BIOLOGY
OF
REPRODUCTION
19,
879-885
(1978)
An in vivo Analysis Influencing the Fertilization H.D.M. Departments
MOORE
and
of Obstetrics Cornell New
of Factors of Hamster
J.
and
M. BEDFORD
Gynecology
University
Medical
1300 York,
Avenue, York
York New
Eggs
and
Anatomy
College, 10021
ABSTRACT Most
current
environment can cricetine oviduct. which oocytes of some aspects
analytical studies be manipulated
As a preferred
are instilled of fertilization
into
of and
are
rodent fertilization also because of
alternative
for certain
the oviducts in vivo. Using
of mated
the
in vitro,
because
the
dimensions
of
murine
and
a technique
has been
conducted
small
questions, hamsters,
thereby
the
allowing
the
devised
in
investigation
ovariectomized native ova, it is shown that neither foilicular fluid, cumulus oophorus nor corona radiata are required for fertilization in vivo. A demonstrated beneficial effect of these products on fertilization rate was not specific, since maximal fertilization of cell free ova occurred when only homologous serum was used as their vehicle. Contrary to conclusions derived from recent in vitro studies, hamster diplotene oocytes are shown to be readily penetrable in vivo. The low rate of polyspermy in cell free ova transferred in serum denies the idea that the cumulus oophorus acts to protect against polyspermy by virtue of its physical presence. The high (23%) incidence in cell free ova transferred similarly but in protein free medium suggests rather that it is the state of the ovum which determines the efficiency of the block to polyspermy in vivo. females
and,
in some
cases,
with
this approach, in intact or unilaterally oocytes to distinguish experimental from
labeled
seems
INTRODUCTION
The
internal of the
ampulla
site of Fallopian
fertilization tube is one
unavoidable.
question
in the of the
to
mammalian
be
Notwithstanding asked
eggs
can
factors which has precluded easy analysis of the physiology of this event in mammals. However,
seems logical to do not been established
the conclusive demonstration zation of rabbit eggs with
support
(Chang, later of ozoa Chang,
of uterine
in
fertilispermatozoa vitro
Thibault and Dauzier, eggs with epididymal
1959;
hamster
1961) and spermat-
in vitro fertilization techniques of mammals. Subsequently, not of the small size of the oviduct in
rat, mouse and hamster, of fertilization in that ducted in vitro. This important
in
environmental
where the inhibitors or of some fertilization
most analytical studies group have been conapproach has proved
elucidating
questions
parameters
are
of
milieu,
component the
is required use
of
in vitro
first place, conditions
of it
it has which
necessarily
reflect
1967;
Chang, attribute
1976) there has been a tendency to it a specific role in fertilization
in the
al.
cells
eggs
absence (Bavister,
of
have
any 1969),
oviduct, 879
in in
been
fertilized
these as have
we
Chang,
have
Hanada
and to and
suggested
surrounding
role
and
Miyamoto and Notwithstanding
June 12, 1978 March 21, 1978
of
have
their
(Yanagimachi, Toyoda
1969;
(1972)
and
essential spermatozoa
hamster
mals
systems
Yanagimachi,
et
have an hamster
thus,
1972;
Accepted Received
so. In the that the
Austin,
follicular
persistent presence of certain enzyme or a controlled level of a certain ion other
analyzed
fertilization in vitro
Gwatkin
where
interest;
this, if the
fertilization in vivo, then
those required in the oviduct and, because of some conflict between in vitro studies, the importance of certain elements as physiological factors in fertilization remains unclear. For instance, in the finding that follicular fluid is supportive of fertilization in vitro (Barros and
capacitated in vitro (Yanagimachi and 1964), laid the way for development
of repeatable for the eggs least because
be
about
that
matrix
the capacitation the oviduct.
of Yet
in vitro
in
the
follicular those
components of other mam-
1972;
Pavbok
and
Chang,
1974;
1972). the small
devised
an
MacLaren,
Harper,
dimensions approach
1970,
of the whereby
880
MOORE
AND
oocytes, in some cases marked to distinguish them from native ova (Overstreet, 1973; Overstreet and Bedford, 1974), are transferred to the tubal has allowed vivo
ampulla in mated hamsters. This an investigation of the possible in of the
role
of the
products
follicle
for
fertilization in this species, of the penetrability of its immature of the bearing ovum on the
(diplotene) of foblicubar incidence
naturally
females.
mated
oocyte in vivo cells around of polyspermy
and the in
BEDFORD
by
dissecting
and
the
stage
To transfer
involved
AND
METHODS
instillation
of ovulated
ova or
and
manipulation
of
oocytes
displayed
be penetrated
vesicle.
Transfer
was
made
either
to intact oviducts or, in some cases, to the oviduct of a hamster from which the ipsilateral ovary had been removed surgically at least 2 cycles prior to the experiment. The natural products of ovulation were therefore absent in the latter case (Fig. 1). Donor female hamsters (80-120 g) were injected with 40 IU pregnant mare serum gonadotropin (PMS) (Sigma) on the day of the postestrus vaginal discharge and 56 h later with 40 IU human chorionic gonadotropin (hCG) (Sigma). About 15-20 ova in cumulus oophorus were recovered from each oviduct 1 5 h after hCG and were placed in TC 199 (Difco Lab) modified and ment, 0.1%
mm;
dilution
Chang,
10:3
1964).
mature ova hyaluronidase
with
0.125
M glycine (Yanagimachi all the cellular investwere placed in medium containing (415 units/mg) (Sigma) for 1-2
To remove
oocytes recovered from follicles were gently shaken for a minute in a small test tube containing 1 ml of 2% sodium citrate. This treatment removed all investing cells and those oocytes containing a germinal vesicle were set aside in modified TC 199. Where there was a need to distinguish native from transferred ova, the latter were first labeled by incubation for 1 mm in Tyrode’s solution containing 0.01% fluroscein isothiocyanate (FITC) (Overstreet, 1973) and
immature
washed
twice
in modified
TC
199.
Both
the
intact
and the unlaterally ovariectomized recipient females were kept on a reverse light cycle (0600-1800 h dark/1800-0600 light) for at least 2 weeks prior to experiments so that estrus was first displayed between 0600-0900 h. These animals were mated 4-7 h before the expected time of ovulation and anaesthetized with nembutal (Abbot) given i.p. 3 h later. Mature ova either in cumulus or denuded of follicular cells or, in some experiments, diplotene oocytes were instilled with a fine pipette through the periovarian bursal membrane to lie at the tubal ostium (Fig. 1). The ova were transferred either in modified TC 199, in follicular fluid collected from preovubatory ovarian follicles or in hamster serum previously heated to 56#{176}C for 1 h. Preliminary studies showed that the transfer of eggs could be completed within 10 mm of their collection and that a majority passed into the ampulla with 15 min. Eggs recovered at various times
first
TC
199
whether
polyspermy
oocytes taken from follicles into the oviducts recipients. Where foilicular oocytes were used they were recovered by pricking antral follicles of females sacrificed in diestrus, 30-56 h before the onset of the cyclic LH surge. The oocytes invariably
by
in
were
examined
of fertilization.
discover
tion
dipbotene of mated
a germinal
oviduct
a Leitz fluorescent phase contrast microscope and their identity as naturally ovulated ova, instilled ova or diplotene oocytes, established according to the fluorescence emitted by the FITC label. They were then mounted on wax spot slides and assessed for penetration before and after fixation in acetic alcohol and staining in lacmoid. In particular, a record was made of the number of polyspermic ova, of the supplementary spermatozoa in the perivitelline space
RESULTS
MATERIALS Egg
the
under
from
maximal
fertiliza-
occur system,
following diplotene
could
the
in
vivo
secondary
follicles
were
used,
for
since the fertilizing spermatozoon elicits no cortical granule reaction, these have either a weak or no zona block to polyspermy and can (Moore 95%
more
Bedford,
68
diplotene
of
when
by
and
recovered
16
than
one
1978).
spermatozoon
In 8 experiments,
oocytes
were
penetrated to the
h after their transfer
oviducts of 2.9
of intact mated (1-8) spermatozoa
through oocytes maturation,
the zona pellucida (Table 1). These had not undergone spontaneous as judged by the presence of a
germinal
vesicle
bodies.
Ninety-seven
ovulated
by in
normally
results allows
and
the
that and in
the
polar
transfer procedure there are sufficient
ampulla
for
if appropriately
of
the native ova were fertilized These initial
of
the that
a mean
penetrated
absence
those recipients the same oviduct.
spermatozoa place
and had
percent
indicated penetration
take
hamsters
polyspermy
receptive
to
oocytes
are
present. in
mass
either
in
their prior and
the
investigate
To cumulus
cumulus
possible
cellular investments to ovulation into unilaterally
ferred
to
intact
recovered invested
recipients
about the same native ova (100%),
rate but
instilled,
there
a small
tion
(8 1%)
rate
Table instilled the
2).
was and
were
a rise
hand,
the
side
percentage
mated
12-16 h ova transfertilized
drop
in the
fertiliza-
in polyspermy
If washed, cumulus into the oviduct of
ovariectomized
the ova of
(91%) as those of the when cell free ova were
of ovulation were absent, maximal (96%) with no other
hamsters
and were Cumulus
at
of
ovulated denuded
were instilled 2-3 h the oviducts of intact
ovariectomized
h previously after ovulation.
4
role
fertilization, or previously
(4.7%,
invested ova mated hamsters
where
natural
were on
products
their fertilization was polyspermy. On the of cumulus
free
ova
EGG
TRANSFER
IN THE
HAMSTER
881
TABLE 1. Sperm penetration rate following transfer of follicular (dipbotene) oocytes into oviducts of intact mated hamsters 2 h before ovulation. Type
of
No.
No. of hamsters
oocyte transferred
No.
of
Penetrated sperm per
of
oocytes instilled
oocytes recovered
108
Fertilization
oocyte
or ovum
rat (%)
68
2.9
..
73
1.0
97
Diplotene oocytes 8 Native
ova
..
aJthough
its
decondense
.
is penetrated
viteilus
in the ooplasm
spermatozoa
by
of immature
penetrated in that milieu, though stillas high as 66%, was significantly lower (P
OF
REPRODUCTION
19,
879-885
(1978)
An in vivo Analysis Influencing the Fertilization H.D.M. Departments
MOORE
and
of Obstetrics Cornell New
of Factors of Hamster
J.
and
M. BEDFORD
Gynecology
University
Medical
1300 York,
Avenue, York
York New
Eggs
and
Anatomy
College, 10021
ABSTRACT Most
current
environment can cricetine oviduct. which oocytes of some aspects
analytical studies be manipulated
As a preferred
are instilled of fertilization
into
of and
are
rodent fertilization also because of
alternative
for certain
the oviducts in vivo. Using
of mated
the
in vitro,
because
the
dimensions
of
murine
and
a technique
has been
conducted
small
questions, hamsters,
thereby
the
allowing
the
devised
in
investigation
ovariectomized native ova, it is shown that neither foilicular fluid, cumulus oophorus nor corona radiata are required for fertilization in vivo. A demonstrated beneficial effect of these products on fertilization rate was not specific, since maximal fertilization of cell free ova occurred when only homologous serum was used as their vehicle. Contrary to conclusions derived from recent in vitro studies, hamster diplotene oocytes are shown to be readily penetrable in vivo. The low rate of polyspermy in cell free ova transferred in serum denies the idea that the cumulus oophorus acts to protect against polyspermy by virtue of its physical presence. The high (23%) incidence in cell free ova transferred similarly but in protein free medium suggests rather that it is the state of the ovum which determines the efficiency of the block to polyspermy in vivo. females
and,
in some
cases,
with
this approach, in intact or unilaterally oocytes to distinguish experimental from
labeled
seems
INTRODUCTION
The
internal of the
ampulla
site of Fallopian
fertilization tube is one
unavoidable.
question
in the of the
to
mammalian
be
Notwithstanding asked
eggs
can
factors which has precluded easy analysis of the physiology of this event in mammals. However,
seems logical to do not been established
the conclusive demonstration zation of rabbit eggs with
support
(Chang, later of ozoa Chang,
of uterine
in
fertilispermatozoa vitro
Thibault and Dauzier, eggs with epididymal
1959;
hamster
1961) and spermat-
in vitro fertilization techniques of mammals. Subsequently, not of the small size of the oviduct in
rat, mouse and hamster, of fertilization in that ducted in vitro. This important
in
environmental
where the inhibitors or of some fertilization
most analytical studies group have been conapproach has proved
elucidating
questions
parameters
are
of
milieu,
component the
is required use
of
in vitro
first place, conditions
of it
it has which
necessarily
reflect
1967;
Chang, attribute
1976) there has been a tendency to it a specific role in fertilization
in the
al.
cells
eggs
absence (Bavister,
of
have
any 1969),
oviduct, 879
in in
been
fertilized
these as have
we
Chang,
have
Hanada
and to and
suggested
surrounding
role
and
Miyamoto and Notwithstanding
June 12, 1978 March 21, 1978
of
have
their
(Yanagimachi, Toyoda
1969;
(1972)
and
essential spermatozoa
hamster
mals
systems
Yanagimachi,
et
have an hamster
thus,
1972;
Accepted Received
so. In the that the
Austin,
follicular
persistent presence of certain enzyme or a controlled level of a certain ion other
analyzed
fertilization in vitro
Gwatkin
where
interest;
this, if the
fertilization in vivo, then
those required in the oviduct and, because of some conflict between in vitro studies, the importance of certain elements as physiological factors in fertilization remains unclear. For instance, in the finding that follicular fluid is supportive of fertilization in vitro (Barros and
capacitated in vitro (Yanagimachi and 1964), laid the way for development
of repeatable for the eggs least because
be
about
that
matrix
the capacitation the oviduct.
of Yet
in vitro
in
the
follicular those
components of other mam-
1972;
Pavbok
and
Chang,
1974;
1972). the small
devised
an
MacLaren,
Harper,
dimensions approach
1970,
of the whereby
880
MOORE
AND
oocytes, in some cases marked to distinguish them from native ova (Overstreet, 1973; Overstreet and Bedford, 1974), are transferred to the tubal has allowed vivo
ampulla in mated hamsters. This an investigation of the possible in of the
role
of the
products
follicle
for
fertilization in this species, of the penetrability of its immature of the bearing ovum on the
(diplotene) of foblicubar incidence
naturally
females.
mated
oocyte in vivo cells around of polyspermy
and the in
BEDFORD
by
dissecting
and
the
stage
To transfer
involved
AND
METHODS
instillation
of ovulated
ova or
and
manipulation
of
oocytes
displayed
be penetrated
vesicle.
Transfer
was
made
either
to intact oviducts or, in some cases, to the oviduct of a hamster from which the ipsilateral ovary had been removed surgically at least 2 cycles prior to the experiment. The natural products of ovulation were therefore absent in the latter case (Fig. 1). Donor female hamsters (80-120 g) were injected with 40 IU pregnant mare serum gonadotropin (PMS) (Sigma) on the day of the postestrus vaginal discharge and 56 h later with 40 IU human chorionic gonadotropin (hCG) (Sigma). About 15-20 ova in cumulus oophorus were recovered from each oviduct 1 5 h after hCG and were placed in TC 199 (Difco Lab) modified and ment, 0.1%
mm;
dilution
Chang,
10:3
1964).
mature ova hyaluronidase
with
0.125
M glycine (Yanagimachi all the cellular investwere placed in medium containing (415 units/mg) (Sigma) for 1-2
To remove
oocytes recovered from follicles were gently shaken for a minute in a small test tube containing 1 ml of 2% sodium citrate. This treatment removed all investing cells and those oocytes containing a germinal vesicle were set aside in modified TC 199. Where there was a need to distinguish native from transferred ova, the latter were first labeled by incubation for 1 mm in Tyrode’s solution containing 0.01% fluroscein isothiocyanate (FITC) (Overstreet, 1973) and
immature
washed
twice
in modified
TC
199.
Both
the
intact
and the unlaterally ovariectomized recipient females were kept on a reverse light cycle (0600-1800 h dark/1800-0600 light) for at least 2 weeks prior to experiments so that estrus was first displayed between 0600-0900 h. These animals were mated 4-7 h before the expected time of ovulation and anaesthetized with nembutal (Abbot) given i.p. 3 h later. Mature ova either in cumulus or denuded of follicular cells or, in some experiments, diplotene oocytes were instilled with a fine pipette through the periovarian bursal membrane to lie at the tubal ostium (Fig. 1). The ova were transferred either in modified TC 199, in follicular fluid collected from preovubatory ovarian follicles or in hamster serum previously heated to 56#{176}C for 1 h. Preliminary studies showed that the transfer of eggs could be completed within 10 mm of their collection and that a majority passed into the ampulla with 15 min. Eggs recovered at various times
first
TC
199
whether
polyspermy
oocytes taken from follicles into the oviducts recipients. Where foilicular oocytes were used they were recovered by pricking antral follicles of females sacrificed in diestrus, 30-56 h before the onset of the cyclic LH surge. The oocytes invariably
by
in
were
examined
of fertilization.
discover
tion
dipbotene of mated
a germinal
oviduct
a Leitz fluorescent phase contrast microscope and their identity as naturally ovulated ova, instilled ova or diplotene oocytes, established according to the fluorescence emitted by the FITC label. They were then mounted on wax spot slides and assessed for penetration before and after fixation in acetic alcohol and staining in lacmoid. In particular, a record was made of the number of polyspermic ova, of the supplementary spermatozoa in the perivitelline space
RESULTS
MATERIALS Egg
the
under
from
maximal
fertiliza-
occur system,
following diplotene
could
the
in
vivo
secondary
follicles
were
used,
for
since the fertilizing spermatozoon elicits no cortical granule reaction, these have either a weak or no zona block to polyspermy and can (Moore 95%
more
Bedford,
68
diplotene
of
when
by
and
recovered
16
than
one
1978).
spermatozoon
In 8 experiments,
oocytes
were
penetrated to the
h after their transfer
oviducts of 2.9
of intact mated (1-8) spermatozoa
through oocytes maturation,
the zona pellucida (Table 1). These had not undergone spontaneous as judged by the presence of a
germinal
vesicle
bodies.
Ninety-seven
ovulated
by in
normally
results allows
and
the
that and in
the
polar
transfer procedure there are sufficient
ampulla
for
if appropriately
of
the native ova were fertilized These initial
of
the that
a mean
penetrated
absence
those recipients the same oviduct.
spermatozoa place
and had
percent
indicated penetration
take
hamsters
polyspermy
receptive
to
oocytes
are
present. in
mass
either
in
their prior and
the
investigate
To cumulus
cumulus
possible
cellular investments to ovulation into unilaterally
ferred
to
intact
recovered invested
recipients
about the same native ova (100%),
rate but
instilled,
there
a small
tion
(8 1%)
rate
Table instilled the
2).
was and
were
a rise
hand,
the
side
percentage
mated
12-16 h ova transfertilized
drop
in the
fertiliza-
in polyspermy
If washed, cumulus into the oviduct of
ovariectomized
the ova of
(91%) as those of the when cell free ova were
of ovulation were absent, maximal (96%) with no other
hamsters
and were Cumulus
at
of
ovulated denuded
were instilled 2-3 h the oviducts of intact
ovariectomized
h previously after ovulation.
4
role
fertilization, or previously
(4.7%,
invested ova mated hamsters
where
natural
were on
products
their fertilization was polyspermy. On the of cumulus
free
ova
EGG
TRANSFER
IN THE
HAMSTER
881
TABLE 1. Sperm penetration rate following transfer of follicular (dipbotene) oocytes into oviducts of intact mated hamsters 2 h before ovulation. Type
of
No.
No. of hamsters
oocyte transferred
No.
of
Penetrated sperm per
of
oocytes instilled
oocytes recovered
108
Fertilization
oocyte
or ovum
rat (%)
68
2.9
..
73
1.0
97
Diplotene oocytes 8 Native
ova
..
aJthough
its
decondense
.
is penetrated
viteilus
in the ooplasm
spermatozoa
by
of immature
penetrated in that milieu, though stillas high as 66%, was significantly lower (P
