or
BIOLOGY
8,
REPRODUCTION
369-377
(1973)
of Sperm
Stimulation
Movement
Fertilization
in the
J.
M.
Worcester
Foundetion
for
Received
Unfertilized
eggs
by injection
of
after
insemination
same
time,
entry
of
junction
of
reduced
the
oviduct
one
oviduct
percentage
similarly
ligated
which
oviduct.
permitted
only
of
eggs
at
the
sperm
of
eggs
of
sperm
Additional
experiments
supposition.
Various
h after
insemination
oviduct
at
the
to
be
was
taken
nail
1.5
junction.
49%
being
a delay
of
to
penetrated,
as
if
indirect
from
the
proximal
isthmus
injection
obtain
in
with
one
HCG,
junction
and
increase
the
have
the
in
to
oviduct
0.5
of
the
site
recipients
h
before
rabbit
the
of
eggs
support
at
to
was
products
fertilization.
to
of
versus
permitted
oviduct
by
evidence
the
transfer
ligated
should
the
of
sperm
egg
the
in
contralateral
after
a stimulation
and
transfer
not
in
eggs,
48
ligation
in
direct
the
of
from
the
h
movement
of
placed
38%
0.5
magnitude
evidence
to
did
this
prevent
transfer
14
Delaying
h
At
ampullar-isthmic
the
h
penetrated
this
sperm
to
the
before
during
9-10.25
HCC.
polish
at
induced
at
with
ligature
0.25 h after
egg
transfer
in the ligated
compared
to
delaying
the ligation
until
were
ampullar-isthmic
with
a
01545
ovulation
recipients
injection
immediately
performed
substances
and
covered
utero-tuhal
movement
were
of
and
penetrated
Since
of
1972
after
oviducts
Placing
penetration,
oviduct.
result
ovulation
17%
However,
nonligated
this
random,
recipient
the
until
was
normal
the
21-29%
oviducts.
of
the
Massachusetts
8,
shortly
rabbits
were
the
of
junction
penetration,
nonligated
in
into
November
spermatozoa
ovaries
eggs
Shrewsbury,
accepted
to
million
to the Site
Oviduct1
Biology,
donor
transferred
200
Isthmus
HARPER
1972;
from
were
recipient’s
own
ampullar-isthmic
48%
recovered
with
the
her
4,
the
Rabbit
K.
Experimental
August
HCG,
from
this
9.75-10.5
ligation
of
both
oviducts.
the
Frozen
rat eggs in cumulus,
or frozen
rabbit
eggs
in cumulus,
or 50 d of Ringer’s
solution
did not enhance
penetration.
However,
after
transfer
of freshly
ovulated
rat eggs in cumulus
29 and
36% of eggs
were
penetrated
in the ligated
and
nonligated
oviducts,
respectively.
These
figures
did not differ significantly.
Fresh
rabbit
cumulus
with
eggs
removed
or rabbit
eggs
with
adherent
corona
cells
following
removal
of the cumulus
with hyaluronidase
had no stimulating
effect.
Thus
the
experiment
with fresh rat eggs provided
further
evidence
for a stimulatory
effect of the products
of
ovulation
or
rabbit
of which
In
previous
on
eggs
component
1961a,
1963)],
mainly
the
to
be
transport
in
cumulus
of the
experiments
it was
observed
that
the period
of ovulation
9.5 h after
injection
were
sperm
without
to
products
(Harper,
the
produce
sperm
in
in
the
the
of ovulation
1972),
isthmus.
more
isthmus
oviduct
isthmic
proximal
tion.
tion
were
transfer
cipients
369
©
of
1973
by
reproduction
The
Society
in any
for
form
the
Study
reserved.
of
Reproduction
failure
effect
produces
in the
1A preliminary
account
of some
portions
of
this work was presented
at the
Annual
Meeting
of the Society
for the Study of Fertility,
Nottingham University,
England
(1971).
Copyright
All rights
The
a similar
at the begiiming
of
in the rabbit
[about
of HCG
(Harper,
found
oviduct.
of
leaves
this
the
had
to
became
moved
the
junction,
cumulus
the
stimulatory
During
oviduct
rabbit
open
question
effect.
next
4-4.5
from
region
i.e.,
of
the
sperm
so that
the
proximal
the
ampullar-
site
As a consequence
of this
of sperm
in the
oviduct
penetrated
in a 1.5-h period
to the oviducts
at 14 h (58%)
h,
redistributed
of
fertiliza-
redistribumore
eggs
following
of inseminated
rethan
at 9.5 h (40%)
370
HARPER
after
11CC
was
injection.
significant,
indicate
at 9.5
that
had
h
ment
of
during
was
this
not
to
next
the
peared
possible
investments
or
large
of
oviduct
move-
Thus,
mouse,
shortly
region.
(1972)
most
after
utero-tubal
the
has
of eggs,
their
fluid
entering
sperm
mating
of further
is
junction
oviduct
isthmus
seen
at
before
the
site
of
are
recent
and
entry
becomes
enter
the
that
ists a mechanism
tozoa
from
reaching
before
arrival
of the
observed
that
sperm
is equal
of
eggs,
by
sperm
ampulla.
which
the
coitus.
and
to
indicate
that
of ovulation
sperm
to migrate
to the site of
ampulla.
Therefore,
ments
were
carried
for
such
a
ejaculates
USP
concentration
was
from the
fertilization
proximal
in the
the
present
out to determine
experiif evi-
mechanism
The
female
rabbits
were of the N.Z.
kg and supplied
mature
males
used
used
in
these
experiments
for
the
collection
of
semen
present
estimated
400
million
given
tory
arrest
one
the
anterior
and
imme-
with
All
in
done
50
IU
groups
Table
of
1,
were
When
the
the
site
nients
is
noted.
utero-tubal
between
these
The
of
entry
oviducts
with
nail
Ponds,
Inc.)
egg
transfers
at
oviduct
than
this,
animal
the
Rabbit
12
recovered
15
with
were
h
the
insemina-
were
incisions
When
done
at
closed
were
the
time
induce
for
re-
eggs
14.5 h after
time
were
HCG
were
still in cumulus,
as possible
after
egg
portion
of the oviducts
were
microsyringe
done
in
Ringer’s
pipet
of
solution.
mare
72
h
133
IU
before
In
the
such
cases,
later,
since
ovuladelayed
following
This
of
50
the
Some
of
serum
Inc.)
and
somewhat
be slightly
in
animals
flushing
pregnant
treatment.
spread
ton
by
superovulation.
superovulatory
fers
injection
Organon
48
were
recovered
appeared
to
most
donor
iv
HCG
Ringer’s
with
between
from
after
(Gestyl#{174},
given
large
or
ligations
(IU)
sterile
treated
gonadotrophin
to
mating
until
and
units
being
into
the
ovaries
Chesebrough1970a).
warm
were
h
international
oviducts
the donors
eggs
(Harper,
the
kept
experi-
incision.
eggs
between
HCG
junc-
individual
own
h after
of
or
later
the
in
recipient’s
9-9.75
injection
These
on the isthmus
In the
text
and
prevented
by
painting
polish
(Cutex#{174}, colorless,
and
hemostat
Scissors.
or
ligations
of the
apart,
a
was
tion
opening
with
of
was
ligated,
ampullar-isthmic
junction,
junctions.
the
distance
with
severed
the
depth
were
short
iv
respira-
ether
oviducts
a
at
the
surgery,
crushed
placed
If
and
placed
completely
mg/kg
minutes.
for
tissue
tables
Nembutal#{174}
50
imminent
were
at the
under
several
insufficient
were
HOG
eggs
tion
Each
into
Laboratories):
seemed
tion,
midway
the
first
over
was
ligations
and
mixed
spermatozoa/mi.
Inc.).
were
slowly
anesthesia
times
by
the
inseminated.
All operations
anesthesia
(Abbott
was
mixed
sterile
Ringer’s
to give
a final
deep
the
used
elapsing
of
aliquot
ml of this suspension
intravenously
(iv)
except
artificially
days
diluted
with
Laboratories)
(Pregnyl#{174}, Organon
and
White
strain,
weighing
over 3.5
by Mount
Hope
Rabbitry.
The
sperm
HCC
general,
METHODS
were
with 0.5
injected
the
AND
with
vagina
diately
females,
2
an
not
ejaculates
inseminated
then
be obtained.
MATERIALS
of
female
generally
An
then
(Cutter
was
solution
was
were
using
The
use of a hemocytometer.
and
they
of
semen
bucks
week,
per
number
am-
the
arrival
of
in the
oviduct
stimulator>’
the
intervening
the
number
of ampullar
to or lower
than the number
products
twice
of
fertile
male
ejaculations.
the
and that
the eggs
are penetrated
as soon
as the latter
reach
the
These
observations
could
also be
construed
than
between
three
Each
ligatures
sugex-
In addition,
more
Collections
or
vagina.
administered.
prevents
spermasite of fertilization
eggs.
artificial
also
These
have
there
breeds.
two
two
Zamboni
(1972)
oviduct
mixed
from
the
are
agreement
with
rabbit
(Harper,
Oura
and
Zamboni
in the mouse
in the
after
good
the
Stefanini,
(1969a),
gested
could
the
Thereafter,
fertilization
in
in
findings
1972).
dence
patent.
the oviduct
sperm
that
times
these
observations
caused
isthmus
in
remain
in the caudal
coils
of
for 5.5-7
h. Very
few
sperm
pulla
the
that
closes,
sperm
into
Most
of the
difficult.
the
to the
enter
the
oviduct
at a time
when
the
junction
utero-tubal
found
of
made
and
it ap-
the
oviduct
in some
way
stimulated
migration
of sperm
from
the isthmic
ampullar
Zarnboni
to
fertilization
hours.
that
the act
of follicular
were
as
in the
gradual
site
several
difference
so
eggs
placed
to await
the
sperm
the
While
it
accounts
recovery
of
recovered
injection.
for
eggs.
between
The
eggs’
In
13.5
which
transferred
as soon
recovery
to the ampullar
of recipient
females.
Transwere
solution
control
using
0010
a Hamil(Hamilton
SPERM
IN
TRANSPORT
TABLE
EFFECT
OF
TIME
AND
SITE
12-14.5
II
OF
OVIDUCT
OF
Oviduct
Transfer
in
to
nation
0
9.5-10.0
2 This
group
Before
4
Brackets
Co.)
of
1-5
None
10
131
30
71
J46
137
10
26
24
9
a glass
which
collected
sacrifice,
the
eggs
quantity
eggs
remained
cases
where
frozen
or
solution
carion
remained
medium
then
was
eggs
in
rabbit’s
in a watch
dioxide.
eggs
rabbit
either
the
eggs
as
other
in
eggs
14
46’
216
13
9
426
fl4.6
15
38’
13
87’-
(36
21
11
0
4
42
6
ff37
13
10
7
7
65
17
46’
13
7fl..6
5
13
54
18
49’
17
94’
middle
=
or
17
of
three
or
egg
right
In
At
tract
or
by
the
hyaluronidase
Co.) and
corona
were
placed
on
2 h
after
cervical
removed,
transfer,
the
eggs
used
for
deducing
that
of
at P
same
is
‘
as
had
an
used
were
of
6, but
not
from
previously
made
Mainland
(Harper,
using
and
.6).
the fourfold
Murray
(1952)
to
after
the
ovimating
test.
RESULTS
When
ducts
of
40%
h,
of
and
eggs
rabbits
the
of
truded
at
eggs
those
the
transferred
9-9.75
Table
polar
1).
have
body
can
(Harper,
be
and
from
and
the
was
were
recipients
were
reproductive
from
the
and
for evidence
same
criteria
had
duction,
the
been
transfer
eggs
4S%
(Table
1).
that
are
and
penetration
percentage
centage
does
on
injection
of
the oviduct
the
the
same
respectively
are
obtained
if
placed
at the utero-tubal
the middle
of the isthmus:
reduced,
sperm
and
showing
are
9.5
just
affect
in
14%,
results
penetrated
Thus
not
penetrated
is significantly
of eggs with
extrusion
oviducts.
proximately
junction
versus
Similar
ligations
junction
body
fixed,
egg
utero-tubal
seen
but that ligation
of the ampuljunction
causes
a significant
re-
ovu-
to
the
It
1.5
ex-
within
47%
time period,
lar-isthmic
recipient
of
h
are penetrated
penetrated
second
and
1972
are
of
adherent
egg
from
been
tests
x’
different
analyses
contingency
the
rabbits.
0.01.
<
percentage
cumulus
flushed
(1972).
and
recovered
the
Harper
prior
(bovine
testes type
the eggs devoid
of
the
from
The
removed
cells
taken
5 mm.
the
the
were
ligation
freshly
dislocation,
and
shown
and
as possible
to
83’-
rapidly
froze
needles
or
in
quantity
were
were
dissecting
with
Fresh
transfer,
experiments,
transferred
still
solution.
cumulus
5
the
after
a small
176
junction.
results
Statistical
left
on
the
random)
1970a,b).
into
in
in
oviducts.
The eggs were then mounted,
stained
with
lacmoid
and examined
of penetration
(Chang,
1952).
The
were
at
penetrated
expressed
until
mechanically
with
but
1.5
and
same
as
utero-tubal
=
those
transferred.
killed
bucks,
significantly
the
possible
rapidly
Chemical
cumulus
UTJ
52
transfer.
chosen
differ
2
isthmus,
fertile
to
rats
at least
cumulus
were
with
Sigma
for
206
31
as
transferred
In
alone
dissolved
frozen
cumulus
cumulus
cumulus
medium
15
1
66
used
glass
The
thawed
oviducts.
lated
eggs
48’
146
smear
contained
portion
of the
eggs
the
22
5
48
treatments,
Ringer’s
rabbit
transfer,
78
30
2
of
medium
this
rats
of Ringers
cumulus
of warm
in
before
soon
in
6
0
476
0
unmated
as
25
9
tube.
vaginal
ampuilar
ruptured
and
a small
the
The
4’
9
were
from
which
cells.
53
6
2
26
or
were
77
0
oviduct.
Eggs
13
17
4
oviducts
random
body
No.
6
(left
to different
at
polar
No.
33
superscripts
capillary
the
trated
>10
23
32
prior
ovidocts
6-10
second
21
two
with
Pene-
(67
immediately
different
subjected
morning
on
only cornified
oviduct
was
the
mated
\Vith
eggs
J67
mid-I
done
\ith
sperm
on
sperm
137
individual
oviducts
control
solid
was
to
were
right
ii
J4o
A!J
with
COLLECTED
6
-
junction,
ligation
in
1.5
EGGS
OVIDUCTS
10
AIJ
animals
connected
RtBBIr
THE
eggs
6
None
indicate
animal
1,
0
Mid-I
in columns
experiments
the
examineil
None
h after
indicates
Figures
the
rabbits’
None
ampullar-isthmic
I
or
liga-
A IJ
0.Shafter
AIJ
I
25
of
#{149}
in
or
lion’
Before
925-10.0
of
UTJ
Before
of
No.
No.
egg
Before’
9.S-10.25
OF
To
FOR
No.
of
9_9755
75
RECIPIENTS
Site
transfer
9. 25-9.
INSEMINATED
PENETRATION
TRANSFERRED
Eggs
relation
insemi-
AND
ligation
Time
H after
ON
INJEcTIoN
371
ovwucr
1
LIG.tTION
IICG
AFTER
THE
not
but
second
different
the
per-
the
polar
between
at this time of transfer
h after
insemination
HCG)
adequate
to ensure
normal
sperm
are
fertilization,
(apand
in
HARPER
372
but
they
mal
are
for
the
portion
of the
If the
ligation
most
part
isthmus.
of the
in the
tested
proxi-
ampullar-isthmic
junction
transfer
in the
was
delayed
until
0.25
h after
of eggs,
the percentage
penetrated
ligated
oviduct
was still significantly
lower
than
in
38,
respectively).
was
repeated,
ligation
one
When
not
the
the
made
this
case,
the
oviducts
were
not
or
that
seen
in
(17
this
in
oviducts.
each
50
with
l
of
transfer.
ment
along
the
The
following
try and
obtain
on the
different
effect.
ments
The
was
oviduct.
experiments
direct
evidence
were
for
rationale
behind
as follows:
since
ampullar-isthmic
transfer
did
junction
interfere
not
but placing
the
before
transfer
ligation
did,
done
such
to
duct,
h after
egg
penetration,
position
to be
OF
PRODUCTS
OF
AND
PENETRATION
OF
OVULATION
RABBIT
TO
TRANSFERRED
Oviduct
Time
Treatment
SOpl
into
ampulla
0.5
h before
AIJ
ligation
Ringer’s
eggs
frozen
Before
Site
Transfer
Before
Before
-
thawed
Before
None
cumulus
eggs
only
with
but
T1
C
=
raband
ovi-
treatment
the
in
that
AMPULLAR-ISTHMIC
H
per-
the ligated
freezing
and
INSEMINATED
JUNCTION
HCG
AFTER
RECIPIENTS
ON
INJECTION
2
FOR
SPERM
AND
u’
of
No.
No.
of sperm
With
(h after
rab-
eggs
insemination)
bits
examined
0
1-5
6-10
>10
(49
40
9
0
0
18’
3
152
16
25
8
3
59..b
24
0
0
11’
3
1 52
13
30
5
4
75’
23
76
43
31
2
0
466.5
22
173
29
40
4
2
605.6
60
34
25
1
0
56
8
29
13
6
ff42
26
16
0
0
386.s
(41
10
24
4
3
70’
15
J
37
15
0
0
29’
8
Au
.
No.
-
97o102o
AlJ
10
25
AU
no cumulus
None
9.lo-lO.25
AIJ
10.00-10,50
AIJ
-
None
Before
of
in or on cells
With
Pene-
sperm
cumulus
control
75-10
free
oviduct
rabbit
10 00-10
AIJ
J
1
‘
8
s
25
50
8
None
-
as Table
6
-
corona
conventions
=
received
This
penetrated
noticed
not
freezing
in one
increasing
lion
Before
None
Same
after
substance
ligation.
ligated
while
was
experiments.
liga-
None
-
None
I
OF
None
-
in cumulus
radiata
On the
penetrated
penetration
control
in
egg
experiment
of
Rat eggs in cumulus-
Rabbit
this
test eggs
None
-
None
Rabbit
h before
oviduct
and
%
second
tested
polar
No.
%
body
No.
%
6’
2
67’
45..b
19
79’
5d
1
33’
44.6
16
70’
296.5
20
91’
27
366
20
71
436.o
15
256.o.d
14
93’
86’
38
68’
34
89’
in cumulus-
None
frozen&
of
of rabbits
of eggs
It was
13.25-15.0
OF
one
Eggs
of
to egg
& thawed
Rat eggs
in
transfer
None
Rabbit
in
of sperm
received
2
LIGATION
OvIDUcTS
group
unsuccessful
centage
oviduct.
COLLECTF.D
THE
hence
rabbits
ligation
relation
solution
EGGS
procedure
stimulation
ligation
in cumulus
as the test
0.5
was
TABLE
EFFECT
next
bit
eggs
thawing
the actual
handling
the
solution
side
previous
to
to
transfer
results
control
from
trans-
causing
oviduct
The
The
an
in the same
the
substance
Ringer’s
before
ovi-
was
that
(Table
2) were as expected.
side very
few eggs
were
these
experi
ligating
the
0.5
with
group
and
the
at
eggs
used
transferred
and
of
motility,
one
h
no tubal
ligation.
This
result
provided
indirect
evidence
for a stimulating
effect
of
the products
of ovulation
on sperm
move-
the
sperm
other
nothing
rabbit
were
themselves
oviduct
0.5
the
on
one oviduct
was ligated
The
but
and
not
movement,
other
in
To ensure
the operation,
of
oviduct,
were
of
animals
handled
mechanics
the
effect
placed
oviduct
junction.
was
both
the
egg
of eggs
nonligated
from
control
stimulating
ferred
to it until
test
for
penetration
dramati-
the percentages
ligated
and
different
duct
experiment
changed
a
was
that
ampullar-isthmic
versus
exception
that
until
0.5 h after
situation
cally.
In
penetrated
from
control
with
was
transfer,
the
for
movement
0.5 h later
.52T1
(SOT,
41
9
0
0
18’
45C
15
25
5
0
57n,b
after
oviduct
ligation,
6
transferred
first,and T,
=
rabbit
eggs
transferred
second,
to same
37b,
15’.”
5
55’
12
80’
8
100’
7
14s.d
6
86’
16
366.5
10
63’
I.
eggs
145,d
oviduct.
SPERM
thawing
caused
consistency
much
more
the matrix
without
a
marked
TRANSPORT
change
of the
cumulus.
sticky
and tightly
was
less able
to
breaking.
The
in
rabbit
eggs
transferred
the six rabbits
in this
the
It became
packed,
and
be stretched
number
of
THE
IN
frozen
to one oviduct
of
group
ranged
from
4-7,
rneiotic
and
metaphase,
the
of
373
OVIDUCT
vitelline
trusion
a second
of
Placement
of
these
rat
there
between
the
percentages
trated
in
the
ligated
the fresh
rabbit
eggs transferred
0.5 h
later
by
the
following
means:
most
of
the frozen
eggs
were
denuded
while
the
ducts.
Penetrated
eggs
ligated
oviduct
of only
others
were
of the
rabbits
second
mally
meiotic
clumped
abnorso dis-
portions
persed
the
attached
was
be
(91%)
the
chromatin
division
was
either
or more
usually
as to
29
and
unrecognizable.
Only
eggs
recovered
the zona
pellucida,
to
had
2 of
a sperm
and
Since
none
to
of
stimulate
lected
as
eggs
test
transferred
being
cumulus
have
eggs
sperm
rat
the
age
could
these
or
in
cumulus
The
varied
from
22.6. In
transferred
were
Most
of
all were
herent
rabbit
them
still
after
tal
environment
usual
Of the
were
were
still
surrounded
0.5 h less
denudation.
the
(70%)
corona
radiata
eggs
recovered
duct
in
by
cells,
from
tried
exposure
brief
rat
249
cumulus,
closely
to the
all
and
ad-
oviduc-
stages
to
exposure
with
did
show
in
of
hyaluroni-
lating
effect
was
thawed,
to rat
eggs.
lus
were
in
transfers
then
rat
although
cumulus
eggs
have
rabbit
nonligated
than
not corona
cells from
the rabbit
eggs.
of the
rat eggs
had
been
penetrated
a rabbit
sperm
nor were
any rabbit
None
by
sperm
sults
adherent
to
found
seen
the
cellular
Despite
this,
in
ance
of
the
zona
the
being
showed
division
clumping
of
pellucida
investments
rat
had
the
activated,
of
the
eggs
or
of the
the
chromatin
since
first
polar
of
the
rat
eggs.
appear-
of
the
eggs
frozen
of
oviduct
that
compared
body,
rat
eggs.
substance
(range
these
to
these
eggs
rat
eggs
had
and
dismatrix
thawing.
experiment
clear
that
are
many
penetrated
ligated
do
penetrated
had
in
oviduct,
not
in
received
that
However,
result
genthe
stimulate
However,
when
the reare compared
with those
eggs
to
of
its
been
the
each
recovered.
had dis-
pellucida
Much
in
ex-
22-29)
zona
intact.
this
it is
given
in cumu-
of 25.4
previous
group
receiving
the stimulating
agent,
that
there
is no difference
centage
crepant
test
as
many
second
the
freezing
of
movement.
of this group
the rat
cumulus
eggs
because
results
isolation,
thus
totally
denuded
the remaining
the
by the
the
in
more
rat
In
presumably
disrupted
frozen
was
rabbits.
surrounding
appeared
When
viewed
being
transferred
still
rabbit
a stimu-
139 (69%)
were
most
of the cytoplasm
appeared
ex-
the
such
and
appeared,
the proof those
body
their
as
were
the
11 rabbits
and
treatment
eight
eggs
oviduct
the
to
used
oviin
polar
frozen
to
pene-
of the remaining
penetrated
in the
produce
an average
frozen
found
6 of the
due
Such
periments,
was
were
a similar
sperm
removed
ovi-
eggs
significantly.
failure
of
to
and
solution
and
the
nonligated
second
eggs,
but
dase
ex-
difference
of
Furthermore,
sperm
not differ
that
the
cumulus
erally
recovered.
in
significant
and
with
eggs
penetrated
test
no
oviduct.
At recovery
number
even
though
the same
ovi-
showed
A
next
general,
the amount
of
was equivalent
in vol-
175
transferred
col-
number
of rat eggs
9-29,
with the aver-
ume to that surrounding
of rabbit
eggs
transferred.
eggs
freshly
eggs
but in three
one egg was
of
and
investments
movement,
agent.
the
destroyed
their
was
group,
only
nonligated
eggs
freezing
ability
of this
truded
To
penetrated.
without
duct
prior
to the ligation
at the ampullaristhmic
junction,
had apparently
stimulated
sperm
movement
to the site of fertilization
because
not,
protrusion
but
body.
polar
with 5 being
the most common
number
(total
32).
On recovery,
the eggs subjected
to freezing
could
always
be distinguished
from
partial
membrane,
is
it
fresh
can
in the
the
frozen
that
had
this
probably
received
rat
be
per-
ligated
rat
eggs
fresh
apparent
dis-
accounted
for
374
HABPER
the
penetrated
fact
by
that
in the
significantly,
being
group
the
the percentages
control
oviducts
which
contralateral
As
higher
rat
much
received
frozen
a consequence
products
for
rabbit
eggs
lus by use
cumulus
the
the
this
test
the
after
duct.
sperm
had
did,
Very
sperm,
few
the
was not
experiments.
in four
of eight
the
In
bit
free
eggs
of
of
of eight
tion
the
covered,
and
belonged
to
eggs transferred
the
this
50
control
of
eight
second
eggs
the
were
T2
only
compares
transferred
oviducts
with
5
at the
of the
increased
one
T,
or
sperm.
rate
pene-
of rabbut
was
liga-
eggs.
For
have
then
not
recovered
same
time
same
rabbits.
of
the
migration
to
they
of
the
do
of
products
stimulates
not
an
sperm
site
from
of fertiliza-
indicate
agent
is, nor
e.g.,
stimulation
how
what
its
of
motility
or chemotaxis.
Despite
the standard
conditions
perimentation-standard
number
effect
oviduct
of
ex-
of sperm
inseminated,
use
of one
oviduct
in each
animal
as
a control,
standard
transfer
media,
and similar
time of experiment
after
insemination-variation
both
between
and
within
groups
was apparent.
To ensure
that
chance
potheses,
groups
results
would
not lead
to false
differences
between
experimental
were
not considered
significant
less
P <0.01,
than
usual.
The
vided
When
junction
transfer,
to
done
those
the T1
recovery,
of
to support
oviduct
isthmus
However,
T2
they
the 53
recovered,
entry
both
the
provide
evidence
the
proximal
this stimulatory
is mediated,
Of
not
It was
usually
easy
to distinguish
from
the T2 eggs
at the time
of
that
into
tion.
first
missing
group.
3 were
while
the
of
experiments
ovulation
had
to oviduct
of
polar
present,
none
than
direct
ovi-
effect
transfer
no
second
sperm
more
and
the
were
transferred
one
oviduct
present
eggs
(range
5-8) such
to one oviduct
prior
The
the
to as T1 and
those
transas T2 in the Table.
It was
all the
T1 eggs
were
reif any
sperm
side.
had
was
DISCUSSION
suggestion
eggs
previous
coneggs
were
ligated
and six
the
showing
than
eggs
there
T1 and T2 eggs
in
percentage
pene-
contrast,
significantly
and were
penetrated
In addition
five eggs
By
treated
the
by corona
cells
a test
substance
h
T
the
sperm,
percentage
more
the
in
indirect
oviducts.
0.5
the
been
referred
ferred
second
assumed
that
out
of
experiment,
rabbits
and
that
An average
6.5
were
transferred
simplicity,
and
percentage
out
had
on or in
only
one
into
more
different
from
Penetrated
surrounded
cumulus
as
eggs
eggs
oviduct
nonligated
next
examined.
rabbit
the
of
control
and
trated
trol
found
out
entry
In the
or
extrusion.
eggs had
the control
on
oviduct
between
with
body
more
always
comof the T2 eggs
in cumulus.
treated
trated,
almost
many
was
were
penetrated,
but
of those
that
were,
most
had the second
polar
body
extruded,
indicating
that
penetration
occurred
fairly
rapidly
be found
the
percentage
cumuand
the
substance,
had
each.
still
In
oviduct
of a group
fewer
eggs
in
that
those
results
eggs,
further
to try to decomponents
from
needles,
as the
oviduct
sperm
the
in
ovulation
might
be reeffect.
Freshly
ovulated
to one
Significantly
ligated
such
would
positive
were
separated
of dissecting
masses,
and
in
eggs
the T1 eggs were
denuded,
while
pletely
difference
of
of
then
transferred
of 8 rabbits.
them,
since
oviduct.
achieved
with
the fresh
rat
experiments
were
undertaken
termine
which
of the possible
of the
sponsible
of eggs
differed
was
positive
the
stringent
un-
requirement
indirect
evidence
is protubal
ligation
experiments.
by
ligation
of
the
ampullar-isthmic
vvas delayed
until
0.25
h after
egg
results
were
no
different
from
obtained
prior
when
to
delayed
occurred.
random
a more
hy-
egg
until
such
transfer.
ligation
When
0.5 h, normal
sperm
movement
some
increase
in
If
was
ligation
penetration
was entirely
penetration
SPERM
would
of the
be expected
ligation,
the
plotted
with
progressive
expected
values
graphically
21-29%
and
and
1 h delay,
would
be,
experiments
already
within
instead
14 and
0.25
that
egg
h
of 1.5 h
ampulla
takes
did
a dramatic
17% to 49%
shift
(Table
from
1)
observed.
to
3.3-13.4
It
is
fimbriated
mm,
ampullar-isthmic
the mean
the
ues
ranging
from
5.0-8.4
mm
1961b;
1965;
Boling
and
Blandau,
in rabbits
10-15
h after
an injection
(i.e.,
during
of any
ligation
ovulation).
the fact
that
for eggs
to
fertilization.
latoiy
effect
exerted
subsequent
rected
to
as opposed
near
port
failure
the
the
the stimuof ovulation
ampullar-isthmic
objective
to indirect,
above
finding.
the experimental
not
responsible
to produce
ous,
since
that
for
of
finding
evidence
It
to support
phenomenon,
a beneficial
the presence
effect
seems
of fresh
rat
to produce
destroyed
rat eggs
the
or
stimulation
and
thaw
for disrupting
both
the
cycles
cells,
consistency
are
and
of
In
the
present
experiments,
fresh
rat
eggs
effect
in
stained
on
If
mo-
cumulus
observed
per,
1961b;
1965),
it was noted
tractions
of the circular
muscle
were
most
frequent
(Harthat
of the
conam-
the
mass
around
of cumulus.
In
view
pected
of
that
be as
cumulus.
culumus
as
was
would
a very
the
not
suggest
cells
may
unlikely
been
alone
that
the
since
eggs
in
and
this
eggs
or
when
a very
quantity
the
Thus,
are
this medium
oviducts
of
of
to
plays
oviducts
solution,
the eggs
donor
small
the
role.
eggs
rabbits,
the
with 1-2 ml of Ringer’s
the cumulus
containing
flushed
only
ex-
would
fresh
rat
the case,
role,
from
licular
dilute
corona
have
play
an important
that
follicular
fluid
significant
recovered
and
it might
effective
This
result
are
this,
rabbit
rabbits.
no rab-
the
to
species-specific.
of oviduct
was
recipient
cumulus
the
eggs
oviduct
the
and the integrity
of the nuclear
structures
of the vitellus
were
disrupted
by this procedure.
rabbit
the
transferred
a classical
it was clear
the
of
through
actually
movement.
had
space
rather
than
chemotaxis,
may
be the
likely
explanation
for this
phenomeIn previous
studies
in which
the trans-
are
penetrafreezing
eggs
attached
is not
stimulation
plus
curieggs
rat
673
stimulatory
since
potential
of the
their
investments
of sperm
of
the
even
atDickman
perivitelline
sperm
clear
were
transfer
medium
by itself
as test
exerted
no effect.
The
failure
of
and thawed
rabbit
and rat eggs
obviously
resulted
in an enhanced
tion of rabbit
eggs.
It may be that
and thawing
rabbit
and
direct,
became
quite
manipulations
this
di-
rabbit
that
corona
It seems
were
1 of
effectiveness
pulla
of this time
the
site of
experiments
the
that
use of the
substance
the frozen
most
reach
It may well be
of the products
is only
junction.
All
that
val-
or were
although
the
in
sperm
movement
this is the case,
tility,
most
non.
(Harper,
1971)
of HCG
the
Thus,
The
indicates
end
stimulating
effect
to be shown
when
was delayed
for only 0.25 h, could
be due to
is required
Freeze
means
known
the
that
sperm
73 had
pellucida.
and
as
change
from
rabbit
a
zona
these
a gradual
was
transport
of the
junction
A delay
penetrated
the rat eggs,
reported
(1962)
0.5
375
OVIDUCT
THE
bit
sperm
tached
to
been
following
course,
equivalent
in
to no ligation.
However,
such
IN
delay
when
have
penetrated
respectively.
of
noted,
not
occur;
between
should
30-38%
TRANSPORT
whatever
fol-
fluid is transferred
must
be (a) very
or (b)
trapped
in the cumulus
and
cells.
In the latter
event,
it might
have
been
either
in
expected
alone
was
the
to
manifest
experiment
transferred
which
eggs
were
transferred.
or
with
its
effect
in which
adherent
In
neither
evidence
of stimulation
ment
forthcoming.
Since
cumulus
in
the
one
in
corona
cells
case
was
any
of sperm
moverabbit
cumulus
cells alone
failed
to cause
sperm
migration,
rabbit
eggs
surrounded
by
corona
cells
were
expected
to be effective.
The
results
achieved
In
were
a
previous
therefore
paper
disappointing.
(Harper,
1970a),
376
HARPER
I
reported
to
determine
lation
tion
experiments
in the
oviduct
eggs
subsequently
of
oviduct.
which
the
could
whether
At that
differences
I was
in experimental
Chang
(1952),
(1962),
and
myself
Chang
(Harper,
garding
of
penetration
eggs.
(Barros
and
1968b;
Bedford,
son, 1969;
Yanagimachi,
sibility
tact
which
1968;
Bedford
1970a)
refree
other
Gwatkin
workers
gave
which
1968a;
and
Chang,
products
and
the
pos-
rise
to
been
in con-
ovulation
might
be
only
partially
might
capacitated”
1970a).
The
results
obtained,
in
not support
this suggestion
(Har-
per,
1970a).
ments
might
The
have
a stimulation
of
manifest.
However
since
the transfer
(13.5-14.75
time
had
the first
An obvious
rabbit
eggs
1969b),
eggs
and
trated
mating)
(1.5
h)
to be
the case,
too late
and too
between
much
entry
lots of eggs.
is to place
fresh
in one oviduct
of
insemination
0.5 h before
and injecligation
of
and
tion
more
troublesome
first sperm
is the
1951;
there
transfer
of
sperm
tected
(as
eggs)
because
to
is
et
a
all
al.,
in
in
the
eggs
rabbit
eggs
were
transferred
with
HCG
injection),
this
experiment
for
0.5
ferred.
before
h before
Despite
penetration
also
of
In
previous
left
in
trated
T,
T2
eggs
the
T1
(Harper,
rabbit
failed
to
mains
obscure,
devoid
stimulate
not
1 to
eggs
might
or
in
(Harper,
be
the
point
from
rat
eggs
failed
rabbit
oviduct,
been
there
tially
denuded.
oviduct.
not
be
de-
of
T2
al-
attached
experiments
to
would
89%
gest
reason
of
of
of
present
layer
in,
inhibited
and/or
or
the
this
the
were
after
caused
but only
the
for
oviduct.
that
HCC
eggs.
This
Stambaugh,
(1969)
that
of
is
cells
cells
in
however,
ion
in rabbit
indirectly
experiments,
or
and
the
not
for
also
the
rabbit
also sug-
concentration
denudation
In addition,
hyaluronidase
injection
par-
recovery,
total
denudaremoved
the
rabbit
corona
bicarbonate
be detected
position
re-
both
and
ion
concentration
and
is primarily
responsible
responsible
after
why
cumulus
egg
period
cumulus
These
results,
that
eggs
pene-
movement
in vitro
the
the rat
reported
not
rapidly
eggs,
from
removal
1 ratio
of
are penenot
rat
layer
Noriega,
poor.
were
only
the
a shorter
Yet
bicarbonate
hyaluronidase,
h
The
with
the suggestion
and Mastroianni
agrees
but
seen,
was
of additional
interest
that
these
experiments
was that
to become
denuded
in the
while
rabbit
eggs which
had
for
hyaluronidase
tion of the
the
stimula-
1970a)
One
emerged
trans-
of similar
sperm
unless
ovi-
enhanced
eggs
2.5
1970a).
eggs
was
35%
for
re-
in the
were
only
experiments
such
eggs
the
after
sperm
left
not
oviduct
cells
h
natural
only
the
of
the
corona
this,
penetration
the
substan-
(10-10.5
were
same
the T2 eggs.
however,
the
adherent
and
the
oviduct
early
distribution,
per-
in
tially
longer
than
1.5 h of
In the present
experiments,
possibly
1969a,b),
movement
above
course,
were
al.,
et
enhanced
penetration
all these
sperm
ready
have
penetrated
the T1 eggs.
Indeed,
zeferred
Of
cumulus
problem
fertilizing
Stefanini
until
(Stefanini
oviduct.
T1
T2
treatment
junction
and
if
sperm
of
hyaluronidase
second
experiment
in cumulus
(Chang,
that
reaction.
ampullar-isthmic
sperm
than
were
greater
sperm
movement
this
was not
was
performed
and
potentially
that
if the
centage
eggs
eggs
significantly
of the
granulosa
fluid
are
involved
of T2 eggs.
One problem
of this approach
is the
difficulty
in
distinguishing
the
T1
from
the T2 eggs
on recovery.
A second,
is
ovulated)
a
cell
types
follicular
rabbits
9.5 h after
tion
of HCG
and
and
(Naturally
format
of these
experibeen
expected
to permit
h after
elapsed
of
T1
penetrated,
duct
and
had
of
Ander-
1969;
capacitated
and therefore
have
an
over sperm
without
such contact
(Harper,
fact,
did
the
achieved
and
Barros,
and
“Sperm
the
results
by
1969)
with
be fully
advantage
to resolve
1967;
Iwamatsu
that
trying
cumulus
Reports
Austin,
the
of ovupenetrathe same
entering
time,
by
denuded
attempted
products
hasten
of
these
activity
is
eggs
in
authors
could
oviduct
fluid
even
mating.
This
sup-
confirmed
by
the
SPERM
TRANSPORT
IN
THE
through
ACKNOWLEDGMENTS
the
investigation
This
Health
was
Service
supported
Research
by
Career
a U.S.
pub-
Development
Award
(No. K4-HD-42,369)
and by a U.S. Public
Health
Service
Research
Grant
(No.
HD-02597)
from
the National
Institute
of Child
Health
and
Human
Development.
Sincere
thanks
are due to
Ann
Elliott
S. W.
for excellent
technical
assistance.
cumulus
through
of the mouse
17, 203-206.
BARBOS,
C., AND AUSTIN,
fertilization
and
the
J. Exp.
J. M. (1968).
in the
hamster.
BEDFORD,
in
capacitation
of
Rec.
Anat.
160,
the
sperm
rabbit.
head
Amer.
J.
vitro
capacitation
with Fallopian
and
J. Reprod.
rat.
C. R. (1967).
sperm
Fert.
In
acrosome
vitro
reaction
fertilization
123,
in
L.,
AND
329-358.
M. C.
CHANG,
revealed
opment
CHANG,
ova
in
(1951).
the
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golden
acrosome
hamster
follicular
the
and
fluid
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pp.
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its
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YANAGIMACHI,
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corona
fluid.
pattern
Ultrastructure
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MA5m0IANNI,
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(1969b).
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of rabbit
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(1969a).
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18, 51-58.
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22, 147-152.
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rabbit
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(1972)
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