BIOLOGY

OF REPRODUCTION

Behavior

16, 3 15-321

(1977)

of Nuclei of Testicular, Spermatozoa Injected T. UEHARA Department

and

R. YANAGIMACHI and

ofAnatomy

University

Caput and Cauda Epididymal into Hamster Eggs

Reproductive

Biology,

of Hawaii School of Medicine, Honolulu, Hawaii 96822 ABSTRACT

Isolated nuclei of testicular, caput and cauda epididymal spermatozoa of the hamster were injected into mature unfertilized eggs by micromanipulation. The nuclei decondensed within the egg cytoplasm regardless of the type of spermatozoa used and whether the eggs were activated or not. Decondensed nuclei of testicular and cauda epididymal spermatozoa could transform into pronuclei in activated eggs, whereas those of the vast majority of caput epididymal spermatozoa failed to do so at least within the time period studied (up to 9 h). The failure of nuclei of caput epididymal spermatozoa to develop into pronuclei could be due to the presence of a factor in the nuclei which prevents transformation of decondensed sperm nuclei into pronuclei. This factor might be absent or inactive in the nuclei of testicular spermatozoa and removed or inactivated again by another factor while the spermatozoa reside within the cauda epididymis.

INTRODUCTION

The “ripening” lian spermatozoa tant with physiological

a variety and

spermatozoa 1975).

Among

sperm

mobility tozoa; sperm

The

into

changes

membrane 3)

membrane;

-SS-

spermatozoa didymal ford 1971; vin, the

changes maturation.

and his Bedford

-SS-tion.

associates

et

1974) have spermatozoa

progressively

Accepted Received

al.,

in

bull

studies

during by

epiBed-

(Calvin and 1971; Bedford

and

extensively during

such

October August

stabilized epididymal

a stabilized

condition,

nuclei. These cleavage of complex

Mahi

and

Yana-

study nuclei

was

initiaim-

of

both

mature spermatozoa decondensing and

are equally subsequently

into

injected

pronuclei

when

into

egg cytoplasm. MATERIALS

The was by

AND

METHODS

medium used for handling and culturing the a modified Krebs-Ringer’s solution develBiggers et al. (1971). This medium supple-

mented with 4 mg/mI bovine V; Armour/Reheis) (called paper) had a pH value of

serum

albumin

BWW

medium

(Fraction

this when equilibrated with 5 percent CO2 in air. Mature unfertilized eggs were collected from the oviducts of unbred golden hamsters between 14.5 and 16 h after an injection of human chorionic gonadotrophin, i.e., about 2 to 4 h after ovulation (Yanagimachi, 1969). The cumulus cells surrounding the eggs were removed by treating the eggs for 10-15 min (25#{176}C)in BWW medium containing 0.1 percent bovine testicular hyaluronidase (300 USP units/mg; ICN). The cumulusfree eggs were thoroughly rinsed with BWW medium and kept in fresh BWW medium under mineral oil

Bedford, and Cal-

shown that nuclear proteins of all eutherian mammals

cross-linking Despite

significantly Extensive

protein

and of

sys-

chromatin

1971;

The present whether

ex-

(enzyme

sperm involve

sperm

sug-

possesses

mechanisms

the

Yanagi-

1972),

cytoplasm

well

the egg transform

1961;

Bedford,

Bedford,

1975). determine

transforming

eggs oped

nuclear

in

and

mature capable

composition

egg

decondense could

bonds

gimachi, ted to

the

the

in and

(Austin, 1970;

efficient

(Calvin

and overlying electrophoretic

condensed at the time of spermiation, does not undergo profound morphological changes during epididymal maturation of spermatozoa. According to Gledhill (1971) however, the the

pronuclei

tems?) to mechanisms

modi-

spermatozoa decondense

Noda,

that

tremely

and lipoprotein contents of sperma4) light-scattering properties of the surfaces; and 5) membrane permeability. sperm nucleus, which is already highly

of

fertilizing rapidly

and

gesting

Bedford, are

of

sperm

machi

shape and internal struc2) cohesiveness between

acrosomal plasma

1967;

reported

the

in 1) the size, the acrosome;

outer

nuclei cytoplasm

of mammais concomi-

of morphological as well as biochemical changes in the

(Orgebin-Crist,

fications ture of the

or “maturation” in the epididymis

in are by

maturathe

12, 1976. 17, 1976.

equilibrated 315

with

5 percent

approximately

CO2

in air. Cauda

in

7.5

epididy-

UEHARA

316

AND

YANAGIMACHI

acetic acid 1) overnight and lacmoid in 45 percent acetic as “activated” when 70-100

stained with 0.5 percent acid. Eggs were recorded percent of the cortical granules had disappeared from the egg cortex (determined with live eggs using a phase-contrast microscope) and the second polar body had been extruded.

RESULTS

The not

micrographs of isolated Unsonicated sperm nuclei; e-f, sonicated sperm nuclei (note that the perinuclear material at the rostal end of the sperm nuclei, indicated by arrows, is not present in sonicated sperm nuclei), a and e, Cauda epididymal sperm nuclei; b, caput epididymal sperm nucleus; c, d andf, testiFIG.

1.

sperm

Phase-contrast

a-d,

nuclei.

cular

sperm

nuclei.

Xl ,1 20.

injection

always

jected the

were

collected

epididymal tubule at needle, and suspended epididymides

and

to remove 0.9

blood

percent

several

with

places

puncturing

with

as possible

a pair

of sharp

and

the

a sharp

in 0.9 percent NaCI. were blotted with filter

testes

as much

NaCI

by

Caput paper

minced

scissors.

in

After

repeated agitations for 3-5 mm, the supernate (containing spermatozoa) were filtered through two layers of tissue paper (Kimwipe type 900-S; Kimberly-Clark) to remove tissue debris. Suspensions of the epididymal and testicular spermatozoa thus obtained were homogenized using a tissue grinder with a Teflon pestle and filtered through 8 layers of tissue paper. Most sperm nuclei, not other parts of the sperm, pass through the paper (Figs. ia-d). Some samples of sperm nuclei thus obtained were sonicated in distilled water for 5 mm at 20 KHz and washed twice with 0.9 percent NaCI by centrifugation (1800 X g for 10 mm each). Some of the perinuclear materials were removed by sonication (Figs. ic-f). Sperm nuclei were sedimented by centrifugation (1800 X g for 10 mm), suspended in 0.9 percent NaCI containing 20 percent (w/v)

nuclei

into

some

cases,

out

of

eggs

the

was

was

the

in-

eggs

at

withdrawn.

less of when they were examined (up to after operation). Such obvious cases were included in the data. Table 1 summarizes obtained 3 and

when the 4.5 h after

were successful

9 h not the

examined injection

eggs

of sperm nuclei. Eight to 39 percent of the eggs injected with sperm nuclei were not activated (Figs. 2a-c), but all of them contained swollen (decondensed)

sperm

nuclei

variation

in

the

incidence

was

probably

due

most

degree of micropipette

stimulation rather

activated ed well

to

2d-g).

unactivated

the

difference

nucleus nuclei

62-90

injected. of cauda

percent

dicating that isolated nuclei of mal spermatozoa were capable into sperm pronuclei. testicular spermatozoa into

in the

eggs

the

were

them contain(Fig. 4), incauda epididyof developing

The isolated were also

pronuclei

the in

When eggs epididymal

of

and 5 5-61 percent of developed sperm pronuclei

developing

The eggs

given to each egg by than to the difference

the type of sperm were injected with spermatozoa,

(Figs. of

nuclei capable

(Fig.

5),

of of

whereas

series of experiments (data are not shown in Table 1), 8 eggs were injected with unsonicated nuclei of caput epididymal spermatozoa and examined 7-9 h later. In spite of such long incubation periods, none of the eggs contained sperm pronuclei (2 eggs were unactivated and 6 were activated; all contained swollen sperm

jection,

cytoplasm, into sperm

and

pyrrolidone

In

(K-90, ICN), and kept for up to 4 days at 4#{176}C. The method used for injec. tion of a single sperm nucleus into an egg by micromanipulation was essentially the same as that previously described (Uehara and Yanagimachi, 1976) except that the egg was first pierced completely through with the tip of the injection capillary, the capillary was left in this position for about 10 seconds, then the tip was slowly withdrawn into the egg cytoplasm before a sperm nucleus was released into egg cytoplasm. After the sperm nucleus was injected, the capillary was quickly withdrawn from the egg, and the egg was cultured at 38#{176}C in BWW medium under 5 percent CO3 in air. Between 3 and 9 h after the inslip

polyvinyl

sperm

successful.

These nuclei lay either in the perivitelline space or on the surface of the vitellus, and never showed signs of nuclear decondensation regard-

between spermatozoa

of

DISCUSSION

nuclei were brought time the micropipette

results mal

AND

eggs were

mounted

between

examined

with

a phase-contrast

a slide

and covermicroscope

for the condition of the cortical granules as well as the sperm and egg nuclei. Sperm nuclei in early stages of swelling (decondensation) were often difficult to examine in living specimens. In such cases, eggs were fixed

in

acetic

alcohol

(100

percent

ethanol

3:glacial

those of caput not in almost

epididymal all

cases

nuclei). Apparently spermatozoa readily

periods The

spermatozoa (Fig.

nuclei

6).

of

decondense

but most pronuclei

of at

them least

caput

In

were a separate

epididymal within the egg

cannot within

develop the time

studied. reason

for

the

failure

epididymal spermatozoa nuclei is unknown at the

of nuclei

to transform present time.

of caput into proIt is pos-

SPERM

NUCLEI

INJECTED

INTO

EGG

317 ‘C

i: “!! C V

Cl)

.

,a

C

‘

r-

©

mN mr-i

NO r-.oo

r-i

0r’ -

NN en

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‘C C

‘0 -

‘C V ‘C

-

&)C..

00

00

0

00

00

0

I .C

‘0

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.

o

.0 ‘C

,

-

V

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C

-

‘z;

0 0.

O0 V

=

..N C

N

“0

0

.

,

I

-

en

mN

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.C

>-

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U

..V

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m.

,.,,.,

,,,

*-

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-

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V’s I..

V

a C

‘C ‘5

en

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1+

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0

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E ‘5

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E

..

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g

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CV ‘

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C.

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‘C’-’>

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. 0

,s

c.

I-’

V

UEHARA

318

FIG. injection, maining nucleus; FIG.

injection.

2. Eggs

unactivated

following

AND

injection

with

YANAGIMACHI

isolated

nuclei

of cauda

epididymal

spermatozoa,

a, A low magnification view of the egg; b, Egg chromosomes at metaphase II; c, Cortical intact; d-g, Swollen sperm nuclei in the egg cytoplasm; o, An oil droplet injected together p1, the first polar body. a, X420; b-g, Xi ,120.

3. Egg activated The egg nucleus

4 h after granules

re-

with sperm -

injection with isolated nuclei of cauda epididymal spermatozoa, 4 h after into a pronucleus, but the sperm nucleus failed to do so. a, A low magnifiof the egg; b, Egg pronucleus; c, Cortical granules are absent from the egg cortex; d-e, Swollen in the egg cytoplasm; 9, Egg nucleus; p1 and p2, the first and second polar bodies, respectively. X480.

cation view sperm nuclei a, X420;b-c, FIG. 4. Eggs

activated

following developed

following

injection. Both egg and sperm and sperm pronuclei in other p1 and p2, the first and second

injection

with

polar

bodies,

isolated

nuclei

of cauda

epididymal

spermatozoa,

4 h after

a, A low magnification view of an egg; b-d, Egg magnifications; 9, Egg nucleus; d, Sperm nucleus; 0, Oil droplet; respectively, a, X420; b-c, X 510; d, X i,000.

nuclei developed eggs under higher

into

pronuclei.

NUCLEI

SPERM

INJECTED

INTO

EGG

319

FIG. 5. Eggs activated following injection with isolated nuclei of testicular spermatozoa, 4 h after injection. 9, Egg nucleus; d, Sperm pronucleus; o, Oil droplet. a, X420; b, X 510. FIG. 6. Eggs activated following injection with isolated nuclei of caput epididymal spermatozoa. a, A low magnification of the egg, 4 h after injection; b-c, Swollen sperm nuclei in the egg cytoplasm, 7 h after injection; d, An early sperm pronucleus found in the cytoplasm of an egg, 7 h after injection; 9, Egg nucleus; n, Small nucleoli in an early sperm pronucleus; o, Oil droplet; p1 and p2, the first and second polar bodies, respectively. a, X420;b-d, X1,i20.

sible

that

the

nuclei

of

caput

epididymal

sper-

spermatozoa.

The

nuclei

of

cauda

epididymal

matozoa were in very unstable condition and the isolation procedures caused severe damage to the nuclei, According to Calvin and Bedford (1971) and Bedford et al. (1971), nuclei of rabbit caput epididymal spermatozoa are less stabilized by -SScross-linking than those of ejaculated spermatozoa. This was deduced from

spermatozoa

their observations didymal spermatozoa

the isolation procedures and should develop into sperm pronuclei. Apparently is not the case as our data indicate. An

dithiothreitol ing agent) an anionic rate than When caput

and

that

(DTT, a specific sodium dodecyl

detergent) those of

hamster and cauda

the nuclei of caput exposed to a solution

disulfide-reducsulphate (SDS,

decondensed ejaculated

spermatozoa epididymides

at a faster spermatozoa. from were

2 mM DTT containing 1 percent densation of nuclei occurred fastest spermatozoa

followed

by

epiof

caput

the testis, exposed to SDS, deconin testicular epididymal

decondensed

(Mahi

and

Thus,

testicular

stabilized the

nucleus,

would

tive

Yanagimachi,

by

cross-linking

sperm

nuclei

-SS-

cross-linking. of

the

most

for

caput epididymal pronuclei is that

slowest

the

testicular severe

failure

spermatozoa caput sperm

to be least If for

in

the to

-SS-

stabilizing

spermatozoa damage during

of

the

fail

to this alternanuclei

of

to develop into nuclei contain a

which prevents the transformation nuclei into pronuclei. This factor

absent or inactive spermatozoa, added of caput spermatozoa,

rate data).

appear

responsible

nuclei

explanation

the

unpublished

is solely

receive

“factor” sperm

at

may

of be

nuclei of testicular or activated in nuclei and removed from or in-

UEHARA

320

activated

again

spermatozoa mis. In any sperm

by

another

nuclei

appear

are sperm

the

nuclei

only

to

epididymal

sites where “post-testicular

sperm changes

the

to

may

their

the

seven

with

does

fertilized

was

presence

of sperm

egg

cytoplasm

and

Smidt

and

(or

caput

produced

among

fetuses

the

egg).

epididymal

live

Holtz 25 gilts

spermato(mean

of

changes protein)

velop

into

(including -SScross-linking and gained the ability to

functional

the spermatozoa or while they epididymal

sperm

pronuclei

reached the resided within

spermatozoa of

the

of the

hamster

are

tal conditions epididymis

rabbit

totally

to complete their maturation testicular changes in the nuclei. interesting

incidental

study was within

the the

and

pig,

incapable

including finding

decondensation cytoplasm

of

post-

in the

pre-

of sperm apparently

of

“unactivated” eggs. Our criteria for egg activation were the breakdown of cortical granules and the resumption of meiotic division of the egg nucleus. These are merely the visible indications of physiological

egg

involved

“unactivated”

densed)

tion

activation. Many other and biochemical events in

egg eggs

sperm been

to of

activation.

conclude egg

swollen

that

“partially”

we

activated.

that

cytoplasm)

invisible must also

Therefore,

containing

nuclei

egg is

the

the

egg

a

cyto-

supported

by

grants

from

U.

S.

might would

activation necessary

REFERENCES Austin,

C.

R.

(1961).

The

Thomas, Springfield. Bedford, J. M. (1972). of sperm penetration

An

Mammalian electron into the

Egg.

C.

C.

microscope study rabbit egg after

mating. Am, J. Anat. 133, 213-254. J. M. (1975). Maturation, transport, and fate of spermatozoa in the epididymis. In “Handbook of Physiology.” Sec. 7, Vol. 5, (R. 0. Creep, ed.), pp. 303 -317, Williams & Wilkins, Baltimore. Bedford, J. M. and Calvin, H. 1. (1974). The occurrence and possible functional significance of -S-S-

crosslinks

reference

to

in sperm

eutherian

heads,

with

mammals,

J.

Genetics

be

(activafor

of the Spermatozoon.”

particular Exp. Zool.

H. 1. (1971). and In

(R. A. Beatty

mem“The

and

S. Glusecsohn-Waelsch, eds.), pp. 69-96, Bogtrykkeriet Forum, Copenhagen. Biggers, J. D., Whitten, W. K. and Whittingham, D. C. (1971). The culture of mouse embryos in vitro. In “Methods in Mammalian Embryology.” (J. C. Daniel, Jr., ed.), pp. 86-116, Table 6-5, Freeman, San Francisco, Calvin, H. I. and Bedford, J. M. (1971). Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during

maturation

in

the

epididymis.

J.

Reprod.

Fert,

Suppl. 13, 65-75. Gledhill, B. L. (1971). Changes in deoxyribonucleprotein in relation to spermateliosis and the epididymal maturation of spermatozoa. J. Reprod. Fert. Suppl. 13, 77-88. Holtz, W. and Smidt, D. (1976). The fertilizing capacity of epididymal spermatozoa in the pig. J. Reprod. Fert. 46, 22 7-229. Horan, A. H. and Bedford, J. M. (1972). Development

of the fertilizing didymis of the 30,

(decon-

observed It

not

was

188, 137-1 56. Bedford, J. M., Cooper, C. W. and Calvin, Post-meiotic changes in the nucleus branes of mammalian spermatozoa.

before

caput epididymis it. Unlike caput

eggs (Horan and Bedconceivable that hamster require some specific environmenwithin the lower parts of the (corpus and cauda epididymides)

spermatozoa

hasty

pronucleus

Public Health Service (HD-03402), the Ford Foundation and the Population Council, We thank Ms. C. A. Mahi and Dr. B. J. Rogers for their helpful suggestions.

of de-

fertilizing (penetrating) ford, 1972). It is

have

of

of

a

natural Bedford,

eight

per gilt, at 3-4 weeks post-insemination). Obviously the nuclei of some caput epididymal spermatozoa of the rabbit and boar had completed a series of post-testicular matu-

ration nuclear

work

the

within

the

that

caput

by

pronuclei of

in

fetuses

be

obde-

epididymal

as evidenced egg

reported

with

two

An

never fully

plasm.

This

species. According out of 47 eggs

cleavage

(1976)

inseminated

sent nuclei

into

activation

at

of the

nuclei complete or maturation”

one

inseminated

spermatozoa

those

say

Transformation

nucleus

“full”

can

necessary for of sperm

We have containing

pronuclei.

require

we

ACKNOWLEDGMENTS

may vary according to to Orgebin-Crist (1967),

zoa,

cytoplasm. eggs

sperm

spermatozoa.

The

but

activation is not of decondensation

sperm

decondensed

in

decondense,

in the egg “unactivated”

veloped

1971; 1976)

occurring

nuclei

that “full” initiation

nuclei served

the cauda cross-linking

passage

sperm least the

epididyof hamster

Bedford, et al.,

changes

during

YANAGIMACHI

as spermatozoa

the testis in the -SS-

protein (Calvin and et al., 1971; Meistrich

unlikely

while

cauda

to change

are transported from epididymis. Changes of nuclear Bedford

factor

reside within the event, the characteristics

AND

Mahi,

ability Syrian

of spermatozoa in the epihamster. J. Reprod. Fert.

416423.

C. A. and Yanagimachi,

R. (1975). Induction of nuclear decondensatjon of mammalian spermatozoa in vitro. J. Reprod. Fert. 44, 293-296. Meistrich, M. L., Reid, B. 0. and Barcellona, W. J. (1976). Changes in sperm nuclei during spermato-

SPERM genesis 99,

and

epididymal

maturation.

Exp.

NUCLEI Cell Res.

embryonic

didyinal Biophys. Uehara, T. injection

INTO

M. (1967). Maturation of spermatozoa rabbit epididymis: Fertilizing ability and mortality in does inseminated with epispermatozoa. Ann. Biol. Anim. Bioch. 7, 3 73-389. and Yanagimachi, of spermatozoa

subsequent transformation male pronuclei. Biol. Reprod.

R. (1976). Microsurgical into hamster eggs with

of sperm 15,467470.

nuclei

into

EGG

Yanaginiachi,

ster

72-78.

Orgebin-Crist, in the

INJECTED

321

R. (1969).

spermatozoa

Fert. 18, Yanagimachi,

275-286. R. and

microscope

studies

hamster

egg.

Am.

In vitro

by

capacitation

follicular

Noda,

Y.

of sperm J. Anat.

fluid. D.

(1970),

incorporation 128,

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Biol.

Electron

into

the

429462.

REVIEWS

Bedford, J. M. (1975). Cited in reference. Orgebin’Crist, M. (1969). Studies on the

the epididymis.

of ham-

J. Reprod.

Reprod.

Suppl.

function

1, 134-1 54.

of