BIOLOGY

OF

REPRODUCTION

20,

247

-252

(1979)

Sperm Forward Motility Distribution and Species

Tissue

TED

S. ACOTT,

HOWARD

Protein: Cross Reactivity

J.

DEBORAH and

BRANDT

Reproductive Oregon Regional

DALE

JOHNSON, D.

HOSKINS

Physiology Section, Primate Research Center,

Beaverton,

Oregon

97005

ABSTRACT Spermatozoa from the bovine caput epididymidis develop progressive motility when they are incubated with a cyclic AMP phosphodiesterase inhibitor and bovine seminal plasma. Using a photomicrographic motility assay, we have determined the distribution of the factor (or factors) that produce this motility in a variety of bovine fluids and tissues. We have shown that the factor is present almost exclusively in reproductive tract fluids. The species cross reactivity of the seminal plasma motility factor was also studied. Bovine caput sperm were stimulated to high levels of forward motility by the seminal plasma of all the mammalian species tested, including monkey, man, dog, rabbit, pig and elephant. This species cross reactivity was also apparent in the ability of bovine seminal plasma to produce forward motility in the caput sperm from a number of mammals, including dog, cat, guinea pig, rabbit, bear and monkey. We suggest, on the basis of these data, that the mechanism of development of the capacity for progressive motility of sperm, which occurs during epididymal transit, may depend upon at least two factors, i.e., increased changes in intrasperm cyclic AMP levels and the binding of a forward motility protein and that these elements are common to many mammalian species.

INTRODUCTION In the

many

mammalian

capacity

their

et

al.,

of

this

progressive

washed midis

from

respond

to

lar,

motion

twitching (Hoskins

et

epididymal

or

elevations

in

coupled al., now in

without

al.,

1975).

cAMP forward

Hoskins

that

at

least

of

the

two

are

named

devoid forward

Forward

The

discovery

of

part cell

of

Motility

This Protein

this

protein

motility

et

1976,

1974).

We

factor lar

difference

which

has

1978;

weight

which

et

on

is

al.,

the

of

sperm

caput

to

Hoskins, July June

here,

28, 1978. 6, 1978.

the

247

we

used

distribution

Acott,

have

in

the

1976).

and bound

postulated

the

that

forward

in

as the

sperm

(Brandt the

various

from

(Acott

experiments

and

reported assay

bovine

develop-

move

epididymis the

this

motility

epididymis

photographic in

The molecu-

equilibrium

called

caudal In

that

Hoskins,

as several

is involved

motility

1976).

and

charac-

covalently

have

and

the

plasma

aggregate

produced

1978a,b)

forward

seminal

contain

we

improved

and

exists

in to

an

purified

and

We

protein,

ment

Accepted Received

species

to

both

sperm

(Acott

that

appears

us

develop with

for

bovine

Hoskins

led

1975).

motility

is a protein

protein, based

in

is by

phosphodiesterase

partially

factor

carbohydrate.

been

FMP. was

incubated

described

have

this

that

caput

is respon-

protein

when

assay

the

initiates

(Garbers

is that

or

terized

to motion

the

This sperm

recently

and

motion

caput

al.,

an

progression

plasma.

et

is

above

this

bovine

photomicrographic

caudal

that

a cAMP

(Hoskins

motion

from

forward

and

have

1973;

twitching

taken

seminal

motility

We

progres-

al.,

types

a protein,

motion.

of

et

of

plasma

inhibitor

vigorous

moderate

that

seminal

respond

progression

1973a,b;

for

by

and

to

phos-

contrast,

sperm

levels

believe

sible

In

epididymis

converted

a flagel-

cAMP

forward

occluded addition

Lindholmer,

a

sperm

forward

that

and that

by human

demonstrate

epididy-

developing

(Elisson 1974)

displayed

the

the

observed caput

with by

a response

sperm

studying development

the

bovine

ejaculated

with 1971,

While

incubation

inhibitors

1975,

in

we

the

phodiesterase

sion

AMP motility,

sperm

(for

Hamilton,

1975).

cyclic

during

epididymis

1975;

observation

Lindholmer,

develop

motility the

Bedford,

involvement

sperm

progressive

through

see:

Orgebin-Crist of

species,

for

transit

reviews

the

to tissues

study and

ACOTT

248

fluids this

as

well

as

the

species

cross

reactivity

AND

METHODS

of

FMP.

MATERIALS

Bovine and pig seminal plasma were obtained from All West Breeders, Burlington, Washington and dog seminal plasma was obtained from Pel-Freez Biologicals. Rabbit and human seminal plasmas were donated by Dr. N. Alexander, elephant seminal plasma was donated by the Portland Metropolitan Zoo and monkey seminal plasma was obtained as previously described (Mastroianni and Manson, 1963). Ejaculates were first centrifuged (800 X g, 15 mm, 25#{176}C)to sediment sperm and the supernatant fraction recentrifuged (178,000 X g, 1 h, 4#{176}C) to remove high molecular weight debris. Those samples which were heattreated (90#{176} C, 10 mm) were centrifuged two more times (178,000 X g, 1 h, 4#{176}C)for removal of the gelatinous material produced. All samples were dialyzed against assay buffer (0.04 M KCI, 0.102 M NaCI, 0.005 M MgSO4, 0.01 M KH2PO4, 0.02 M Hepes IpH 7.51) prior to use. Bovine testes and serum were purchased from local abattoirs; horse, dog and cat testes were donated by Dr. Kenneth McGough and bear testes were donated by the Portland Metropolitan Zoo. Sperm were removed from the caput portion of the epididymis and suspended in 5 ml of assay buffer (25#{176} C) as previously described (Hoskins et al., 1975). Sperm in these suspensions were then washed by centrifugation through 4 volumes of 8% Ficoll in the same buffer. Centrifugation was carried out in two steps, the first at 500 X g for 5 mm and the second at 1,150 X g for 5 mm. The resultant sperm pellet was resuspended in 1 ml of assay buffer and 20 pI aliquots were used in the motility assay. These operations were carried out at room temperature. Bovine liver, heart, adrenal, spinal cord and vitreous humor were purchased from Pel-Freez Biologicals. Tis.sues were disrupted with a Brinkmann sonic disruptor in an equal volume of assay buffer at 4#{176}C, centrifuged (178,000 X g, 1 h, 4#{176}C) and dialyzed with or without prior heat treatment (90#{176}C, 10 mm) and further

centrifugation

were

(178,000

collected

dialyzed

without

with

or

further

centrifugation.

similar

manner

epididymal and

(1942)

containing fluid collected

by

Forward graphic

Hoskins, caput buffer glucose

blue

the the

clot

dye

method as

was

of was previously

retracted.

to

testicular

in a

AL.

buffer. The final sperm concentration sperm/mi. This mixture was incubated mm with gentle agitation. The activated then placed on a thermostated (37#{176} C) slide (0.1 mm depth) and a 5 second oid

photograph

The

forward

taken motility

multiplication of

in

lengths

curves curve,

i.e.,

for

a

each

the

take

the

or

a bovine

or

Henle

completed

linear were

of

then

a

the

compared to

protein. the

of

portion

determine

Protein

method

concen-

of

Lowry

et

(1951).

RESULTS

Tissue

AND

DISCUSSION

Distribution

The

data

caput

in

sperm

in a

of

Each

and

Table

bovine

for

the

tration

from

tration.

fluid

were

Maximum region

are

presented

in

it

since

tion

is

or

the

reflects

the

were

each

curve.

is

and

whether

quite

taken

from

The

data man-

the

concentra-

activity FMI

in

clear

(FMI/pg

attainable

FMP

seminal

on

concen-

cumbersome

maximum

it fluid

concenboth

values

of

(or

(FMIs) of

protein

specific

amount

Nonetheless,

indices

rather

fluid),

protein)

dymal

this

FMPs

tabulated

of

unclear

(FMI/pl

of

concentration

and

FMI

plateau

homogdialyzed

portion

volumes

the

tissue

a wide

linear

and

of

and

motility

the

curves basis

of activity

presence

forward

taken

distribution

heat-treated,

over

Relative

were

ner

fluids was

[FMFs))

range.

the

motility-inducing

sample

assayed

factors’

the

1 show

forward

number

enates.

elute

the fluid

of

FMFs,

that

solution neat was

that

plasma

best

these

fluids.

caudal are

epidi-

rich

sources

that

the

amounts. and

rete

fluid

other

contains

sources

The

specific

seminal

much

contain activities

plasma

are

of

also

much

less

and

only

trace

caudal

fluid

higher

than

(1973).

assessed described

by

those

photomicro(Acott

and

1976, 1978). An aliquot (20 zl) of washed sperm was added to 0.28 ml of 37#{176}Cassay containing 33 mM theophylline, 10 mM Dand 10 mg of bovine serum albumin/mi of

fluid, this

for

is as of

in of

motility factor is used here the FMP in bovine seminal but not elsewhere.

fluid

plasma column,

a

as

seminal

given

volume, of

this We

the

(Brandt “Maximum

previously

al., FMI,”

present

observation

in is

not

demonstrated the

FMF fluid

in caudal

et

fluid

approximately

between

FMP

on

rete

protein

latter

have

differences

Rete

plasma

Since

amount

plasma,

and

active

fluids.

comparison.

the

incongruous. major

nonreproductive

however,

contains, 1/20

the

basis

seminal

The term forward since we have characterized plasma and caudal fluid,

saturation

standard

by

of

Triplicates

the

FMI/g

determined

veloc-

were

curves,

fluid were

average

a normal

homogenate.

plasma

sperm

Concentration

of

from

seminal

FMl/.tl

trations al.

tissue

by

motile measurement

hyperbola,

concentration

with

their by

form

chosen

averaged

obtained

photographs.

rectangular

fluid

was

by

Polar-

illumination.

of

determined

on

concentration,

(FMI)

sperm) as

exposure

field

percentage

tracks/100

which

the

the

nm/second

track

dark

index

of

(number ity

with

was 2.5 X 106 at 37#{176}C for 20 sperm were hemacytometer

Caudal by

sucrose

used

Casillas

and

prepared

described

a 55%

Rete

and

treatment

was

had as

that

tubules.

motility method

heat

serum

collected

except

dextran from

the

was

4#{176}C).Fluids

centrifuged

prior Blood

after

1 h,

dilution,

without

fluid

Zittle

X g,

ET

1978a,b). in

Table

of or

rete

seminal

The 1 demon-

last

SPERM

TABLE

1. Tissue

distribution

FORWARD

of forward

MOTILITY

motility

factors

in bovine

Relative Source

Per

plasma fluid fluid

Seminal

Caudal

249

tissues

and

fluids.

FMIb

MI o f fluida

100 174

PROTEIN

Per

9.6 17 1

g

Maxim

of proteina

100 470 105

630 630 310

30 24 34

Serum Liver

0.52

0.1

0.48

0.09

300

Heart Spinal

0.37 0.00

0.19

0.24

0.12

175

0.04

0.00

0.28

humor

cord

aVolumes homogenates bM

protein

values

(see

Materials

and

one

very

of

these

high

duce

maximum

those

seen

plasma

with

All

FMP. in

considerably

Table

caudal

being

fluids

3

300

51

0.5

0.13

280

22 39

to

the

the

neat,

of

(e.g., allow

activity

any

case,

fluids

ant

question, of

other

ly

also

or

26

supernatants

sperm

of

the

tissue

are

Table

is

nonreproductive Table

show

is surprisingly

a

the

species,

ty

special

the

in

seminal

data

Species

Cross

Seminal

Plasmas

The

extent

several

species

bovine

caput

is

Reactivity:

Various

with to

Bovine which

Caput

shown

in

plasma

forward in

the

Table

from

motility

presence

2.

Species Bovine

Cross Seminal

in the

small.

these

These that

species

FMFs to

diversi-

variation

relatively

of

species

wide

evidence

all

that

pro-

variation

these

the

The

also

those

The

of

nondialyzable

plasma.

produces to

in

light

in either

found

the

contain

with

at in

least bovine

Sperm

seminal

stimulates sperm

of

bovine

shown plasma,

near

protein

is

the

tissues

plasma.

in

identity

seminal

studied

significantly

treatment,

convincing

stable,

functional

species

seminal

patterns.

fluid

plasma

heat

activity.

the

by

very

of

provide

possibly

and

seminal

reproductive

seminal

particular-

of

and

values

the

small of

the

these

all

heat

FMI/mg

FMI/pl

import-

of

values

bovine

in

for

mechanisms

produced

after

by

evidence

FMI

cases,

FMI

epididy-

equally

all

or

duced

maximum

species.

those

In

maximal

produced

The

from

fluids

1.

values

those

the

plasma

than

before

clearly

sperm

that

FM!

to

provide

maximum

higher

the

2)

the

studied

itself.

between

mammalian

present

species factors

equal

plasma

seminal

predom-

whether

FMP

all The

the

not

that

maximum

are

Table

of

caudal

An

mammalian

similarity

produce

completely.

suggests bull.

(see

is found and

the

FMls

remains studying

1

or

(maxiare

clear

nondialyzable

seminal

basic

is

of

eliciting

bovine

activities) It

all

with plateau

(specific

stable,

approach

from

the

slope

of

bovine

the

observed

of

heat

activation

motil-

pattern although

identical.

capable

by

radioummunoassay)

activity

contains

of

question

in

then,

plasma man,

in

property

tissues,

observation

FMP

the

of

plasma

This the

of

which

and

plasma

that

not

minimal

this

data

are

produce

or

resolve

assay

which

and

FMI)

contain

data,

and

chemical

seminal

fluid. for

the

mum

are

seminal

factors

and

motility

in

these

the

plasma,

seminal FMI

and

follow

quantitatively

seminal

plasma

from fluid

methods

the

forward

inantly

of

many

fluids

a

seminal

induction

factors,

us to

maximum

than

on

to

Alternative

FMP

seminal

homogenates

show

nature

i.e.,

body

unclear.

curves

pro-

50%

or

and

caudal

complex

of

other

than

fluid

effects

these

amounts

phylline

71 2.6

nonheat-treated

even to

FMP.

susceptible

nature

fluid,

unable

conclusion only

The

is

role

other

Our

measured,

ity,

mal

39

12.9

greater

caudal

nonspecific

surprising.

values

lower

contain

The

that

53

260

Rete

the

is that

plasma

In

350

5.4

section).

is

1 similarly

fluid.

therefore,

will

5.3

17.9

Methods

differences.

FMI

values

in

21.2

0.19 0.2 0.39

correspond

concentrations,

shown

or

0.09

± SEM.

strates at

and

FMIb

9.6 46 19

5.6 0.35 0.64 0.86 1.5

Rete

Testis Vitreous Adrenal

um

of

Concentration

in theo-

and

the The

Sperm observation

Reactivity Plasma of

Several

that

Species the

seminal

plasma

of

250

ACOTFETAL.

TABLE

2. Stimulation

of bovine

caput

Heat treatment

Source

Bovine

Monkey

660

26

1.40

0.11

25.50

721

48

1.80

0.18

68.9 46.1

5.2 4.6

7.30 4.80

725 622

41 13

3.70 5.80

0.17 0.08

27.2 28.0

1.20 0.05

34.40 0.60

592 384

71 132

0.04 5.00

0.03 1.25

13.2 3.0

1.10 0.75

8.90 2.70

530 473

28 36

1.80 3.50

0.12 0.60

16.2 9.6

3.80 1.60

53.3

-

22.00

34

421

28.8

570

19

2.40

0.10

449

56

1.40

0.12

-

2.14

495

38

3.60

+

1.39

613

12

12.70

species

could the

which

bovine

seminal

activate

the

mammalian

sperm This

bull

in the

is

any

of

this

TABLE

3. Activation

that to

study

are

of caput

data

is

used

to

produce

other suscep-

plasma the

FMI

0.29

0.29

7.8

0.90

2.40

17.4

3.30

the

FMF-sperm species.

compiled

in

from

it

The Table

several

for

bovine are

3.

species

source

Bull Dog Cat Guinea Rabbit Bear Monkey

Maximum

pig

aMe

sperm

from

is noteworthy

produce twitching,

of bovine

FMIs

approaching

sperm. strictly

because

of the

that

the

various bovine

forward

vigorous

FMI/Mg

% motile

These

wide species. seminal

plasma

motility

in caput

can these sperm.

plasma.

(gm/sec) Literature

Caput

5.97

40

88

12Oc

18

42

45C

450

10

4

3.70

14

60

520 330

52 23

3.3 10

5.80 0.70

23 11

46 38

4.40

16

50

3.30

19

54

6.7

in

However,

3.60

83

across

differences

5

25

to

maximum

4

109

able those

30 49

577

(ref.)b

ooc 20_35c

± SEM.

are for

cNelson

ejaculated

sperm.

L. (1975).

dDetermmned

used

FMIa

these

is

comparable

theophylline-stimulated

seminal

from

630 500

790

bValues

was

BSP #{174} #{189} max.

lines

drawn plasma

caput not

be

seminal

M1 of

Sperm

2.10

3.3

to

bovine

values

species

and

conclusion that

maximum

found

the

uniquely

many

sperm

first

in

eliminate

seminal

idea

The

study

several

from

are

common

results

caput

was

would

sperm

factors

reinforce

bovine

plasma

caput

the

interaction

activate complementary

species. that

would

359

1.90

2.40

suggested

to

0.57

+

sperm

tible

5.97

27.50

SEM.

!

possibility

30 21

FMI/Mla

48.60

+

several

FMI/Mga

+

-

aM

FMla

-

+

Elephant

Maximum

630 630

-

Pig

plasma.

60.10 15.00

+

Rabbit

seminal

+

-

Dog

by

protein

Lowry (mg/mi)

-

Man

sperm

as the

with

diluant.

photographic

assay

(see

Materials

and

Methods

section)

in which

monkey

seminal

plasma

SPERM

The

last

other and

it

clear in

sperm the

are

pig

ejaculated

to

are

that,

minimized.

average

and

they

are

caput

percentage sperm shown

would

be

The

expected

3G motile

sperm

shown

either

the

motion most

twitching The

FMP

flagellar

Casillas, 1975).

In

every

and

when

caput

studied,

when

the

1974,

sperm

were strong

with

good

theophyl-

forward

motility

theophylline

and

synergistic

The

to

The

be

effect

common

conservation of

of

FMP

man

and

and

tissues

FMP

across its

as

bo-

Forward

Motility

biological

event,

understood. indirectly

sulfhydral

a

ion

production reproductive

We whereby

are the

detailed

of

the

tract.

The and

is

membrane

ATPase

activity,

distribution currently FMP

or

itself

some

studying exerts

its

within

the

Oregon

male

BioI.Chem.

253,

6744-6750.

fluid

protein(s).

10th

Annual

Meeting

the Society for the Study of Reproduction. p. 29 (Abstr.). Casillas, E. R. (1973). Accumulation of carnitine by bovine spermatozoa during maturation in the epididymis. J. Biol. Chem. 248, 8227-823 2. Eliasson, R. and Lindholmer, C. H. (1973). Effects of human seminal plasma on sperm survival and transport. In: Transport, survie, et pourvoir fecondont des spermatozoids. (E.S.E. Hafez and C. G. Thibalt, eds.). Inserm, Paris. pp. 219-230. Garbers, D. L., Lust, W. D., First, N. L. and Lardy, H. A. (1971). Effects of phosphodiesterase inhibitors and cyclic nucleotides on sperm respiration and motility. Biochemistry 19, 1825-1831. Garbers, D. L., First, N. L., Gorman, G. K. and Lardy. H. A. (1973). The effects of cyclic nucleotide phosphodiesterase inhibitors on ejaculated porcine spermatozoa metabolism and fertility. Biol. Reprod. 8, 599-606. Garbers, D. L., First, N. L. and Lardy, H. A. (1973). of

The stimulation of bovine epididymal sperm metabolism by cyclic nucleotide phosphodiesterase inhibitors. Biol. Reprod. 8, 589-598. Hamilton, D. W. (1975). Structure and function of the epithelium lining, the ductuli efferente, ductus epididymidis and ductus deferens in the rat. In: Handbook of Physiology. Sect. 7. Vol. 5. American Physiological Society, Washington, D. C. pp. 259-303.

Henle, the

characterization.J. J. M. (1975). of spermatozoa of Physiology,Sect.

epididymal

the effects

of

understanding FMP

not

directly

act in

the

complex

motility, may

changes

bonding,

more

epididymis.

sperm

fluidity,

the

maturation

of cAMP in this

substances

through

fluids that

in the

the

Protein

mechanism.

mechanism and

transit

of

including

contention

involvement

permeability

characterislines

role

i.e.,

FMP

studied.

nonreproductive

our

These

and

species

species in

they

the

cAMP

the

functional

important

of sperm nature of

other

the

absence

an

all

these

reinforce

plays

of

to

the

Maturation, transport, and fate in the epididymis. In: Handbook 7. Vol. 5. American Physiological Society, Washington, D.C. pp. 303-317. Brandt, H., Acott, T. S., Johnson, D. J. and Hoskins, D. D. (1978a). Evidence for an epididymal origin of bovine sperm forward motility protein. Biol. Reprod. 19, 830-835. Brandt, H., Acott, T. S., Johnson, D. J. and Hoskins, D. D. (1978b). The initiation of forward motility in bovine caput spermatozoa by rete testicular and

and

al.,

exhibited

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with

sperm

(Hoskins et

washed,

exhibited

values

FMP.

appears

or

twitching caput

Hoskins

species

incubated

vine

a levels.

low

transit

being

activity

line

the

motile

1975;

on

displaying

The

of

is

there FMP

cAMP

converts

epididymal

after

flagellar

sperm

motility.

1974,

immotile

that

Bedford,

have

theophylline-stimulated

forward

during

we

plasma

of

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system

1975)

seminal

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for

al.,

This

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since

of

T. S. and Hoskins, D. D. (1976). Characterization of factors which initiate forward motility in sperm from the bovine caput epididymidis. Fed. Proc. 35, 1617 (No. 1314). Acott, T. S. and Hoskins, D. D. (1978). Bovine sperm forward motility protein partial purification and

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1010

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monkey less

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This

Regional Primate Research Center. The research described here was supported by National Institutes of Health Grants HD05969, RR00163 and RR05694. We wish to thank Dr. Roy H. Hammerstedt for suggestions and for providing the reproductive tract fluids that were used in the pilot studies. We also wish to thank Miss Beth Jeppesen for her assistance with the manuscript.

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3 are, within

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guinea

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our

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The

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Orgebin-Crist, M.-C., Danzo, B. J. and Davies, J. (1975). Endocrine control of the development and maintenance of sperm fertilizing ability in the epididymis. In: Handbook of Physiology. Sect. 7. Vol. 5. American Physiological Society, Washington, D.C. pp. 3 19-338.

RECOMMENDED

REVIEWS

Hoskins, D. D. and Casillas, E. R. (1975). Function of cyclic nucleotides in mammalian spermatozoa. In: Handbook of Physiology. Sect. 7. Vol. 5. American Physiological Society, Washington, D.C. 453-460. Hoskins, D. D. and Casillas, E. R. (1976). Hormones, second messengers and the mammalian spermatozoa. In: Advances in Sex Hormone Research. (J. A. Thomas and R. L. Singhal, eds.). Vol. 1. University of Paris Press, Baltimore, MD. pp. 284-324. Hoskins, D. D., Brandt, H. and Acott, T. 5. (1978). Initiation epididymis.

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sperm motility in the Fed. Proc. 37, 2534-2542.

mammalian