BIOLOGY
OF
8,
REPRODUCTION
Fertilization
362-368
(1973)
of Frog
Body-Cavity
and
Rana
of Zoology,
Received
Ovulated
are
amphibian
known
as
of Toronto,
11, 1972;
that
eggs
body-cavity
Toronto
accepted
have
eggs.
Eggs
pipiens
P. ELINSON
University
July
Rana
Sperm
clamitans
RICHARD
Department
Eggs:
not
November
been
Body-cavity
5, Oniario,
8,
coated
eggs
1972
with
are
Canathz
jelly
usually
not
in
the
oviduct
fertilized
when
with
sperm.
However,
a low frequency
of fertilization
of body-cavity
eggs
of the frog Rana
pipiens
was
obtained
when
the
eggs were inseminated
by high
concentrations
of R. pipiens
sperm
in the presence
of a water
extract
of jellied
eggs (egg-water).
The frequency
of fertilization
was not predictable
from the sperm
concentration.
Although
most
of the resulting
embryos
had
difficulty
completing
gastrulation,
a few were
raised
to metamorphosis.
‘When R. pipiens
body cavity
eggs
were
inseminated
in R. pipiens
egg_water
by sperm of the frog
R. clamitans,
high
frequencies
of fertilization
were
obtained.
Sperm
concentrations
greater
than
2.5 x 10#{176}
sperm/nil
generally
led
to frequencies
of fertilization
greater
than
60%.
Most
of the
resulting
embryos
were
diploid,
and
all
arrested
at
gastrulation,
a
characteristic
of the pipiens-clamitans
hybrid
cross.
These
results
indicated
that
the R. clamitans
sperm
penetrated
into
the
R. pipiens
body-cavity
egg
and
that
syngamy
occurred.
The
results
demonstrate
that
R. pipiens
eggs
need
not
pass
down the oviduct
or acquire
a jelly coat in order to be fertilizable.
placed
by
Amphibian
a jelly
coat
when
placed
Rugh,
1935;
1953;
bara,
eggs that are
are normally
with
Good
1965;
Katagiri,
Shivers
and
Su
Barbieri
James,
Wolf
kinds
and
Hedrick,
of eggs that
Eggs
that
and
Aplington,
Subtelny
1960;
not
surrounded
fertilized
and
Kam-
tion
some
1956;
Wang,
and
Raisman
have
had
are
known
whereas
eggs
that
as
1971;
1966).
have
jelly
coat
dejellied
never
In
eggs,
high
reeggs,
had
and
eggs
in
the jelly.
contrast
to
it has
not
frequencies
experiments
eggs using
the same
are successful
on dejellied
1965;
Elinson,
1971;
Wolf
they
had
It has been
possible
to fertilize
eggs
by means
of a variety
of
(Kambara,
1953; Katagiri,
1965,
Barbieri
and
changes
1969;
Raisman,
eggs.
362
Copyright
All rights
#{174}
1973
of
by
reproduction
The
in
Society
any
for
form
the Study
reserved.
of
Reproduction
associated
with
were
inseminated
previous
contact
Despite
high
some
for
fer-
(Shaver,
on
been
possible
of fertilization
the
egg.
dejellied
techniques
of
from
that
dejellied
to obtain
of body-
techniques
that
eggs
(Katagiri,
and
Hedrick,
cavity
a jelly
1971;
the
most
insemina-
the presence
It is clear
experiments
1971).
Shivers
and James
that Rana
pipiens
body-cavity
1967;
For
the
the jelly are important
the
amphibian
egg
coat are called
body-cavity
eggs. Body
cavity eggs
have
not travelled
down
the oviduct
where
the jelly
is deposited
around
1966a,b,
Elison,
1971).
involve
other
of
Elinson,
their
of dejellied
extract
of
in
tilization
Pisan#{243}, 1970;
Hedrick,
methods
molecules
1969;
1971).
There
are two
do not have
a jelly coat.
moved
and
these
these
1957;
Shaver
Bradt,
1961;
Raisman,
1970;
and
Wolf
part,
1919;
sperm
(Bataillon,
and Daniel,
1943;
Tchou
1957;
Nadamitsu,
and Barch,
not
(1970)
reported
eggs showed
fertilization
when
sperm
that had
with
fully
jellied
with
frequencies
of
activa-
OF
FERTILIZATION
tion,
the
frequency.
eggs
failed
to cleave
Subtelny
and Bradt
at
(1981)
FROG
body
cavity
eggs
an additional
with
do
so
in
James
whether
the
(1970)
the
R.
can
cleave
nucleus
of
R.
clamitans,
and
the
R. clamitans
sperm
(Elinson,
is a water
extract
sumably
contains
in
Based
on
fertilizing
R.
to
paper
to
(Elinson,
The
report
the
MATERIALS
Sexually
Minnesota.
R.
con-
of
outcome
R.
10%
Ringer’s
of
the
tray.
of
pipiens
females
Schettle
Sexually
Frog
Co.,
Farm,
R.
mature
with
day,
no
pignient
sult
this
develop.
the
eggs
cup
pipi ens
solution
added
sperni
in
one
suspension
The
inseminated
1#{176}C.
the
eggs
were
removed
cleaved
or
data
have
eggs
if
in
decay.
they
from
the
Eggs
were
disrupted
that
animal
were
Cy-
pigmentation
and
activated
but
not be determined
activated
by
not
cytolyzed.
were
It could
were
as such
R.
Ringer’s
added.
or
a
had areas that
were
poorly
the vegetal half. Eggs were scored
if they
were
uncleaved
and had
the
eggs
to
into
were
completely
in
in
from
10%
of
±
disruption
included
1971)
placed
some
often
unfertilized
19
insemina-
a wide-mouthed
pipette
and scored
Eggs
were
scored
as fertilized
complete
or partial
blastulae.
Most
although
tolyzed
was
at
For
unactivated
in
drop
ml)
following
cleaved
was
addition
one
Haven,
previously
(Elinson,
five
before
as
1971).
rinsed
were
half
re-
failed
whether
handling
and
rough
that
unfertilizable
or whether
they
had
been
fertilized
but
did not develop.
Usually,
about
5% to 10% of the number
of eggs in an experi-
those
males
Since
Alburg,
were
chromosome
tinguish
Stillwater,
clamitans
a
micro-disNew
used
egg-water
Next,
placed
Linbro
mental
series were cytolyzed.
Chromosome
squashes
of embryos
pared
by the method
of DiBerardino
and
and
appearing
Co.,
procedures
solution
Finally,
were
in
(Elinson,
of
eggs
embryos
METHODS
J. M. Hazen
from
J.
of
The
1971).
purpose
AND
mature
ml
the cups with
for fertilization.
if they
were
experiments.
were obtained
Vermont
and
the
eggs
0.05
eggs
it was decided
to try
body-cavity
eggs with
sperm.
is
dejellied
drop.
Egg-water
eggs
and premolecules
im-
fertilization
normal
determined
Chemical
following
immediately
green
sperm
unpublished).
of jellied
the jelly
this result,
R. pipiens
clamitans
for
(Linbro
body-cavity
sistenfly
fertilized
R. pipiens
dejellied
eggs
in the
presence
of R. pipiens
egg-water
at a higher
frequency
than
did
R. pipiens
portant
Conn.)
tions,
between
pipiens
with
wa
performed
was
trays
(0.045-0.055
cross-fertilization
sperm
suspension
Insemination
poso
and
raises
the
question
as
eggs
were
actually
fertilized
active
a
in
363
EGGS
hemocytometer.
provided
failed
to
Shivers
of
heads
of a
fact that
when
yet
experiments
in the latter
case.
In a study
of
the leopard
frog
frog
transplantation
the eggs.
The
CAVITY
number
a high
suc-
ceeded
in getting
R. pipiens
body-cavity
eggs to develop
normally
through
the biastula
stage
following
foreign
nucleus
into
BODY
plates
counts
with
24
were
haploid
between
to
27
were
pre(1962).
made
and
only
diploid
chromosomes
were
to
dis-
embryos,
considered
males
diploid.
A diploid
cell is expected
to have
28
were
obtained
from
Lake
Champlain
Frog
Farms,
chromosomes.
The
discrepancies
were
probably
Alburg,
Vermont.
All
frogs
were
stored
at 4#{176}C due
to
misinterpretations
when
counting
overuntil
use.
lapped
chromosomes.
However,
aneuploidy
cannot
Body cavity eggs from R. pipiens
females
were
be ruled
out especially
since
the embryos
produced
obtained
as described
previously
(Elinson,
1971).
were
lethal
hybrids.
The
eggs
to
3
of
the
were
hours
Ringer’s
kept
before
in
Ringer’s
solution
insemination.
solution
was:
The
6.6
g
for
NaCI,
0.15
KCI, 0.21 g CaCb-2
11,0, and 0.20 g NaHCO,
made to 1 liter with distilled
water.
Sperm
were
obtained
by crushing
the
testes
in
10% Ringer’s
solution.
Large
pieces
of testis
debris
were
removed
by filtering
the suspension
through
with
Nitex
(Tobler,
Ernst,
and
1
composition
Traber,
Inc.)
a mesh
opening
of 2Om.
The
suspension
was
centrifuged
for 1 to 4 mm
at
top
speed
(1470g)
in an International
Clinical
Centrifuge.
The supernatant
was
discarded,
and
the pellet
was resuspended
in fresh
10% Ringer’s
solution.
This
suspension
was
used
in the experiments.
The
RESULTS
g
Fertilization
Eggs
of R. pipiens
by
R. pipiens
When
inseminated
R. pipiens
body
cavity
eggs
in 10% Ringer’s
solution
pipiens
sperm,
no
eggs
When
they were
inseminated
no eggs
from
about
half
used
centage
Body-Cavity
Sperm
were
fertilized,
of the
eggs
and
from
were
by R.
were
fertilized.
in egg-water,
of the
females
only
most
a low
of the
perother
364
ELINSON
100
80
w
interior.
External
present
on the
neural
third
died
#{149}
Many
#{149}
but
several
tadpoles
were
These
appeared
normal,
morphosed
frogs
have
been
60
I.0)
embryos
differentiation
these
was
day after
insemination.
within
the first week,
maintained.
and
metaraised
from
tadpoles.
40
Fertilization
Eggs
20
2
4
6
ib
8
1.
by
the
Fertilization
R.
sperm.
insemination
from
one
were
in
female.
egg-water
Eggs
inseminated
so that
the
from
20
fertilization
from that
R. pipiens
body-cavity
Each
point
represents
of
pipiens
from
at
points
of
some
two
of the
figure
represent
Points
not
included
were
fertilized
eggs
females
concentrations,
females.
were
female
25
least
sperm
in this
different
at
at
no
some
eggs
another
sperm
concentration.
quency
of
fertilized
correlation
(Fig.
1).
between
There
the
and
concentra-
fertilization
tion of sperm.
males,
however,
frequency
the
Eggs
from
a few
were
fertilized
(Fig.
1).
For
was
fre-
in
eggs
used.
dependent
on
the
(Fig.
above
R.
2.5
produced
sperm
of
X
few
90%
trial,
eggs
from
R.
clamitans
led
(3.4
X 10#{176}and
both
of the
The
greater
The
S
#{149}
S
of
concentration
groups
of
1.8,
late,
porate
eggs.
jellied
embryos
required
eggs
that
all
most
the
to
used
obtain
.
U,
S
40
.
#{149}
vegetal
:
.
20
S
#{149}#{149}
#{149}.
a
10
sperm
90% of
1.5, 1.8,
Fic.
eggs
2.
Fertilization
the insemination
from
one female.
from
began
the
to
failed
cells
fer-
gastru-
to
into
incor-
the
that
the
25
different
tion
that
at
points
were
female
concentration.
egg-water
Eggs
several
concentrations,
figure
represent
Points
indicating
included
were
from
sperm
in the
pipiens
body-cavity
point
represents
of at least
25 eggs
some
females
were
Each
sperm.
in
females.
not
R.
of
by R. clamitans
4
6SPERM/ML
comparison,
inseminated
resulted
embryos
yoiky
S
60
were
fer-
R. pipiens
to fertilize
gave
0.44,
eggs
#{149}
.
U.’
eggs
at
sperm/nil.
body-cavity
but
S
#{149}#{149}
For
of the
1.9 X 10
and
The
tilized
jellied
determinations
S
S
#{149}
concentrations.
concentrations
those
needed
#{149}
S
S
one
of the
fertilized
sperm
.#{149}
sperm/mi),
one-quarter
female
were
sperm
sperm
than
tilization
five
10.2 X 106
about
other
to fer-
more
of the
eggs,
below
1 X 10#{176}/mi
100
were inseminated
using
the same egg-water
and the same
sperm
suspension
at two dilutions.
All the eggs
from
one female
were
fertilized
at
either
sperm
concentration
whereas
from
the
sperm
concentra-
fertilizations.
females
two
the
of fertilization
2).
Sperm
10#{176}/miusually
of 60% or
concentrations
eggs
In all
than
80
experimental
were
clamitans
from
more
The frequency
concentration
tilization
whereas
body-cavity
frequency.
trials,
eggs
females
of the feat a high
instance,
R. pipiens
at a high
experimental
tions
females
were
little
if any
body-cavity
in 10% Ringer’s
solution
by R.
sperm,
3 eggs
out of 675 were
When
the insemination
was done
fertilized
was
Body-Cavity
Sperm
pipiens
in egg-water,
were
fertilized
the
eggs
indicating
unless
R.
inseminated
ciamitans
fertilized.
“i
10 6SPERM/ML
Fic.
R. clamitans
When
..
-a--.
eggs
of R. pipiens
by
unless
fertilized
some
at
so
eggs
from
no fertilizaeggs from
another
sperm
OF
FERTILIZATION
concentrations
used
body-cavity
required
For
eggs
obtain
to
comparison,
clamitans
R. pipiens
0.84,
the
sperm
jellied
and
2.9
Eggs
to
the
to
together
swollen
fertilization
stick
unfertilized
eggs,
and
of
1971).
In
to six cups
A
eggs
same
fertilization
scoring
flaccid,
cup.
dejellied
of
fertilized
and
all five
eggs
cups
not being
fertilized.
average,
three
eggs
of the
the five cups
were
fertilized
quency
of fertilization
of
embryos
was
eggs
also
through
two
Rather,
on the
five in each
of
to give a fre-
of 60%.
of the
in the
developing
It is possible
activated
and
R. clamitans
develop
through
haploids
(Nace
R.
the
sperm’s
velopment.
R. clamitans
pipiens
perimental
but
caused
irradiated
the
was
deex-
haploids
in
the
the
egg
to
begin
developing
rule out these
two
.parthenogenetically.
possibilities,
two
studies
were
whether
the
done
clamitans
development
body-cavity
numbers
to
see
nucleus
of
of
was
the
or
not
present
embryo
egg.
First,
15 blastulae
To
during
from
the
were
body-cavity
radiated
the
the
develop,
males
The
3a).
When
were
sperm,
at the yolk plug
although
there
result
if syngamy
pronuclei
had not
appeared
somes
in
each
of
The
of the
occurred.
to be
male
diploid
remaining
numbers
the
arrest
of R.
develop-
plates
of
and
Eleven
and
three
emchromo-
counted,
and
began
trulate
nucleus
light.
dose
The
gynogenetic
used
R.
to inpipiens
by unirallowed
blastulae
what
began
appeared
a dorsal
remained
eggs
to
lip, whereas
as blastulae
the same
feirradiated
R.
44 of 52 healthy
blastulae
from
by
Most
stage
was
(Fig.
3b).
eggs
clamitans
demonstrated
was
present
of
at the
variation
body-cavity
R.
were
was
fertilized
sperm
and
2 had
gastrulation.
of gastrulation
R. pipiens
unirradiated
sperm
suspension
were
fertilized
and
one was haploid.
bryos
had
different
as
1970).
R. clamitans
R. clamitans
When
of 54 healthy
2
88%,
96%. Haploidy
sperm
eggs.
be the beginning
of
the other
52 embryos
(Fig.
chromosome
determined.
the
tilization
embryos
they
is inacti-
100%
eggs
were
clamitans
R.
gastrulation.
clamitans
of the
ultraviolet
jellied
clamitans
of the
gas-
that
early
to produce
when
seminate
to
R.
with
sufficient
three
separate
exfrequencies
of fer-
would
female
the
during
R.
half
from
the
69%,
by
that
present
The
embryos
were
periments
in which
were
eggs
gastrulation
et al.,
nucleus
fertilized
indication
is
egg
that
did not participate
is also possible
that
conditions
sperm
eggs
is an
in
of
vated
by the ultraviolet
light.
The
at the
end
of the
blastula
stage
Accordingly,
prepared
and
sperm
body-cavity
development
nucleus
It
sign
ment.
the
R.
initiated
R. pipiens
clamitans
arrests
no
Ting,
1951).
When
irradiated
with
ul-
light,
The
pipiens
normally
but
or
traviolet
nucleus
nucleus
embryo
that
little
the
sperm
When
a
a R.
stage
with
pipiens
sperm
clamitans
light.
fertilize
gynogenetic
sperm
Presence
blastula
the
pipiens
develops
trulation
(Moore,
1949;
R. clamitans
sperm
are
not a
being
other
the
the
in
R.
fertilizes
embryo
syn-
show
inseminate
sperm
the
development
to
with
R.
ultraviolet
eggs
that
chromosomes
to
with
egg,
of chromosomes
indicated
paternal
clamitans
jellied
to either
the
The
pres-
occurred.
experiment
presence
was
fertilization
in the
probably
The
second
R.
were
number
embryos
gamy
This
of 60% for a given
trial
was usually
result
of all five eggs
in three
cups
diploid
of the
irradiated
a typical
experimental
of five eggs each
were
frequency
of the
in most
body-cavity
Blastulae,
cytolyzed
in the
inseminated.
cups
for
and
had
membranes.
together
for
ence
R.
X 10/ml.
tended
(Elinson,
trial,
four
of
365
EGGS
CAVITY
the counts
did not correspond
haploid
or the diploid
number.
those
eggs.
needed
to fertilize
90% of
eggs were
0.45, 0.62, 0.78,
from
true
fertilized
than
jellied
concentrations
removed
found
obtain
were
higher
fertilized
BODY
FROG
that
in
the
44
were
time of scoring,
in the extent
The
failure
of
fertilized
sperm
by
to
gas-
the R. cla7nitans
development.
366
ELINSON
Fic.
3.
embryos
radiated
by
not
of
the
R. pipiens
body-cavity
eggs
fertilized
were fixed 42 hours
after
insemination.
R. clamitans
sperm.
The
embryos
are
absence
moved
of
into
this
embryo
a
blastopore
the
interior.
has
not
(a),
but
were
inseminated
clear
yolk
plug
indicating
The
fifth
egg
and
The
by
embryo
cleaved.
(b)
with
the
the
at
These
sperm
near
that
completion
by
The
eggs
arrested
presence
the
R. clamitans
uv-irradiated
(a)
in
of
lower
late
left
is
were
from
had
been
irradiated
a
gastrulation
is
DISCUSSION
fertilized
R.
use
of
not
sperm
requirement
for
since
fertilization
present
important
in
body-
R.
demonstrates
pass
down
coat
in
fertilization
measures
the
order
of
not
fertilization
of jellied
eggs,
presence
of egg-water
and the
higher
egg-water
presumed
pipiens
here
oviduct
or acquire
a jelly
to be fertilizable.
However,
the body-cavity
eggs required
for
the
concentrations.
egg-water
of dejellied
was
eggs
The
expected
required
(Elinson,
to contain
1971).
Egg-water
molecules
normally
the
jelly
for
egg’s
coat,
which
is
be
blastula.
with
ultraviolet
on
as
the
have
A
female
seen
seen
which
same
R.
pipiens
frequency.
pipiens
sperm
with
not
respeot
known.
four
portion
those
in
light.
A
embryos.
are
concentrations
of
R.
to some
fertilizations,
of R. clamitans
sperm
body-cavity
The
eggs
differences
R. clamitans
and
to their
fertilizing
It is interesting
sperm
ability
to note
are possible
American
between
Ranidae
ported
exception
that
with
sperm
are
clamitans
the
unable
to
(Moore,
fertilize
1949,
at
between
cross-fertilizations
species
of North
all
re-
foreign
eggs
1955).
are
that
of
Since
R.
R.
clamitans
sperm
can
fertilize
eggs,
they
must
be different
from
sperm
of other
Rana.
R. ciamitaris
in some
way
One
possibility
for
be
this
difference
clamitans
fertilization.
Although
high
pipiens
sperm
led
high
concentrations
unir-
can
cells,
partial
a high
needed
namely,
The
with
(12x).
is unfertilized
The
fertilization
of
cavity
eggs
reported
that
the frog egg need
as
vegetal
the
sperm.
inseminated
blastula
yolky,
eggs
of
were
coat
easier
sperm
(vitelline
than
other
would
that
the
penetrate
the
vitelline
membrane)
frog sperm
of
can.
can
sult,
they
can
get
past
vitelline
coat more
readily
egg
plasma
membrane.
the
As
the
extracellular
and contact
R.
egg
a rethe
OF
FERTIUZATION
The
fertilization
cavity
peared
of
R.
normal.
eggs
to be
probably
could
arise
diploids.
by
syngamy
or
and
a doubling
some
set
(diploid
suits
the
R.
of
radiated
however,
diploid
pected
would
were
be
in
sperm
Second,
of
it is not
unirradiated
would
affect
the
gastrulate
diploids.
if
Three
uv-irradiated
ability
of the
they
arisen
lowed
by
The
the
by
that
would
L.
Hall
be
R.
supported
and
To
does
polyspermy
tions
the
after
fertiuization
nuclei
more
eggs
for
not
negate
of body-cavity
would
decrease
studies
more
on
folthe
bryology
R.
used
ments
cavity
F.
D.,
Bufo
of
capacity
“If
it becomes
technique
sperm
cannot
be
by
used
for
the
technique
can
since
or to
cleave.
insemination
amphibia.
5.
the
be
This
(1989).
fertilization
de
nus
et
10,
l’activation
la polyspermie
(1962).
Ann.
Sd.
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The
investigation
of
its
of
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Develop.
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P.
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Zool.
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the
Embryologia
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A.
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coelomic
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of treaton the
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Na-
a Pure
from
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the
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the
101-126.
Calif.
the
a lethal
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body-cavity
Butler
by
J.
in
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M.
pipiens.
without
em-
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studies
However,
to be
it
of fertiliza-
body-cavity
The
RAISMAN,
(1919).
pipiens
during
eggs
normally
to experimental
from
and
eggs
involved
arenaruin
E.
tion
body-cavity
L.
manuscript
This
work
Grant
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AND
Ia technique
GOOD,
the
A6356
Canada
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factors
ELIN5ON,
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Dr.
363-372.
5,
fertilization
laboratory
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a boon
in
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Ohio J. Sci. 57, 91-99.
Aiworn,
J. F., AND SHAVER
J. R. (1962).
Interfemale
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of eggs
and ovaries
in the frog.
Exp. Cell Res. 27, 150-153.
Rana
division
into
obviously
that
No.
and
DIBERARDINO,
pro-
his
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on
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thank
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Natur.,
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but
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105)
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pipiens-clamitans
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of
clamitans
most
fact
fertilize
have
them
it will be
condi-
shortly
than
to
Research
H. W.,
body
par
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to
tion
the
normal
(1965,
possible
and
jelly,
these
examined
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has
the value
of
Rugh
text
under
be
et
Toronto.
Non-gametic
whether
or at first cleavage
for
than
two cells. Polyspermy
would
in
occur
must
Smith
reading
the
assistance.
of
Scientific
of
BAnajnssI,
have
of
determine
Grant
Council
Applied
University
of
classified
fertilization
distribution
like
by
Research
APL1NGTON,
to
may
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