evident in a substantial number (>50%) of acrosome reacted cells in Ca”' stimulated cultures. No detectable binding of SBA was to

zones

presence

WGAF,

postacrosomal

cells

com-

appears

equatorial

the

of

in

“band”

acrosome reacted cells some cases the depleted

some

postacrosomal

CAF

unlabeled

however,

be minor

variability

characteristics.

traverse

in In

to

(Fig.

overlying

exhibits

conspicuous

cells;

appear

surface

membrane

region

epididymal X 2000.

in

(asterisk).

pig

distribution

spermatozoa

of lectin observed

binding in

the

on present

guinea study

LECTIN

BINDING

TO GUINEA

PIG SPERMATOZOA

provides

additional

existence

of

hydrate

evidence this

1977;

glycocomponents lectins

displayed

surface

showed

a

to

the

Similarly, ing man

and

serpm

show

butions

may

exhibit

been

al.,

anterior

the

of

in

1974; rabbit

The

present distrivariations.

entire

surface

whereas

has

found of

mouse

agglutinin,

present

the

of

it

is primarily

tail regions Soy bean

localization

the

study

on

the

to most

acrosome.

intensity

diminishing

al., and

species

lectin

is shown restricted

portion

The

et

sperm, this

bind(Edel-

mouse

receptor

certain

that

flagellum.

1975)

labeled

precise

lectin

1977).

over the middle piece and sperm (Sela et al., 1975). in contrast, have a very

the of

lectin

epididymal

reported

of

al.,

that

WGA

pig

in

Gall

et

et

also

the cells

acrosomal

in

1971; Sela

example,

anterior

piece

Millette,

(Nicolson

For

binding

the

patterns

studies

guinea

over

CAF

described

1977;

Surface

fluoresceinated

Unreacted

principal

previously

Millette,

by

from

nonuniform

were

1977).

carbo-

nonuniformly

in

order

segment

al.,

membrane.

decrease

descending

surface

plasmalemma

et

detected

were

sperm

of continuous

Nicolson

the

suggesting

regionalization

within

(Millette,

1301

of

Con

A

and

fluorescence discernible in transition from the middle to principal piece is similar to the reduction in density of Con A-hemocyanin

WGA

label

occurring

hamster

1976). to

the

surface

result

macromolecules

lymphoid

and

et

al.,

1974;

because

postfixation similar to

butions

This that

of

Singer

surface

those

redistri-

other

seen

together

as that

cells

somatic

Nicolson and

to

appears

such

and

yielded in with

at higher

Yanagi-

1972)

Nicolson,

labeling

reincubation

Koehler, binding

lingand-induced

1972;

inference,

epididymal

lectin

sperm

the

machi,

FIG.

and

of

(Karnovsky

vation

in

be

on

seen

annulus (Kinsey of

on

regions

bution

live.

the

restriction

The

discrete not

at

spermatozoa

sperm the

distrilabeled obser-

temperatures

10. Phase contrast micrograph of guinea pig following incubation for 24 h in CAand glucose-free Tyrodes medium. The morphology of the cells (labeled with soybean agglutinin) is unchanged from that of normal epididymal cells and no acrosome reaction has occurred. X 2000. FIG. 11. After incubation in Ca’ and glucosefree Tyrodes medium for 24 h, a high concentration of SBA binding can still be seen over the anterior portion of the acrosome. In addition, brightly fluorescent aggregates are discernible over the middle and principal pieces. X 2000. sperm

SCHWARZ

1302

AND

KOEHLER

r

12

FIG. 12. Phase contrast photomicrograph addition of 2-5 mM CaCI2 (CAF labeled). comparison to epididymal cells. X 2000.

(3 7#{176}C)of

labeled

cells

tures

(10#{176}C)did

bution,

suggests

ity

may

be

complex. of

of

patchy

terns

on

O’Rand

initially cause

important

in

result

O’Rand

plane

of

the

tempera-

prior these

degree membrane

of

mobil-

ences

the

noted

labeled

tation.

membrane

with who

obserpat-

which

as

mobility

in

decreased

FIG. 13. The fluorescence image of the CAF medial acrosomal region (asterisk). X 2000.

noted

rather

labeled

than

1974). the

to Species manner

cells reveal many

in vivo

account

our

work

and

is from

lectin

specific

to

a

native

variety

in

which

cells which

differthat

different

sugar

residues

valencies

(Sharon may

of

binding

antibodies.

many

of

antibodies differences

capaci-

for

surface

bind appropriate

exhibit

may

following

may

used

10 mm following the remains unaltered in

may

data

containing

proteins

in

present

lectins

contrast

rigid)

reasons between

The

and

capacitation.

observations

lateral

Several

studies The

more

became

O’Rand.

a variety

antibody to

(i.e.,

redistrifree

sperm

contrasts

sperm

a high

to

the

(1977)

flourescent

rabbit

at low significant

constraints

interpreted

indicating the

that

This

vations

not

of preincubated guinea pig spermatozoa fixed As previously described, the overall morphology

also

such

surface

no longer

bind

and

play

in

Lis, a role

displays

lectin

in the

LECTIN

BINDING

TO

GUINEA

PIG

1303

SPERMATOZOA

-

-‘

-F-

FIG.

14. Phase contrast

micrograph

of cells fixed

and treated

with

WGAF

10 mm

after

CaCl2

addition.

X 2000.

FIG. 15. Ten mm after addition of Ca+*, zones “cleared” of several WGAF treated cells (asterisk). X 2000.

are

distributed.

sperm

for

cells

often

Finally, labeling

of

label.

and

prefixed

The

in

present

cells

considerable

constraints

uncapacitated

cells

with

a patch-

with

both

free

does

live

there

are within

mobility membrane

plasma not

nonmobile

the

and

work

seem

nature

and

to of

modify

the

alterations in

under

pig

capacitating

confined

the

to

ously al.,

in

1975;

sperm

tated

pig

guinea and

of in

noted;

however,

binding

was

The

helical

fluorescent

over

the

no

similar

and

the

an

piece

fluorescent principal

are

seen

(Koehler, clusters

pieces

in

these

in

cells probe

SBA. dase

(500

over

may

spermatozoa Therefore,

(1978a,b)

incubated

with

middle specimens

bations

and

and

Cohen,

of

and

in

neural 1978)

and

possible

arise

the

principal

manner cells human

in

1972).

via

a

has piece

incu-

other

sites.

binding

shown undergo

pig

Franklin

mechanism of

in

guinea

and

Talbot

by SBA

increase

of

unmasking work

a

capacitating

by the

of

Hermann,

that

or

used

crest and

agglutinability

could

as

binding

surface, in

overt

WGA

neuramini-

mm) the

accompanying

Freeze-fracture midpiece

with

(Uhlenbruck is

no A,

Con

epi-

of the epididy-

of

of

noted

of

part

yielded

30-60

indicated

that

than

As

but

binding

sperm

that

mediated

SBA

out,

to

described

trypsinization

for

entire

a sequel

(1978b)

enhanced

it

material

the

p/mI

reported

As

pretreatment

to

with

intensely

the

(1973) the

recently

sperm.

carried

However,

spermatozoa

seen

pig

in

with Con A or WGA to the distribution The

SBA

elevated of capaci-

capacitation-accompanying mild proteolysis

mild

was

(Sieber-Blum

However,

such by

study, cells

similar

sometimes

the

of

Nicolson

flagellum.

these

of

few

increase the

1973). along

to

receptors.

the

1978a).

Franklin

and

differences

distributions

staining.

associated

fracture

Prior

pattern

corresponds

hamster

reaction,

over

of

effects

and

mal

spermatozoa

guinea

present

(Gordon

appreciable

mitochondrial

membrane

freeze

previ-

WGA

evident

middle

pattern

been

rabbit

1978).

and

internalization

subsequent

as has

acrosome

A

SBA

seen

didymal

1976)

the

Con

were

suggest

solely

of

SBA

regions

of flagellar

account for agglutinability

Franklin,

Talbot

not

Koehler,

and

stimulation

differences

studies

Koehler,

(Kinsey

Ca”'

head

sperm

emphasized

of

might mediated

were

conditions

of

enhancement

capacitation level of SBA

mimicking agglutinability

components

spermatozoa

aggregation the

incubated

surface

in

guinea

in the acrosomal

binding could result from the removal of certain “coating” substances, allowing cryptic galactosyl residues to be exposed. This difference in lectin labeling accompanying in vitro

this,

The

et

represent

(Talbot

sperm

surface.

observed

sites are evident

Alternatively,

such

damage

that

to

sperm

capacitation

basically

experience,

indicates

could

living

utilized

our

membrane

resulting

work

that

in

undergo

fragmentation

the

O’Rand

and,

of binding

that

the altera-

1304

SCHWARZ

AND

KOEHLER

LECTIN

tions in

in vitro

internal

and

tempting

to

cules

membrane

capacitation

Koehler

in

modifications

in the

fashion

can

changes

same

regions

mole-

the

be

is

in

determined

tem,

After

addition

acrosome

of

reaction

lation)

are

WGA.

This

anterior

no

to

rabbit

sperm

following

tonic

medium

and

clearance

or

binding

sites

(Gordon

et

(Kinsey

and

pothesize

or

1975)

analogous

to

membrane

1976,

al.,

surface

sperm

head

(Con

A)

prior

to

also

could

Price

and

surface

antigens)

capacitation

has

matozoa necessary (Gordon 1978;

of

Oliphant,

al.,

of

postulated

been

several

prelude et

as a consequence in

mammalian to 1975;

1976).

the

species acrosome

Kinsey

However,

and

earlier

acrosomal

The

(Kinsey of the

membrane

for

all

Con

on

this

were

acrosome

the

A

3

lectins.

labeling

surface

has

after

Koehler, reacted sperm

traversing location of

the this

the

1976).

and

is

the

postacrosomal carbohydrate

for

guinea Such

speras

Koehler,

morph-

spermatozoa

nagimachi and In summary, The surface

vitro

reaction

pig

alterations

in

(Friend

et a!.,

surface

and

1977).

membrane

morphology in the postacrosomal region be significant considering the role that region ultimately plays in gamete fusion

sites

the

to appear

whereas

labeling

the

surface

in

shown

of

flagellum,

only

in

“unmasking”

WGA

sites

study,

change

deficient zone is similar to the intramembranous particle-free regions observed in freeze-fractured specimens of acrosome reacted

Baldwin,

of

this

“band”

sperm

binding

to

unlabeled region.

1977).

The loss, removal, or modification associated macromolecules (lectin

inner

reaction.

major

of

binding

the

of of

the

the

reaction feature

in

the

or

A

be

fusion

acrosome

the

over

surface

may

membrane

the

completion

acrosome Another

account

over

After

been

a shedding

binding

minor.

the

the

Con

hy-

demonstrated

to

sites

in

region

acrosomal

involved

in

percentages

after

stimulation

demonstrated

pro-

regions

Ca”'”'

binding

Prior

could

the

the

sys-

distributions Ca” stimu-

reductions

foreshadowing

reaction,

rabbit

(Papahadjopoulos

1976;

(Oliphant,

a

dependent

Alternatively,

lectin

SBA

spermatozoa

labile

antecedent

variations

rearrangements

Ca””'

particularly

in

hyper-

in

One

those

membrane

reduction

lectin

hamster

systems

1977).

from

on

Such

shown

immediately are

a necessary

distribution

in

molecular

over

lectin

made

receptors

carbohydrate

to

higher altered

specific

Prior

and/or

to observe

lectin

events

A

hemocyanmn

1978).

reaction

material

the

and

such

redistributions

artificial

or

of

sperm

Utilizing

capacitation

displaying

These

Con

1976).

been

Koehler,

that

cesses,

et

loss

of

1976).

vitro

changes

subpopulation

able

(>40%)

in

surface

synchronous,

we were

postacrosomal

with

of

also

the

poststimu-

incubation

(Koehler,

has

to

observations

redistribution

al.,

acrosome

by

labeling

antibodies

coupled

the

stained

similar

prior

mm to

longer

is

but

(10-15

regions

region

or

Ca”,

small

more

lation.

work.

standard

indicated

(Koehier,

of cells

further

by

rather

a

present,

some

employing

systems,

(10-25%)

however,

coordinated

in

1305

evidence

capacitation

underlying

structure;

are

only

It

surface

PIG SPERMATOZOA

ological

1974;

1975). in to

membrane

2 events

during

Rudolf,

Gaddum-Rosse, associate

TO GUINEA

structure

and

(Friend

these

whether

BINDING

a

spermatozoa segregated ologically quantity

Noda, 1970). our findings carbohydrates

indicate on

regions. distribution

that: guinea

are arranged in distinct, zones corresponding separate and/or

1) pig

partially to

2) Differences of

may this (Va-

these

morphin the surface

16. Phase contrast photomicrograph of guinea pig spermatozoa fixed while undergoing the ac?osome (20 mm poststimulation with CaCI2). Vesiculated acrosomal caps can be clearly seen. X 2000. FIG. 17. Fluorescence micrograph showing that the vesiculated acrosomal caps label quite intensely with CAF. Posteriorly, an unlabeled “band” can be seen traversing the postacrosomal region (arrows). X 2000. FIG. 18. An acrosome reacted cell 20 mm after addition of CaCI2. The putative inner acrosomal membrane is now exposed and labels with CAF. The unlabeled “band” appears across the equatorial segment-postacrosomal boundary (arrow). FIG. 19. Phase contrast photomicrograph of an acrosome reacted guinea pig spermatozoon 20 mm after Ca stimulation. The acrosome has dissipated. FIG. 20. As with CAF, wheat germ agglutinin binding sites are evident over the presumptive inner acrosomal membrane and a “band” devoid of WGAF labeling is seen over the equatorial segment-postacrosomal boundary (arrows). FIG. 21. SBA labeling of acrosome reacted cells faintly reveals some receptor sites on the presumptive inner acrosomal membrane. The postacrosomal region is unlabeled by this lectin, whereas the punctate fluorescence on the midpiece of incubated cells is retained after the acrosome reaction. FIG. reaction

SCHWARZ

1306

carbohydrates In the case increase

of

in

a punctate lectin

accompany soybean

binding

to

fashion.

Some

(Con

binding

acrosomal

the of

3)

cells

over

the

4)

a beltlike

zone

equatorial

segment

in

The

these

the

lectins

cleared

medial of loss

of

fraction

aspect

of

often

the show

carbohydrate

at

the

postacrosomal

acrosomal

shows

this

in

the

a major

anterior

inner cells

in of

extensive in

of

and

often a loss

stimulation

spermatozoa

putative

used

over

medial

Reacted

reacted

show

Ca””'’ an

observed

acrosome,

region.

WGA)

After

was

capacitation. there is an

flagellum cells

reaction,

binding the

the

A,

region.

acrosome

lectin

in vitro agglutinin

AND

membrane

binding

sites

for

all

study.

REFERENCES Aketa,

K, (1975). Physiological studies on the spermegg binding in sea urchin fertilization. II. Effect of Concanavalin A on the fertilizing capacity of sperm. Exp. Cell Res. 90, 56-62. Austin, C. R. (1951). Observations on the penetration of the sperm into the mammalian egg. Austr. J. Sci. Res. B4, 581-589. Barros, C., Berrios, M. and Herrera, E. (1973). Capacitation in vitro of guinea pig spermatozoa in a saline solution. J. Reprod. Fert. 34, 47-549. Barros, C. (1974). Capacitation of mammalian spermatozoa. In Physiology and Genetics of Reproduction. Part B. (E. M. Coutinho and F. Fuchs, eds.). Plenum Press, New York. pp. 3-24. Barros, C., Garavagno, A. and Berrios, M. (1974). What is capacitation? Excerpta Medica International Congress Series No. 394, Biological and Clinical Aspects of Reproduction. Bedford, J. M. (1970). Sperm capacitation zation in mammals. Biol. Reprod.

128- 158. Brackett, B. G. and Oliphant, of rabbit spermatozoa 260-274. Chang,

M. C. (1951). matozoa deposited

and fertiliSuppl. 2,

G. (1975). Capacitation in vitro. Biol. Reprod. 12,

Fertilizing capacity of sperinto Fallopian tubes. Nature

168, 697-698. Edelman, G. M. and Millette,

Molecular Proc.

Nat.

Acrosomal of altered

membranes. J. Cell Biol. 63, 466-479. Friend, D. S., Orci, L., Perrelet, A. and Yanagimachi, R. (1977). Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa. J. Cell Biol. 74, 561-577. Gabel, C. A., Eddy, E. M. and Shapiro, B. M. (1979). Regional differentiation of the sperm surface as studied with ‘251-diiodofluorescein nate an impermeant reagent that

Gall,

labeled

W. E., Millette, Chemical and

allows

J.

components.

C. F. and Edelman, structural

spermatozoa.

In:

Reproduction. eds.). Plenum

(E. M. Coutinho Press, New York.

analysis

Cell.

isothiocyaisolation

Biol.

G. M, (1974). of

mammalian

In

Physiology

and

Genetics

and pp.

of

F, Fuchs, 241-257.

Goldman, M. (1968). Fluorescent Antibody Methods. Academic Press, New York. Gordon, M., Dandekar, P. V. and Bartoszewicz, W. (1975). The surface coat of epididymal, ejaculated, and capacitated sperm. J. Ultra. Res. 50, 199-207. Gwatkin, R.B.L., Anderson, 0. F. and Hutchinson, C. F, (1972). Capacitation of hamster spermatozoa in vitro: The role of cumulus components. J. Reprod. Fert. 30, 389-394. Gwatkin, R.B.L, (1977), Fertilization. In: The Cell Surface in Animal Embryogenesis and Development. (G. Poste and G. L. Nicolson, eds.). North Holland, New York. pp. 1-54. Karnovsky, M. J., Unanue, E. R. and Leventhal, M. (1972). Ligand-mnduced movement of lymphocyte membrane macromolecules. II. Mapping of surface moieties. J. Exp. Mcd, 136, 907-930. Kinsey, W. H. and Koehler, J. K. (1976). Fine structural localization of Concanavalin A binding sites on hamsetr spermatozoa. J. Supramol. Struct. 5, 185- 189. Kinsey, W. 1-I. and Koehler, J. K. (1978). Cell surface changes associated with in vitro capacitation of hamster sperm. J. Ultra. Res. 64, 1-13. Klugerman, M. R. (1966). Chemical and physical variables affecting the properties of fluorescein isothiocyanate and its protein conjugates. J. Immunol. 95, 1165-1173. Koehler, J. K. (1973). An unusual filamentous component associated with the guinea pig sperm middle piece. J. Microscopic 18, 263-266. Koehler, J. K. (1976). Changes in antigenic site distribution on rabbit spermatozoa after incubation in capacitating media. Biol. Reprod. 15, 444-456. Koehler, J. K. and Gaddum-Rosse, P. (1975). Media induced alterations of membrane associated particles of the guinea pig sperm tail. J. Ultra. Res. 51, 106-118. Millette,

C. F. (1977). Distribution lectin binding sites on mammalian In: Immunobiology of Gametes. M. H. Johnson, eds.). Cambridge Cambridge. pp. 51-71.

Nicolson,

C. F. (1971).

probes of spermatozoan structure. Acad. Sci. USA 68, 436. Friend, D. S. and Rudolf, I, (1974). disruption in sperm: Freeze fracture

of the Press.

KOEHLER

G.

L.

(1973).

Neuraminidase

and

mobility of spermatozoa. (M. Edidin and University Press, “unmasking”

and failure of trypsin to “unmask” B-D-galactoselike receptor sites on erythrocytes, lymphoma, and normal and transformed fibroblast cell membranes. J. Nat. Cancer Inst. 50, 1443-1451. Nicolson, G. L. (1974). The interaction of lectins with animal cell surfaces. Int. Rev. Cytol. 39, 89-120. Nicolson, G. L. (1974). Mobility and restriction of mobility of plasma membrane lectin binding components. Science 184, 1295-1296. Nicolson, G. L., Usui, N., Yanagimachi, R., Yanagimachi, H. and Smith, J. R. (1977). Lectin binding sites on the plasma membrane of rabbit spermatozoa. J. Cell Biol. 74, 95 0-962. Oliphant, G. and Brackett, B. G. (1973). Immunological assessment of surface changes of rabbit spermatozoa undergoing capacitation. Biol. Reprod. 9, 404-414. Oliphant,

C. (1976).

Removal

of sperm-bound

seminal

LECTIN

plasma of the

BINDING

TO

GUINEA

components as a prerequisite to induction rabbit acrosome reaction. Fertil. Steril. 27,

28-38. O’Rand,

M. C. (1977). antigen’s mobility Biol. 55, 260-270.

Papahadjopoulos,

Restriction of a sperm surface during capacitation. Develop.

D. W., Vail,

J. W., Pangborn,

W. A.

and Poste, G. (1976). Studies on membrane fusion. II. Induction of fusion in pure phospholipid membranes by calcium ions and other divalent metals. Biochim. Biophys. Acta 448,

265-283. Papahadjopoulos, D. W., Vail, J. W., Nir, S., Jacobson, K., Poste, G. and Lazo, R. (1977). Studies on membrane fusion. III. The role of calciuminduced phase changes. Biochim. Biophys. Acta 465, 579-598. Poo, M. M., Poo, W. J. and Lam, J. W. (1978). Lateral electrophoresis and diffusion of Concanavalin A receptors in the membrane of embryonic muscle cells. J. Cell Biol. 76, 483-501. Price, M. R. and Baldwin, R. W. (1977). The shedding of tumour cell surface antigens. In: Dynamic Aspects of Cell Surface Organization. (Poste, G. and G. L. Nicolson, eds.). pp. 423-471. Sela, B. A., Wang, J. L. and Edelman, G. M. (1975). Isolation of lectins of different specificities on a single affinity adsorbent. J. Biol. Chem. 250, 75 35-75 38. Sharon, N. and Lis, H. (1974). Use of lectins for the study of membranes. In: Methods in Membrane Biology. Vol. 3, Chap. 4. (E. D. Koru, ed.). Academic Press, New York. pp. 147-199. Sieber-Blum, M. and Cohen, A. M. (1978). Lectin binding to neural crest cells: Changes of the cell surface during differentiation in vitro. J. Cell

PIG

SPERMATOZOA

Biol.

1307

76, 68-638.

Singer,

S. J. and Nicolson, C. L. (1972). The fluid mosaic model of cell membranes. Science 175, 720-73 1. Talbot, P. and Franklin, L. E. (1978a). Surface modification of guinea pig spermatozoa during in vitro capacitation: An expression using lectin-induced agglutination of living sperm. J. Exp. Zool. 203,

1-14. Talbot,

P. and increases capacitated

Franklin, L. E. (178b). Trypsinization lectin-induced agglutinability in unguinea pig sperm. J. Exp. Zool. 204,

291-297. Uhlenbruck, C. and Hermann, W. P. (1972). Agglutination of normal, coated, and enzyme treated human spermatozoa with heterophile agglutinins. Vox Sang. 23, 444-451. Veron, M., Foerder, C., Eddy, E. M. and Shapiro, B. M. (1977). Sequential biochemical and morphological events during assembly of the fertilization membrane of the sea urchin. Cell 10, 321-328. Vretblad, P. (1976). Purification of Iectins by biospecific chromatography. Biochim. Biophys. Acta 434, 169-176. Yanagimachi, R. (1969). In vitro capacitation of

hamster Reprod. Yanagimachi, ological

spermatozoa by follicular fluid. J. Fert. 18, 275-286. R. and Noda, V. D. (1970). Physichanges in the postnuclear cap region of mammalian spermatozoa: A necessary preliminary to the membrane fusion between the sperm and egg cells. J. Ultra. Rca. 31, 486-493.

Vanagimachi, dence guinea

of

161-174.

R. and Usui, N. (1974). Calcium depenthe acrosome reaction and activation of pig spermatozoa. Exp. Cell Res. 89,