BIOLOGY

OF REPRODUCTION

47, 588-597

Binding JAN

for

of Epididymal

VREEBURG,23

TM.

Department Center

(1992)

MICHAEL

of Endocrinology

Reproductive

Proteins

to Rat Spermatozoa

K. HOLLIND,4

and

and Reproduction,3 Rotterdam, The

Biology

Research,4

MAR1E-CLAllE

Faculty Netherlands

Vanderbilt

In Vivo1 ORGEBIN-CRIST4

of Medicine,

University

School

Erasmus

of Medicine,

University Nashville,

Tennessee

ABSTRACT The secretion the superior and

inferior

luminal

markedly

fluid

was

epididymal

proteins

of epididymal

with

minces

purified

spermatozoa.

quential

extraction

epididymal

their

binding

with

methionine.

proteins

of spermatozoa

Of the

were with

buffer,

the radlolabeled were extracted

proteins could be extracted with with Triton X-100 or SDS, consisted

Analysis

of

sperm-associated

proteins

to spermatozoa

the

proteins

occurs

with

associated

During

their

process, be of volved

transit

through

the

epididymis,

undergo a series of changes of the ability to fertilize eggs.

cific

intraluminal

this

epididymal membrane they may structure

times

isotope

after

mammalian

in the composition by exposure to the

secreted

existing

arrangement

epididymal

of the

studies

secretory

have

reported

proteins

on

provided

techniques direct

epididymal the

evidence

proteins

specificity is still not

[19, 20].

of this known

using

in vitro

to spermatozoa binding

a few

has

binding

[13,21-25]. been

questioned

March

Most

the

while

by NIH grant

HD03820

and

to

regional

secretion

spermatozoa

into

in

the

of proteins

the

rat,

epididymal

after

and

their

injection

of

artery.

AND METHODS

studies

were of analytical reagent quality or better. Brilliant Blue R250 dye, BSA, amino acids, PMSF,

and

have

were from for PAGE

Sigma Chemical Co. (St. Louis, were from Bio-Rad (Richmond,

Ci/mmol)

(1000

L-[35S]TRAN

Biomedicals, a mixture

was purchased

MO). CA),

from

ICN

Inc. (Costa Mesa, CA). L-[35S]TRAN consists of that is dominantly-L-[35S]methionine (about 70%),

L-[35S]cysteine

(about

25%)

with

traces

of other

com-

ponents.

of labeled However, [26],

Incoporation Vivo

and

a grant

from

of [35SWethionine

Mature (300-350 tained from Sasco

The

Andrew

W. Mellon Foundation. J.T.M. Vreeburg, Department of Endocrinology & Reproduction, Faculty of Medicine, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. ‘Correspondence:

[35S]methionine

perior

epididymal

injected period.

to all regions 588

rats

on the surface enters the tunica

pCi then mm

into

the Epididymis

g) male Sprague-Dawley Inc. (Omaha, NE). The

of ether-anesthetized

9, 1992. was supported

can be followed [27]. Most of in vivo injection of

All chemicals

Accepted June 9, 1992. investigation

of

investigated

direction initially ‘This

binding

we were able to examine the binding of secretory proteins with spermatozoa under conditions [20]. In the present study, we have

Coomassie and Percoll All reagents

it which of these proteins bind to the maturing spermatozoa in vivo. Recently, we have shown that after intrajugular injection of [355]methionine into mice, the regional secretion and

Received

tight

Che,nicaL

presence

spermatozoa.

Only

proteins method

MATERIALS

studies have used indirect approaches such as coincident migration of epididymal and sperm proteins on polyacrylamide gels [2-6], immunochemical techniques [7-18], or radiolabeling

that

[35S]methionine, epididymal physiological

[35S]methionine

membrane the

indicated

of epididymal with the

in

or

the

administered

by the

may change the in several ways: and/or modify the

Numerous

was

binding

of spe-

components

proteins

are

isotope

transport important,

that result in their In this maturation

Important

environment

the

which 32 kDa.

injection.

epithelium. These proteins properties of the spermatozoa bind to the sperm surface

molecules. of

environment.

intraluminal

examined after retrograde perfission of the pattern of radioactive proteins In the proteins obtained by in vitro incubation of

of secretory into the lumen, about 1% were associated with Percollthe spermatozoa in the corpus epididymidis than in the caput. Sea high-salt buffer, Triton X-100, and SDS revealed that almost half of

remodeling of the plasma membrane is thought to critical importance since the sperm surface is inin interand intracellular aspects of the fertilization

process [1]. It is likely that changes the sperm membrane are induced

that

pattern

INTRODUC11ON

spermatozoa acquisition

revealed

the isotonic buffer. The firmly bound radioactive proteins remaining, of one major band of 25 kDa and two minor bands of 30 Wa and

at various

3 h after

within

PAGE secreted

proteins

an isotonic

in rats were

to spermatozoa

[‘5Slmethioninc.

from the well-characterized

different labeled

More

and

arteries

was

cannulated

In

rats were obtesticular artery in a retrograde

of the testis near where the vessel albuginea. Radioactive isotope (750

in a total

in a series The isotope

volume of 700 Ill saline) was of about 20 pulses over a 10-15back-perfused the inferior and su-

arteries,

of the epididymis

which

in the

except

rat

supply blood cauda. Af-

the distal

EPIDIDYMAL

SECRETORY

PROTEINS

ter administration of isotope, the testicular artery was ligated at the testicular surface, and the testis was removed. The epididymis was returned to the scrotum, and the wound was closed. Rats were allowed to regain consciousness and were returned to their cages. Al various times after administration of isotope, the animals were killed by cervical dislocation

under

moved

to a Ringer

0.5

mM

anesthesia,

and

phosphate

the

buffer,

epididymis pH

was

7.4(28],

re-

of T&ue,

The

organ

Fluid,

was

and

Spermatozoa

into

five

divided

segments

with

1, 2, and

3 being the proximal, mid, and distal caput; 4 the corpus; and 5 the cauda (Fig. 1). Each segment was minced into 1.5 ml of Ringer phosphate buffer containing PMSF and shaken for 10 mm to permit tent. The tissue pieces were

dispersal allowed

the upper fraction containing was collected. This fraction

of the luminal to settle, after

spermatozoa was layered

and over

conwhich

luminal fluid a discontin-

uous Percoll gradient consisting of 10%, 40%, and 65% Percoll. The gradient was spun at 500 X g for 45 mm at room temperature.

The

aration

upper

of lummnal

100 000

x g for

20 mm

on

a Sephadex G-25 (pH 7.2), freeze-dried, Spermatozoa

layer,

fluid,

were

consisting

was

of a crude

removed

at 4#{176}C. The

M column, and

and

from

in the

at

was placed 20 mM Iris

with

dissolved

recovered

prep-

centrifuged

supernatant

eluted

distilled

didymal

cells.

The

bottom

with

fraction

Ringer

phosphate

with

the

buffer, and 0.1%

between

0.25 M sucrose, 1.5 mM MgC12, centrifuged at 100 000 x g for 20 mm.

The

tissue

by

lyophilization

before

being

supernatant

The

sperm

pellet

buffer, controls,

of washings tion with

was

buffer,

spermatozoa

either

Triton were

in distilled

of proteins sequential

pending

the

sperm

(Gibco).

The

sperm

resuspended

in a double-

lysis buffer containing 0.125 20% glycerol, and 10% 3extracted with a low-salt X-100,

subjected

with a low-salt SDS. The control

for any release ing itself. The

or

(high-salt

1 ml Tyrode’s

wash).

After

pernatants

the suspensions were were treated as described

extracted

with

the

high-salt

of Tyrode’s solution room temperature; cubated

in 1 ml

gation,

the

of Tyrode’s

spermatozoa

ble-strength

were buffer.

Laemmli

Incoi’poration In Vitro The minced

buffer

with 0.1% the control

solution 20 mm

contain-

at room

tem-

the

su-

centrifuged,

and

above.

The spermatozoa

were

incubated

Triton X-l00 spermatozoa

in 1 ml

for 20 mm at were again in-

buffer. Finally, after centrifuincubated with 100 .tl dou-

of [35SjMethionine

into

Epididymal

Tissue

procedure of Brooks [28] was followed. Essentially, tissue (20 mg) was incubated with [35S]methionine

(100 p.Ci) in 1 ml of Ringer phosphate buffer (pH 7.4) containing glucose and a mixture of amino acids for 5 h at

34#{176}C. The media vested, and the

containing the released proteins were collected

electrophoresis

as described.

proteins and

were separated

harby

Estimation Proteins

of the Incoporation

of [355]Methionine

into

5%

filters

Corp., TCA

Bedford,

containing

were

MA)

washed

twice

with

methionmne. in 5 ml of toluene

After

drying,

10

counted

and

mM

2,5-diphenyloxazole and 0.1 g/L lyl) benzene in a liquid scintillation

of

containing

20

ml

the 4 g/L of

1,4-bis(5-phenyloxazospectrometer.

PAGE

electrophoresis 6.8), 2% SDS, or sequentially

a high-salt

control)

M NaC1

lipore

of Spermatozoa

strength Laemmli M Tris-HCI (pH mercaptoethanol,

wash,

0.5

M column, resuspended

was

water. Extraction

ing

centrifuged

in and

fragments

homogenized Triton X-100

dissolved

(low-salt

temperature, and cenresulting supernatants (100 000 X g, 20 mm, 4#{176}C), and lyophilized. The sperm in 1 ml Tyrode’s solution

For estimation of the amount of [35S]methionine incorporated into proteins, small aliquots (5-20 pA) of the luminal fluid fractions, tissue homogenates, or sperm extracts were added to 0.5 ml distilled water containing 50 .tg BSA and 10 mM methionmne. Proteins were precipitated with tnchloroacetic acid (TCA) at a final concentration of 10% (wI v). The precipitate was collected on Millipore filters (Mil-

placed on a G-25 M column to remove any unincorporated label and to desalt the fluid. The eluant was then concentrated

20 mm at room at 500 X g. The

parts,

trifuged

water.

interface

the 40% and 65% Percoll layer. The sperm suspension was washed by diluting with 2 volumes of Dulbecco’s medium (Gibco Labs., Grand Island, NY) and centrifuging at 500 X g for 10 mm. Microscopic examination revealed that the spermatozoa were almost free of contamination with epiwas washed

left for for 5 mm (low-salt wash) were desalted over a G-25 pellets were gently

equal

589

SPERMATOZOA

perature,

containing

PMSF.

Isolation

AND

and

SDS

to the

[29].

same

In the number

buffer before the final extracwashings were done to allow caused extraction

pellet suspension

in

by the process was started

2 ml

of

Tyrode’s

was

then

divided

Electrophoresis was (15 x 13 x 0.15 cm) amide gradients using

Gels

were

macia-LKB,

performed consisting the buffer

calibrated

with

Piscataway,

NY)

in denaturing slab gels of linear 10-15% acrylsystem of Laemmli [30]. a commercial mixture (Pharof proteins spanning the range

from 12 kDa to 78 kDa. Gels were Coomassie Brilliant Blue R250 dye water (1:3:6) and destained in the

stained

with

in acetic acid: same solution

0.1%

(v/v)

methanol: with dye

solution

omitted. The gels were impregnated according to the procedure of Skinner and Griswold [31], dried, and then exposed at -70#{176}Cto Kodak X-omat AR-5 film (Eastman Ko-

into

dak,

of washby resustwo

Rochester,

NY).

VREEBURG

590

TABLE 1. Quantity of radiolabeled the luminal fluid and tissue of the of the control side.

RESULTS Protein

Synthesis

and

Secretion

In Vivo and

ET AL.

In Vitro

proteins (cpm/mg tissue)’ present in injected side and in the luminal fluid

Control

Injecte d side

The

quantities

of in vivo

labeled

proteins

present

lummnal fluid fraction and the tissue fraction are Table 1. As can be seen, [35S]methionine injection in the presence of almost 10 times more labeled in the

control proteins

fluid

fraction

side.

This

of the

indicates

originated

the first

passage

Seven

hours

from

[35S]methionine

into

that

estimated

been

90%

proteins

in that

of the taken

of the

labeled

up

during

the

Region

Tissue

1

9628

1153 7302 ± 1895 6727 ± 1622 2626 ± 770 radiolabeled

4

5 ‘Proteins

of

750

were

9578

±

1585

1027 1194 1071

8670

±

2219

961

±

3080 retrograde

±

1050

579

±

1028b*

±

8803

Fluid

Fluid

2 3

±

by

8936

±

1026”

12482

±

1469

into the testicular

are the mean

±

SEM

± ± ±

78* 172 172 219 126

injection of

135S)methionine “Values

injection

750

p.Ci

of

artery.

for 4* or 8**

animals.

.tCi

testicular

artery, 5-10 x 10 cpm into soluble epididymal Taking into account a 70%

label and an 80% recovery of the injected [35S]methionine incorporated

into

proteins. From these proteins, 40-50% tissue fraction and 50-60% in the luminal

ble

than

given in resulted

the organ.

efficiency of the about 0.5-1.0% to have

about

retrograde

of methionine were incorporated proteins at the injected side. counting proteins,

side

[35S]methionine

through after

injected

Side

in the

of the is

TCA-precipitable were found in the fluid fraction (Ta-

When

and

tissue

present barely

in the detectable

the lummnal files

decided

fluid

fluid fraction were not detectable or only in tissue. Since the profile of proteins in

fluid

published

ididymal

1).

the profiles of labeled proteins present in the fluid were compared (Fig. 1), many of the proteins

fraction as the

to compare regions

after

result

was strikingly

different

of in vitro

studies

tissue

66-’ 45-4

30-’

17-’

FIG. 1. Regional distribution after retrograde injection of 750 regions as illustrated.

12345

of radiolabeled secretory proteins present in fluid Ci InSimethionine into the testicular artery. The

prowe

the protein patterns of the various administration of [35S]methionine

kDa

12345

from [28,32],

and tissue epididymis

from was

regions divided

1-5, into

7 h five

epin

EPIDIDYMAL

SECRETORY

PROTEINS

SPERMATOZOA

AND

in vivo

591

in vitro

kDa

66-’

45-’

30-’

17-’

123412345 FIG.

2.

Radiolabeled

proteins

present

in

luminal

(“Slmethionine into the testicular artery or released epididymal tissue from regions 1-5. The arrowhead 34 kDa.

vivo

with

those

epididymal many

obtained

tissue

secretory

with

by an in vitro [35S]methionine.

proteins

migrating

incubation

with

Figure similar

fluid into the indicates

of minced 2 shows that electropho-

in vivo and in vitro labeling with isotope. There were, however, some clear differences. First, a major protein of 27 kDa was present in segment 1 in vivo, but not in vitro. Second, a protein of about 34 kDa was secreted by all epididymal segments in vitro. In vivo, this protein was cleanly absent from segment 1, but a protein with a similar electrophoretic mobility appeared in the more distal epididymal segments. retic

mobility

Binding

were

of Epididymal

present

after

Secretoiy

both

Proteins

to Spermatozoa

from

regions

spermatozoa timated

associated

extracted didymidis

1, the

number

of spermatozoa

present

in the

spermatozoa. gel

injection

of

with minced are present at

luminal

fluid,

can

it

be es-

1% of the secretory proteins were To examine more specifically

proteins

were

profiles

were

with SDS. Figure of the rat, only

bound

to spermatozoa,

made

3 shows that a few proteins

of sperm

proteins

in the were

caput epiassociated

with spermatozoa. While in segment I no major binding proteins could be observed, in segment 2 a protein of 47 kDa was bound to spermatozoa. In segment 3, two additional proteins of 100 kDa (estimation) and 33 kDa were found to bind to spermatozoa. In segment 4, five major proteins

the

In segment

for a reliable estimation. From the numbers and the quantities of proteins bound to

with

electrophoretic

37, respectively.

was too low of spermatozoa

retrograde

approximately

epididymal

Among

quantities

7 h after

or that

which

of epididymal secretory proteins associated with Percoll-punified spermatozoa (cpm/106 spermatozoa, mean ± SEM of five experiments) in segments 2, 3, 4, and 5 were 391 ± 63, 393 ± 47, 514 ± 117, and 92 ±

The

1-4,

medium during a 5-h in vitro incubation a protein of 27 kDa. The small arrows

were

found

to be

associated

seen

them were the two proteins in segment 3 and three new

kDa,

and

esting

25

since

kDa.

sperm-associated

in luminal

binding

The

this protein fluid

characteristics

spermatozoa.

25-kDa

protein

is especially

is one

of the most

prominent

epididymal only

with

of 100 kDa and 47 kDa proteins of 54 kDa, 30

in low

proteins,

amounts.

of this

protein

yet

The

it is present

prominent

became

interamong

even

spermmore

VREEBURG

592

S

F

S

F

S

ET

AL.

F

S

F

S

F

kDa

66-.

45-.

I

a

30-’

17-.

1

2

FIG. 3. Radiolabeled secretory proteins associated with spermatozoa of 36Sjmethionine. Arrowheads indicate major sperm-associated proteins,

3

4

(5) or present in luminal described in the text.

evident when was investigated

kDa 66-.

five epididymal

regions,

the

the

with

caudal

Sequential

Extraction characterize Percoll-purified

25-kDa

protein

was

and times)

SDS

protein

of Spermatozoa which

type of spermatozoa

(experimental and

major

spermatozoa.

binding from

pooled, split into two fractions, and with either low-salt buffer, high-salt

(three

injection

secretory proteins of isotope (Fig.

time,

X-100,

7 h after

binding of epididymal 5 days after administration

associated

4 were tracted

30-’

the

(F) from

4). At this

To volved,

45-’

fluid

5

SDS

series),

(control

periments, 38 X 106 and tracted. The total quantities

series).

might segments

be in3 and

sequentially buffer,

exTriton

on with

low-salt

In two

buffer

separate

48 X 106 spermatozoa of extracted radiolabeled

ex-

were exsperm-

associated proteins were 439 cpm/’106 spermatozoa and 501 cpm/106 spermatozoa, respectively. Of the sperm-associated proteins, about one third were present in the first low-

17-.

salt buffer extract (Table 2). Subsequent extraction with a high-salt buffer resulted in removal of 6-7% of labeled proteins. This was similar to the amount removed by the lowsalt buffer wash in the control. The third extraction, carried out with Triton X-100, however, removed almost half of the labeled proteins originally present in the sperm fraction, whereas in the control spermatozoa washed for the third

4 from

FIG. 4. Radiolabeled epididymal regions

[Slmethionine.

Arrowhead

5

secretory proteins 4 and 5, 5 days indicates

associated with spermatozoa after injection of 750 1&Ci of

a protein

of 25 kDa.

time

with

low-salt

buffer

only

extracted. The fInal extractions 10-18% of the labeled proteins and approximately 50% in the

3-6%

of the

label

could

be

with SDS removed a further in the experimental series control series. These results

EPIDIDYMAL TABLE from

2.

Percentage

Percoll-purified

of radiolabeled

proteins

SECRETORY

sequentially

extracted

was

Exper iment

Low salt I-sigh salt Triton X-100 SDS

32% 7% 44%

1

Experi ment

(38%)’ (8%) (6%) (47%)

18%

37% 6% 47% 10%

X-100, and SDS. in parentheses represent by control extractions with

the percentage low-salt buffer

2 (33w) (8%) (3%) (56%)

‘Spermatozoa from segments 3 and 4 were pooled. bproteins were sequentially extracted with low-salt buffer,

AND

in the

spermatozoa.’

Extraction”

Triton ‘Values tained SDS.

PROTEINS

high-salt

obby

luminal

fluid

administered.

593

fraction

within

At 3 h, all

1 h after

proteins

[35S]methionine

appeared

to be

se-

creted. Some proteins, however, disappeared after their secretion. In segment 1, for example (Fig. 7), proteins of 33 kDa and 35 kDa were present at 1 and 3 h, respectively, but could not [35S]methionine.

buffer,

of labeled proteins (three times) followed

SPERMATOZOA

Shortly associated

be

detected

after

their

with

spermatozoa.

7

secretion,

h

after

the

injection

the sperm-binding In segment

the 30-kDa proteins were found tozoa at 3 h, but not at 1 h (Fig.

to be 6).

4, the

of

proteins 25-kDa

bound

and

to sperma-

DISCUSSION demonstrate ated proteins

that about 40% of the labeled were loosely bound. However,

half of the sperm-associated requiring solubilization X-100

or

with

the

SDS

to be

low-salt

tained

of the

extracted.

buffer,

sufficient

proteins sperm

of

the

extracts

X-100,

or SDS

labeled

proteins

The

It has

were firmly bound, membrane by Triton

Only

Triton

amounts

sperm-associapproximately

obtained

(control)

further

analysis

by gel

electrophoresis.

low-salt

buffer

resulted

ticularly protein

in the profiles

range above 30 kDa (Fig. 5). Analysis of the of the Triton X-100 extracts revealed the

in a large

protein

con-

for

number

extraction

with

of proteins,

par-

presence of a major band of 25 kDa and two bands of 30 kDa and 32 kDa. Similar protein also visible in the SDS extract protein, which tightly bound arated

from

the

more

loosely

results Brilliant of

of

well

established

that

bind to spermatozoa Evidence for this

specific

epididymal

as they migrate process is based

PAGE of membrane proteins using Blue dye or silver staining detection,

membrane

proteins

in

intact

membrane fractions or in extracts; or polyclonal antisera elicited to

spermatozoa,

se-

through upon the

Coomassie or labeling in

sperm

or by using monoclonal specific sperm proteins

kDa

barely visible bands were

of the controls. to spermatozoa,

abundant

been

cretory proteins the epididymis.

The 32-kDa was just sep-

bound

33-kDa

pro-

60-’

tein.

Secretion

and

The

Binding

profiles

binding

of Proteins

of epididymal

to spermatozoa

were

and 24 h after administering tained in segment 4 are most prominent associations spermatozoa most striking

with

Time

secretory

proteins

investigated

and

their

labeled methionine. Data given since in this segment between secretory proteins

took place. As can be changes in the profile

45-’

at 1 h, 3 h, 7 h, obthe and

30-’

seen in Figure 6, the of secretory proteins

occurred between I h and 3 h after injection. For instance, at 1 h, a protein with of more than 80 kDa was completely absent, and another protein of 34 kDa was present in very low

concentrations.

At 3 h, the

concentration

teins was markedly increased. The 34-kDa ever, seemed to be replaced by proteins with molecular mass at 7 h and 24 h. This might

of both

protein, howa slightly lower have been due

to intraluminal modification of the 34-kDa protein, or to simultaneous transport of proteins with a lower molecular mass into segment 4 and movement of the 34-kDa protein out mal

of segment 4. Transport of proteins from more proxiregions was clearly demonstrated by the appearance of

a 20-kDa isotope.

protein in segment 4 at 24 h after injection of This protein was a major secretory protein in the

caput

epididymidis.

other

segments

Analysis confirmed

of the that

many

protein proteins

17-’

pro-

profiles were

of the present

LF FIG.

Radiolabeled

T

SDS

proteins present in luminal fluid (LF) or of segments 3 and 4. Spermatozoa were either extracted with a low-salt buffer, high-salt buffer, Triton X-100 and SDS; or with a low-salt buffer (three times) followed by SDS. Only the first extract with low-salt buffer (LS), and the extracts with Triton X-100 (T) in the experimental series and SDS in the control series contained enough labeled protein for further analysis. Arrowheads pointing toward lane LS indicate proteins of 54 kDa, 47 kDa, and 33 kDa, extracted with low-salt buffer. Arrows pointing toward lane T indicate minor proteins of 30 kDa and 32 kDa, while the arrowhead indicates the major 25-kDa protein. associated

5.

LS

with

spermatozoa

secretory

VREEBURG

594

ET

AL.

fluid

sperm

kDa

I L‘

* .I, a-

66-’ 45-,

30-’

i

17-. 1

3

7

24

1

3

7

24

FIG. 6. Radiolabeled secretory proteins present in the luminal fluid or associated with spermatozoa from segment 4, at 1, 3, 7, or 24 h after lSlmethionine injection. Arrowheads indicate proteins of 34 kDa and of more than 80 kDa in the luminal fluid 3 h after labeling; a protein of 20 kDa is present at 24 h. Sperm-associated proteins of 25 kDa and 28 kDa are indicated by arrowheads; those of 47 kDa and 54 kDa are indicated by stripes.

(for has

reviews see the potential

[1,331). None of these methods, however, to follow the total process of epididymal

eral proteins creted. More

need more time intriguing, however,

protein secretion and the subsequent selective binding between some of these secretory proteins and spermatozoa. Recently, we have attempted successfully to address these

teins, for example the segment 1, disappeared cretion. It is likely that

questions

down and/or taken up removal by transportation

using

the

technique

[35S]methionine into the limitation of this approach isotope study,

appearing we directly

of

in epididymal back-perfused

proteins

taken up represents labeled istration.

that

about was

about

during the first passage a substantial increase

90% through in the

proteins recovered compared Just as in mice, more than

teins were found The synthesis to be

and

a fast

tration presence fraction agreement

vivo

injection

of

of mice [20, 27]. is the low amount

proteins. isotope

ma! artery of rats. We found that injected quantity of [35S]methionine epididymal

in

jugular vein in the rat

The of

In the present into the epididy0.5-1.0% incorporated of this

of

amount

34-kDa

was

tro

to peripheral admin50% of the labeled pro-

in the luminal fluid fraction [27]. and secretion of proteins in vitro is known

process,

beginning

of [35S]methionine of various

secretory

at about

[28,34].

Our

proteins

1 h after injection of labeled with these in vitro data, but

1 h after adminisdata showing the in the luminal fluid

methionine also

show

are in that sev-

be synthesized is the fact that

and sesome pro-

35-kDa and the 33-kDa proteins in within a few hours after their sethese proteins were rapidly broken since a period of 2-4 h for their seems too short [35]. Moreover,

there is no evidence for their appearance in segment possible synthesis of proteins of such molecular the proximal epididymis is quite conceivable since

the into

the organ. This amount of radio-

to

proteins

5-h incubation survival

teins and

were

abundantly

period. of these

present

A possible proteins

at the

explanation might

be

that,

end

Alternatively, undergone

in vitro,

responsible between

for their in vivo and

breakdown. in vitro data

concerns the absence of a 27-kDa protein in vitro ment 1. We have no explanation for this difference.

be

it demonstrates that with our in vivo method information about epididymal protein secretion

obtained.

pro-

secretion influ-

it is possible that the extensive washing by tissue in the in vitro incubation stud-

ies removes components Another striking difference

theless, ditional

of the

for the in vi-

leave the tubular fragments soon after their thus are no longer accessible to intraluminal

ences. process

2. The mass in in vitro

in segNeveradcan

EPIDIDYMAL

SECRETORY

PROTEINS

kDa

AND

teins after

SPERMATOZOA

595

of 25 kDa, 30 kDa, and washing with a high-salt

binding

proteins,

tein, may coprotein

the

32 kDa remained associated buffer. Two of these sperm-

32-kDa

protein

protein

DE [2, 3] is 32 kDa.

66-’

protein in the caput, mains associated with pacitating media [14].

45-.

fertilizing [5] have ponents

kDa

This

7

13

ment

protein

ISlmethionine. proteins

fluid

Arrowhead

of 33 kDa and

Comparison and those

indicates

27-kDa

protein.

Small

arrows

indicate

35 koa.

secretory proteins with spermatozoa

in the luminal showed that

only a few proteins bound to spermatozoa in the caput epididymidis. In segments 2 and 3, a 47-kDa and a 33-kDa protein were clearly bound to sperm. In contrast to the 33kDa

protein,

the

counterpart

of the

47-kDa

protein

could

not be found in the luminal fluid. all the secreted 47-kDa protein

The reason might be that binds to spermatozoa or

that the 47-kDa protein is derived weight form that is trimmed upon

from a higher molecular attachment to the sperm

surface. Such a process has been described for a mouse sperm surface antigen (SMA 4) that is secreted as an 85kDa protein but is modified to a 54-kDa form upon binding to the spermatozoa [36]. In the corpus epididymidis (segment 4), more proteins were found to be associated with the spermatozoa; among them, five proteins of 100 kDa, 54

salt

buffer.

Of the

in segments

various

3 and

proteins 4, only

atpro-

in the

study

belong

epididymal

to

the

fluid.

Re-

that

only

a small

per-

in the present study may be similar to a 23-kDa recognized by the 2D6 monoclonal antibody deby Jones et a!. [33,38,39] and/or the 26-kDa mat-

uration become

antigen bound

corpus

was

MEP 7 is tightly bound. the major sperm-binding

described by Olson et al. [23]: both to spermatozoa during their passage

epididymidis,

only

detergents

are

proteins through

effective

in

abundant

the 21)6 protein, and the 2D6 protein is more on spermatozoa than in epididymal fluid. More-

over, our spermatozoa and their

25-kDa protein remains during their transport storage in the cauda,

by the

2D6

firmly associated with the through the epididymis a characteristic also dis-

antigen.

Besides the above-mentioned proteins, several others have been reported to bind tightly to spermatozoa. Sulfated glycoprotein-2 (SGP-2) is a major protein secreted by Sertoli cells and reported

epididymal cells in culture [40]. SGP-2 has been to bind to spermatozoa in the testis and to dis-

sociate from spermatozoa in the and be replaced by an epididymal The

epididymal

fide-linked

form

monomers

rete testis and efferent ducts form more distally [16,41].

of SGP-2,

which

of 40 kDa

and

consists 29 kDa,

of 2 disulbinds

to sper-

matozoa in the proximal epididymis and remains associated with spermatozoa despite extensive washing (six times) with PBS [18,40]. This suggests a tight binding of SGP-2 to spermatozoa. However, after in vivo metabolic labeling, we were unable to detect binding of SGP-2-like monomers to spermatozoa in the caput epididymidis. This discrepancy difficult to explain since we could clearly demonstrate binding of SGP-2 to testicular spermatozoa after in vivo

in high reported

isotonic

present

that 33-

protein antigen scribed

tion

to the spermatozoa

One may speculate the loosely bound

It is interesting

sperm-associated protein that

beling with [35S]methionine sults). Another discrepancy ing of a secretory glycoprotein

in an

secretory

that the 32-kDa rat protein present related to the mouse epididymal

kDa, 47 kDa, 30 kDa, and 25 kDa were clearly present. Most of the sperm-associated proteins, however, seemed to be loosely attached since they could be removed by incuba-

tached

is a major

of the 25-kDa

played

of labeled associated

and

present

7 [29].

MEP

solubilizing

FIG. 7. Radiolabeled proteins present in the luminal fluid from seg1 of the rat epididymis at 1, 3, and 7 h after injection of 750 Ci

pro-

centage The

the

hours

in the

complex

has been found fluid is probably

it

protein

17-’

observed

protein

cently, in caudal

25-kDa

ability of the spermatozoa [37]. Olson and Hinton suggested that protein DE consists of various comof different charge and size and that only some of

protein

same

the

binds to caput spermatozoa, and respermatozoa during incubation in caAntibodies to this protein inhibit the

these variants bind to spermatozoa. the tightly bound 32-kDa protein

30-’

and

be similar to previously identified proteins. A glyknown variously as AEG [8], protein IV [32], or

quantities to bind

is la-

(Vreeburg, unpublished reconcerns the absence of bindof 17 kDa, which is secreted

in the caput epididymidis in high amounts to testicular

and has been spermatozoa

596 [24]. tein

This

difference

does

not

could

possess

position. 20 kDa

In the secreted

protein

to spermatozoa,

basis of mulation

be

explained

methionine

present in the

study, caput

sylated

[28]

binding

and

however,

been

pro-

5. Olson

17-kDa

was

site not

not

found.

of secretion, shown), we

reported

com-

a major protein of Binding of this

in our studies is similar unlike the 17-kDa protein, has

ET AL.

in its amino-acid

we found epididymidis.

its molecular mass, in caudal fluid (data

the 20-kDa protein [42]. This protein,

if the

VREEBURG

to be

On

and assume

the accuthat

to protein BC is not glycoa retinoic

acid-

protein

[43,44]. binding of secretory epididymal has also been studied in vitro

The matozoa

epididymal tubules dymal spermatozoa In genera!, these

proteins by culturing

to sperwhole

or by incubating testicular and epidiwith labeled epididymal proteins [19,26]. studies show more sperm-binding pro-

teins than were found in the present investigation, in particular in the caput epididymidis. One explanation for this difference might be that in these studies the sperm-washing procedure of the spermatozoa was not sufficiently effective to remove all loosely bound proteins from the sperm surface: the incubation time in the washing buffer might have been

too

and/or

short

is supported trifugation in buffer

at room

could

extracted

be

ditional

the

buffer

too

by the present data, of spermatozoa through temperature,

loosely

by incubation

explanation

might

cold.

that

our

ponents studies

was eliminated. is that secretory

Another proteins

can

buffer.

sperm

by mincing epididymal tissue, gradients. In this way, contamination

binding,

proteins

in isotonic

be

possibility

that after cenwashing them

bound

obtained Percoll

spermatozoa

This

which show Percoll and

purified by by tissue com-

of

the in associate

vitro with

[25, 26]. Definitive conclusions about in vivo cannot be drawn from these studies since differ in composition from the intraluminal fluid.

however,

media

used

Moreover, the interactions between proteins and tozoa in vivo occur in a highly ordered sequential In conclusion, in vivo labeling teins

after

matozoa, only

An ad-

preparations,

were

finding rapidly

still

secretion, can

a limited

matozoa 3 h after

the present of epididymal be

study describes proteins. The

especially

whether

established.

number

and that a high their secretion.

It was

of proteins level

they found bound

of binding

was

spermafashion.

a method fate of the bind that firmly reached

for pro-

to sperin the

rat

to sperwithin

EM.

The

of Reproduction.

spermatozoon. New

York:

In: Knobil Raven

Press;

E, Neil 1988:

1985;

differences

fluid polypeptides

of the

gel electrophoresis.

J An-

in luminal

by two-dimensional

revealed

6:20-34.

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