BIOLOGY
OF
45, 245-251
REPRODUCTION
Overcoming
the 2-Cell
(1991)
Block
by Modifying
Standard
Culture J. A. Laboratory
of Human
J.
and
and
Boston,
in a Mouse
Embryo
Medium1
LAWIUS
Reproduction
Components
D.
BIGGERS2
Reproductive
Biology,
Massachusetts
Harvard
Medical
School
02115
ABSTRACT The Almost when KH2PO4,
2-cell all
zygotes
cultured
embryo
may
be
obtained in
glucose,
or KCI depend in several
block
on
the
with
development
by
Whittingham’s and
media
caused
by
fertilizing
CF1
medium
pyruvate,
either
concentration lowered since
inappropriate
the
ova
M16.
hybrid
2-cell
of commonly B6D2F1/CrIBR
block
was
in the
medium.
of certain
of blastocysts
preimplantation undergo blocks the block occurs
species
mouse
its occurrence
served
as
it
has
only
effects
the
media
of embryo
constituents
did
by
a high
Although
not
develop
beyond
lowering
the
concentrations
of changing
the
concentration
percentage not
are
of embryos optimal
for
grew complete
culture the
media.
2-cell of NaCI,
of either to the
4-cell
stage KCI, NaC1 stage
preimplantation
is low.
the appropriate adjustment of the concentrations of NaCI, KC1, Kl-12P04, pyruvate, glucose, and NaHCO3. These findings provide direct evidence that blocks to development are caused primarily by inappropriate concentrations of some common constituents of media in current use.
embryos of several mammalian in development in vitro [1]. In the at the 2-cell stage [2, 3]. However,
is strain-dependent, and inbred
The
components,
INTRODUCTION The
used sperm
overcome
or in combination.
of NaHCO3 yield
with
This
individually
concentrations
concentrations
been
ob-
in random-bred
strains but not in F1 hybrids between inbred strains [4,5]. The stage when the block occurs has been correlated with the time the embryonic genome takes over control of development from the maternal genome, which occurs in the mouse at the 2-cell stage [6]. In the absence of direct evidence that blocks to development occur in vivo, it is likely that the blocks to development observed in vitro are caused by adverse culture conditions, such as imbalances in the concentrations of certain constituents [5]. These adverse conditions then result in either impaired transmission of inherited maternal information [7] or defective activation of the embryonic genome [8]. While using simplex methods for optimizing the composition of culture media for the culture of zygotes from random-bred mouse preimplantation embryos [9], we observed that passage through the 2-cell block occurred in media with low concentrations of several components commonly found in culture media, particularly NaC1, KH2PO4, glucose, and pyruvate. We have therefore examined the effects of lowering the concentrations of several constituents in a commonly used medium, M16 [10], in which randombred mice experience the 2-cell block. The results show that the 2-cell block can be overcome in this medium by
MATERIALS
AND
METHODS
Animals Random-bred hybrid male
female mice (Crl:CF1 BR, 6-8 wk old) and mice (B6D2F1/CrIBR) were maintained on 14L: 1OD (lights-on, 0500 h). Female mice were superovulated by i.p. injection of 5 IU eCG (Sigma, St. Louis, MO) at 1530 h, followed by an i.p. injection of hCG (Sigma) 47 h later. After hCG injection, females were placed in cages with males and examined the following morning (Day 1) for the presence of a vaginal plug. Preparation
of Culture
Media
prepared from individual stock solutions of of M16 (Table 1). All components were Sigma Most of the components were stored in 0.1 M solutions, with the following exceptions: NaCI and NaHCO3 were stored at 1.0 M, whereas BSA (fraction V) was stored at a concentration of 100 mg/mI, CaC12 at 0.171 M, and penicillin-G and streptomycin sulfate were stored as one solution at concentrations of 10 000 U/mI and 5.0 mg/ ml, respectively. All cQmponents were stored at 4#{176}C, in 50ml conical centrifuge tubes (USA Scientific Plastics, Ocala, FL) except for the antibiotic mixture, which was stored at 15#{176}C in 5-mi polypropylene tubes (#2063, Falcon Plastics, Los Angeles, CA). Fresh solutions of NaHCO3, pyruvate, and lactate were prepared weekly. BSA was prepared every time media were made. Ten milliliters of each medium was prepared on the day Media
were
each
component purchased from
-
April 1, 1991. January 14, 1991. ‘The work reported in this paper was part of the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Development, grant number HD-21988. 2Correspondence: Dr. John D. Biggers, Harvard Medical School, LHRRB, 45 Shattuck St., Boston, MA 02115. FAX: (617) 566-7980. Accepted
Received
prior 245
to the
start
of each
trial.
The
components,
except
for
246
LAWITF
S AND
stage, bator
TABLE 1. Composition of medium M16 (Whittingham (101). Component
Concentration
NaCI KCI KH2PO4 MgSO4 Lactate Pyruvate Glucose BSA NaHCO3 CaC12 *BSA
E.tperimental Four
4.00 25.00 171
dia Peni-
respectively.
CaCl2, were added to culture tubes (Falcon #2051), distilled H20 was added to bring the volume of each tube to 9.0-9.5 ml, and then CaCl2 was added. Finally, distilled H20 was added to bring the final volume of each medium to 10 ml. Each tube was inverted twice to mix the components and then the contents were drawn into a disposable syringe (Becton-Dickinson, Rutherford, NJ) and the medium was pushed through a 0.2-p.m filter (Millipore, Bedford, MA) into a second tube. Collection
Mice were killed between 1530 and 1545 on Day 1. Pronuclear stage zygotes were flushed from each oviduct with 0.1 ml of Dulbecco’s phosphate-buffered saline (dPBS). Pooled zygotes from several donors were washed through one drop of dPBS containing 0.65 mg/mI hyaluronidase (Sigma) to remove any cumulus cells, followed by two drops of dPBS. Embiyo
conducted.
Each
used
in each
experiment.
Three
During into each trials
each trial, of the me-
were
conducted
Methods
The experiments were analyzed by an analysis of variance after transformation of the data by a two-term inverse sine transformation, proposed by Laubscher [11], given by: n”2 sin’
=
+
where
x n
= =
The
advantage
used
angular
portions distribution
(n
+
(x/n)h/’2
sin1
1)h/’2
[(x
+
3/4)/(n
+
the number of responders, the group size. of this
over
is that
it is more
0.9. The theoretical approaches 1 as n increases.
transformed
responses
components polynomial
of the coefficients
on
the
media
were
[12].
sion coefficients are those variables are transformed
The
obtained
3/2)1h/2,
and
transformation
transformation
Culture
Zygotes were cultured in tissue culture dishes (#3060, Costar, Cambridge, MA) in 50-p.l drops of medium under 5 ml of silicone oil (50 centistokes; Sigma). The dishes were made up the day before the start of each trial and were equilibrated overnight in an atmosphere of 6% C02, 5% 02, and 89% N2 in a plastic culture chamber (Billups-Rothenberg, Del Mar, CA) inside a 37#{176}Cincubator. Each culture dish contained 4 drops. Ten zygotes were transferred from dPBS into drop 1. Any remaining dPBS in the transfer pipette was expelled and the pipette was refilled from drop 2. The ten zygotes were then transferred into drop 2, and this procedure was repeated with drop 3. Drop 4 was a spare. The culture dishes were returned to the culture chamber at 37#{176}C, which was gas-equilibrated and then sealed. Developmental progress was recorded, as described below (statistical methods), at 24 h, 48 h, and 96 h after the embryos were placed in culture. When the culture dishes were outside the culture chamber and not on the microscope
experiment
at three different 27 media were com-
for each experiment, for a total of 30 embryos per treatment group. The responses to the media were assessed as the proportion that were 4-cells on Day 3 post-hCG, and the proportion that reached the blastocyst stage on Day 5. Statistical
Embryo
were
pared in a single trial in each experiment. 10 zygotes were transferred at random
cillin and streptomycin were also added at concentrations of 100 U/mI and 50 p.g/ml,
incu-
Design experiments
compared three different components levels, in a 33 factorial design. Thus,
5.56
is in mg/mi.
they were maintained at 37#{176}Cin a bench-top (Baxter Scientific, McGaw Park, IL).
(mM)
94.70 4.78 1.19 1.19 23.30 0.33
concentration
BIGGERS
the
commonly
stable
concentrations fitted when
of this of the of
using
estimates
to the scale
for pro-
variance Regressions
specific
orthogonal
of the the
regres-
independent
-1,0,1.
RESULTS Development
to the 4-Cell
Stage
Effect of NaC1, K!-12P04, and glucose. All combinations of three concentrations of NaCl, KH2PO4, and glucose were tested. The concentrations used were NaCI: 75, 85, and 95 mM; KH2PO4: 0, 0.6, and 1.2 mM; and glucose: 0, 2.75, and 5.5 mM. The analysis of variance (Table 2) showed that all three main effects, from NaCI, KH2PO4, and glucose, were highly significant, while all the interactions were not significant (p > 0.05). Thus, the effects of the three compounds can be considered to be independent. Regression analysis orthogonal
of the
main
polynomials,
effects
of the confirmed
three
components, that
only
using the
linear
components were significant over the concentrations studied. All three linear regressions had significant negative slopes (Fig. la,b,c). Therefore, increasing the concentration of any of the three components independently depressed the proportion of embryos reaching the 4-cell stage. The apparent
OVERCOMING
TABLE
2.
Source
Analysis
of variance
THE
of the data
of Variation
df
Between
replicates
2
Between
media:
different
medium,
having the concentration
same
bryos reaching was very low two
suggested but this
significance at the p = 27 cell (treatment) means,
statistical The
were
the that
ering
of the
cell
means
a variety
concentrations
in three
0.041
1 1
41.364
to 4-cells. to the
involving
4 4 4 8
0.723
KH2PO4,
other
the and
low-
glucose
100
0.92
30.87 0.030
0.040
3.986
0.462
0.672 1.913 1.34
could
-
90
pyruvate
-
80
Regression
-
70
-
60
-
50
the effects of NaCI and ative linear regression inhibited development
-
40
-
30
-
20
-
10
#{149} MEAN RESPONSE
0
a:
p 0.0006
0.010
8.898
(M16) With
medium
-
z
F 8.54
66.02
1 1
each
(c)
(b)
square
88.471
10
(a)
1.
11.448
1
to a
groups,
corresponding of changes of NaC1,
Mean
1
that at 5.5 mM failed to reach
the 4-cell stage in the control (3%); 29 of 30 failed to grow
showed
247
in Figure
52
0.05 level. each corresponding
plotted
summarized
concentration of NaCl (Fig. la) or the same of glucose (Fig. ic). The percentage of em-
exceptions,
media
also
BLOCK
(26)
NaCI: Linear Quadratic KH2PO4 Linear Quadratic Glucose Linear Quadratic Interactions: NaCI x KH2PO4 NaCI x glucose KH2PO4 x glucose NaCI x KH2PO4 x glucose Error
curvature in the effect of glucose the inhibitory effect was maximal,
2-CElL
analysis
0.05).
Thus,
considered using
the
effects
independent
orthogonal
of NaCI (Fig.
polynomials
shows
pyruvate can be summarized lines. The results confirm as the concentration was
and
2a,b,c). that
by negthat NaCl increased
0
Ui
a:
N
4
0
LL
3
(I)
z
2
The medium,
a: I-
from 75 to 95 mM, and that pyruvate was also the concentration was raised from 0.13 to 0.33 lactate, however, had no effect.
1
M16
-
Ml 6
0#{149}
1
75
85
NaCI
95
I
I
0.0
0.6
KH2PO4
CONCENTRATION
1.2
0.0
2.8
5.5
GLUCOSE
(mM)
FIG. 1. The proportion (expressed either as the transformed proportion or a percentage) of CF1 mouse zygotes that develop to at least the 4cell stage in media containing combinations of different concentrations of NaCI, KH2PO4, and glucose. (a) The main effect of NaCI. (b) The main effect of KH2PO4. (c) The main effect of glucose. Note that the symbols show each cell (treatment) mean (n = 3) for the transformed proportions of each of the 27 media tested.
27
cell are
ing the same bryos reaching
means,
plotted
each
corresponding
in Figure
concentration the 4-cell
stage
2a in three
inhibitory as mM. Varying to
a different
groups,
each
of NaCI. The percentage in the control medium
hayof em(M16)
was somewhat higher than in the first experiment (23%), due mainly to four embryos that developed in the third replicate. Nevertheless, the cell means shown in Figure 2a demonstrate that a variety of changes in the composition of medium M16 produced by adjusting the concentrations of NaCl and pyruvate overcame the block to a lesser greater degree. Figure 2a (solid circles) also shows that ducing the pyruvate fect to that of reducing ing the block.
concentration the NaCI
had a clear concentration
or re-
additive efin overcom-
248
LAWITfS
AND
when
the
100
rather
than
90
In experiment three concentrations regression lines.
10 (a)
z
9
#{149} 0.13 0.23
mM P’YR mM PYR
8
o
mM
0
a:
0.33
#{149} MEAN
0
(c)
(b)
PYR -
RESPONSE
80
7
3-
#{149}
0
a: a-
.1 5
0
#{149} I
1-±
4
-f
M16-
0
o
3
U-
50
N
Ui
a:
60
-
z
a:
m
10
5.8
NoCI
11.6
23.3
0.13
LACTATE
0.23
PYRUVATE
CONCENTRATION
of NaCI, were
centrations periment
KC1, and
done
(mM)
not
Two all combinations
of NaCl, KCI, and A, the concentrations
summarized in Figure effect for NaHCO3 was
NaCI
NaHCO3.
in which
95 mM; KCI: 0, 2.4, and and 25.0 mM. The results
KCI was was not
marginally significant.
x NaJ-ICO3 significant.
factorial experof three con-
NaHCO3 were tested. In exused were NaC1: 75, 85, and
4.8 mM; and NaHCO3: 6.25, and the analysis of variance
3, a and highly
b, and
Table
significant,
significant, However,
the
3. The main
12.5 are main
effect
of
and the main effect for NaC1 there was a very significant
interaction. In experiment
All the other interactions B, the concentrations
were of NaC1
and KCI remained the same as in experiment A; however, the two lower concentrations of NaHCO3 were raised to 10 and 17.5 mM. The results and the analysis of variance are summarized
in Figure
to experiment
The
3, c and
A, where
of all three components nificant. Furthermore, NaHCO3 interactions
teractions
were two-way
KCI X NaHCO3
NaCI
had
d, and
Table
no effect,
3. In contrast the
main
effects
in experiment B were highly sigthe NaCl X NaHCO3 and KCI X were highly significant. All other in-
not significant. tables
showing
interactions
lustrated in Figure 3, a-d. cations and slopes of the NaCl and KCI concentrations of NaHCO3 in the ments reveals that
5b
medium. the overall
the from
NaCI
both
X
the
NaHCO3
experiments
and are
mM
df the
df
52),
=
80%
indicating
that
of NaC1 did mM NaHCO3,
stage. The data corresponding of KCI is also represented The the
of the
=
the
under
not the
embryos
regression NaHCO3
0.263, df 6.25 mM,
=
regression
nificantly
coefficient
different
=
developing
from
zero
(b
mM
the
was
0.49,
signifi-
was 25 mM concentration coefficient was 5b
Sb
=
the
0.263, con-
Similarly,
was not sig0.263, df =
NaHCO3
=
be-
to the three by linear regres-
coefficient concentration
52). When regression
at 12.5
these
affect the reresponse was
not significantly different from zero (b = 0.02, df = 52), indicating that under these conditions centration of KCI did not affect the response. the
df
NaHCO3 (b = 1.07, 5b = 0.26, negative at 25 mM NaHCO3 (b
about
Sb
mM,
data, corresponding to the is represented by linear coefficient was signifi-
with
was
17.5
52). Ax a concentration of 12.5 mM regression did not differ from zero
=
0.263,
=
10 to
=
52). In experiment B, the NaC1 data corresponding and 17.5 mM NaHCO3 can be adequately
by linear regression cient at the lower nificantly different 52), again indicating centration concentration
coefficient df = 52).
lines (Fig. concentration
3c). The regression coeffiof NaHCO3 was not sigfrom zero (b = 0.28, 5b = 0.24, df = that under these conditions the con-
of NaCl did not of NaHCO3
affect the was 17.5
was significantly negative At 25 mM NaHCO3, the
but significantly was very similar
to 10.0 represented
quadratic. to those
response. mM, the (b
When the regression
-1.03,
=
regression
s
Overall, the pattern found in the previous
of responses experiment.
The data corresponding to the three concentrations can also be adequately represented by linear regression (Fig. 3d). The regression coefficient was significantly ative
when
-1.45, NaHCO3
5b
the
NaHCO3
0.242, df 10,0 mM,
=
was
concentration =
was
52). When the regression
significantly different from zero (b = 52), indicating that under these
0.24,
=
negative
was
25
of KC1 lines neg-
mM
(b
the concentration coefficient was -0.12, conditions
5b
=
=
the
=
of not
0.242, df concen-
tration of KC1 did not affect the response. Similarly, the regression coefficient when the NaHCO3 concentration was 17.5 mM was not significantly different from zero (b = -0.78, =
0.242,
df
=
52).
il-
The main result was that the loregressions of the response on were dependent on the amount A comparison of both level of response was
The
3a) the of NaCI, regression
2-cell
= -0.81, NaHCO3
(b of
mM
Effect
A (Fig.
sion lines (Fig. 3b). cantly negative when
0.33
FIG. 2. The proportion (expressed either as the transformed proportion or a percentage) of CF1 mouse zygotes that develop to at least the 4cell stage in media containing combinations of different concentrations of NaCl, lactate, and pyruvate. (a) The main effect of NaCI (the symbols show each cell Itreatmenti mean In = 3) for the transformed proportions of each of the 27 media tested). (b) The main effect of lactate. (c) The main effect of pyruvate.
iments
0.10,
from
ranged
25 mM.
=
concentrations
Ho 95
NaHCO3
or
0.263, however,
5b
maximal,
F-
85
=
yond
0
of
6.5
conditions the concentration sponse. Furthermore, at 12.5
20
a
75
-1.15, NaHCO3, (b
L3Q
0
2
;t C)
,1f
(I)
level
cantly positive at 6.25 = 52) and significantly
70
6
BIGGERS
Blastocyst
Development
In all experiments
experi-
to blastocysts was fore not described
greater
cysts)
were
obtained
the
proportion
of zygotes
developing
generally very low. The results are therein detail. The best yields (43% blastowith
a modification
of M16
in which
OVERCOMING
109-
9
H3-
0 ci_
f
a)
‘
\
6.25 12.5
mM mM
NoHCO NoHCO
9
-
25.0
mU
NoHCO,
H-
I
6-
OF
45, 245-251
REPRODUCTION
Overcoming
the 2-Cell
(1991)
Block
by Modifying
Standard
Culture J. A. Laboratory
of Human
J.
and
and
Boston,
in a Mouse
Embryo
Medium1
LAWIUS
Reproduction
Components
D.
BIGGERS2
Reproductive
Biology,
Massachusetts
Harvard
Medical
School
02115
ABSTRACT The Almost when KH2PO4,
2-cell all
zygotes
cultured
embryo
may
be
obtained in
glucose,
or KCI depend in several
block
on
the
with
development
by
Whittingham’s and
media
caused
by
fertilizing
CF1
medium
pyruvate,
either
concentration lowered since
inappropriate
the
ova
M16.
hybrid
2-cell
of commonly B6D2F1/CrIBR
block
was
in the
medium.
of certain
of blastocysts
preimplantation undergo blocks the block occurs
species
mouse
its occurrence
served
as
it
has
only
effects
the
media
of embryo
constituents
did
by
a high
Although
not
develop
beyond
lowering
the
concentrations
of changing
the
concentration
percentage not
are
of embryos optimal
for
grew complete
culture the
media.
2-cell of NaCI,
of either to the
4-cell
stage KCI, NaC1 stage
preimplantation
is low.
the appropriate adjustment of the concentrations of NaCI, KC1, Kl-12P04, pyruvate, glucose, and NaHCO3. These findings provide direct evidence that blocks to development are caused primarily by inappropriate concentrations of some common constituents of media in current use.
embryos of several mammalian in development in vitro [1]. In the at the 2-cell stage [2, 3]. However,
is strain-dependent, and inbred
The
components,
INTRODUCTION The
used sperm
overcome
or in combination.
of NaHCO3 yield
with
This
individually
concentrations
concentrations
been
ob-
in random-bred
strains but not in F1 hybrids between inbred strains [4,5]. The stage when the block occurs has been correlated with the time the embryonic genome takes over control of development from the maternal genome, which occurs in the mouse at the 2-cell stage [6]. In the absence of direct evidence that blocks to development occur in vivo, it is likely that the blocks to development observed in vitro are caused by adverse culture conditions, such as imbalances in the concentrations of certain constituents [5]. These adverse conditions then result in either impaired transmission of inherited maternal information [7] or defective activation of the embryonic genome [8]. While using simplex methods for optimizing the composition of culture media for the culture of zygotes from random-bred mouse preimplantation embryos [9], we observed that passage through the 2-cell block occurred in media with low concentrations of several components commonly found in culture media, particularly NaC1, KH2PO4, glucose, and pyruvate. We have therefore examined the effects of lowering the concentrations of several constituents in a commonly used medium, M16 [10], in which randombred mice experience the 2-cell block. The results show that the 2-cell block can be overcome in this medium by
MATERIALS
AND
METHODS
Animals Random-bred hybrid male
female mice (Crl:CF1 BR, 6-8 wk old) and mice (B6D2F1/CrIBR) were maintained on 14L: 1OD (lights-on, 0500 h). Female mice were superovulated by i.p. injection of 5 IU eCG (Sigma, St. Louis, MO) at 1530 h, followed by an i.p. injection of hCG (Sigma) 47 h later. After hCG injection, females were placed in cages with males and examined the following morning (Day 1) for the presence of a vaginal plug. Preparation
of Culture
Media
prepared from individual stock solutions of of M16 (Table 1). All components were Sigma Most of the components were stored in 0.1 M solutions, with the following exceptions: NaCI and NaHCO3 were stored at 1.0 M, whereas BSA (fraction V) was stored at a concentration of 100 mg/mI, CaC12 at 0.171 M, and penicillin-G and streptomycin sulfate were stored as one solution at concentrations of 10 000 U/mI and 5.0 mg/ ml, respectively. All cQmponents were stored at 4#{176}C, in 50ml conical centrifuge tubes (USA Scientific Plastics, Ocala, FL) except for the antibiotic mixture, which was stored at 15#{176}C in 5-mi polypropylene tubes (#2063, Falcon Plastics, Los Angeles, CA). Fresh solutions of NaHCO3, pyruvate, and lactate were prepared weekly. BSA was prepared every time media were made. Ten milliliters of each medium was prepared on the day Media
were
each
component purchased from
-
April 1, 1991. January 14, 1991. ‘The work reported in this paper was part of the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Development, grant number HD-21988. 2Correspondence: Dr. John D. Biggers, Harvard Medical School, LHRRB, 45 Shattuck St., Boston, MA 02115. FAX: (617) 566-7980. Accepted
Received
prior 245
to the
start
of each
trial.
The
components,
except
for
246
LAWITF
S AND
stage, bator
TABLE 1. Composition of medium M16 (Whittingham (101). Component
Concentration
NaCI KCI KH2PO4 MgSO4 Lactate Pyruvate Glucose BSA NaHCO3 CaC12 *BSA
E.tperimental Four
4.00 25.00 171
dia Peni-
respectively.
CaCl2, were added to culture tubes (Falcon #2051), distilled H20 was added to bring the volume of each tube to 9.0-9.5 ml, and then CaCl2 was added. Finally, distilled H20 was added to bring the final volume of each medium to 10 ml. Each tube was inverted twice to mix the components and then the contents were drawn into a disposable syringe (Becton-Dickinson, Rutherford, NJ) and the medium was pushed through a 0.2-p.m filter (Millipore, Bedford, MA) into a second tube. Collection
Mice were killed between 1530 and 1545 on Day 1. Pronuclear stage zygotes were flushed from each oviduct with 0.1 ml of Dulbecco’s phosphate-buffered saline (dPBS). Pooled zygotes from several donors were washed through one drop of dPBS containing 0.65 mg/mI hyaluronidase (Sigma) to remove any cumulus cells, followed by two drops of dPBS. Embiyo
conducted.
Each
used
in each
experiment.
Three
During into each trials
each trial, of the me-
were
conducted
Methods
The experiments were analyzed by an analysis of variance after transformation of the data by a two-term inverse sine transformation, proposed by Laubscher [11], given by: n”2 sin’
=
+
where
x n
= =
The
advantage
used
angular
portions distribution
(n
+
(x/n)h/’2
sin1
1)h/’2
[(x
+
3/4)/(n
+
the number of responders, the group size. of this
over
is that
it is more
0.9. The theoretical approaches 1 as n increases.
transformed
responses
components polynomial
of the coefficients
on
the
media
were
[12].
sion coefficients are those variables are transformed
The
obtained
3/2)1h/2,
and
transformation
transformation
Culture
Zygotes were cultured in tissue culture dishes (#3060, Costar, Cambridge, MA) in 50-p.l drops of medium under 5 ml of silicone oil (50 centistokes; Sigma). The dishes were made up the day before the start of each trial and were equilibrated overnight in an atmosphere of 6% C02, 5% 02, and 89% N2 in a plastic culture chamber (Billups-Rothenberg, Del Mar, CA) inside a 37#{176}Cincubator. Each culture dish contained 4 drops. Ten zygotes were transferred from dPBS into drop 1. Any remaining dPBS in the transfer pipette was expelled and the pipette was refilled from drop 2. The ten zygotes were then transferred into drop 2, and this procedure was repeated with drop 3. Drop 4 was a spare. The culture dishes were returned to the culture chamber at 37#{176}C, which was gas-equilibrated and then sealed. Developmental progress was recorded, as described below (statistical methods), at 24 h, 48 h, and 96 h after the embryos were placed in culture. When the culture dishes were outside the culture chamber and not on the microscope
experiment
at three different 27 media were com-
for each experiment, for a total of 30 embryos per treatment group. The responses to the media were assessed as the proportion that were 4-cells on Day 3 post-hCG, and the proportion that reached the blastocyst stage on Day 5. Statistical
Embryo
were
pared in a single trial in each experiment. 10 zygotes were transferred at random
cillin and streptomycin were also added at concentrations of 100 U/mI and 50 p.g/ml,
incu-
Design experiments
compared three different components levels, in a 33 factorial design. Thus,
5.56
is in mg/mi.
they were maintained at 37#{176}Cin a bench-top (Baxter Scientific, McGaw Park, IL).
(mM)
94.70 4.78 1.19 1.19 23.30 0.33
concentration
BIGGERS
the
commonly
stable
concentrations fitted when
of this of the of
using
estimates
to the scale
for pro-
variance Regressions
specific
orthogonal
of the the
regres-
independent
-1,0,1.
RESULTS Development
to the 4-Cell
Stage
Effect of NaC1, K!-12P04, and glucose. All combinations of three concentrations of NaCl, KH2PO4, and glucose were tested. The concentrations used were NaCI: 75, 85, and 95 mM; KH2PO4: 0, 0.6, and 1.2 mM; and glucose: 0, 2.75, and 5.5 mM. The analysis of variance (Table 2) showed that all three main effects, from NaCI, KH2PO4, and glucose, were highly significant, while all the interactions were not significant (p > 0.05). Thus, the effects of the three compounds can be considered to be independent. Regression analysis orthogonal
of the
main
polynomials,
effects
of the confirmed
three
components, that
only
using the
linear
components were significant over the concentrations studied. All three linear regressions had significant negative slopes (Fig. la,b,c). Therefore, increasing the concentration of any of the three components independently depressed the proportion of embryos reaching the 4-cell stage. The apparent
OVERCOMING
TABLE
2.
Source
Analysis
of variance
THE
of the data
of Variation
df
Between
replicates
2
Between
media:
different
medium,
having the concentration
same
bryos reaching was very low two
suggested but this
significance at the p = 27 cell (treatment) means,
statistical The
were
the that
ering
of the
cell
means
a variety
concentrations
in three
0.041
1 1
41.364
to 4-cells. to the
involving
4 4 4 8
0.723
KH2PO4,
other
the and
low-
glucose
100
0.92
30.87 0.030
0.040
3.986
0.462
0.672 1.913 1.34
could
-
90
pyruvate
-
80
Regression
-
70
-
60
-
50
the effects of NaCI and ative linear regression inhibited development
-
40
-
30
-
20
-
10
#{149} MEAN RESPONSE
0
a:
p 0.0006
0.010
8.898
(M16) With
medium
-
z
F 8.54
66.02
1 1
each
(c)
(b)
square
88.471
10
(a)
1.
11.448
1
to a
groups,
corresponding of changes of NaC1,
Mean
1
that at 5.5 mM failed to reach
the 4-cell stage in the control (3%); 29 of 30 failed to grow
showed
247
in Figure
52
0.05 level. each corresponding
plotted
summarized
concentration of NaCl (Fig. la) or the same of glucose (Fig. ic). The percentage of em-
exceptions,
media
also
BLOCK
(26)
NaCI: Linear Quadratic KH2PO4 Linear Quadratic Glucose Linear Quadratic Interactions: NaCI x KH2PO4 NaCI x glucose KH2PO4 x glucose NaCI x KH2PO4 x glucose Error
curvature in the effect of glucose the inhibitory effect was maximal,
2-CElL
analysis
0.05).
Thus,
considered using
the
effects
independent
orthogonal
of NaCI (Fig.
polynomials
shows
pyruvate can be summarized lines. The results confirm as the concentration was
and
2a,b,c). that
by negthat NaCl increased
0
Ui
a:
N
4
0
LL
3
(I)
z
2
The medium,
a: I-
from 75 to 95 mM, and that pyruvate was also the concentration was raised from 0.13 to 0.33 lactate, however, had no effect.
1
M16
-
Ml 6
0#{149}
1
75
85
NaCI
95
I
I
0.0
0.6
KH2PO4
CONCENTRATION
1.2
0.0
2.8
5.5
GLUCOSE
(mM)
FIG. 1. The proportion (expressed either as the transformed proportion or a percentage) of CF1 mouse zygotes that develop to at least the 4cell stage in media containing combinations of different concentrations of NaCI, KH2PO4, and glucose. (a) The main effect of NaCI. (b) The main effect of KH2PO4. (c) The main effect of glucose. Note that the symbols show each cell (treatment) mean (n = 3) for the transformed proportions of each of the 27 media tested.
27
cell are
ing the same bryos reaching
means,
plotted
each
corresponding
in Figure
concentration the 4-cell
stage
2a in three
inhibitory as mM. Varying to
a different
groups,
each
of NaCI. The percentage in the control medium
hayof em(M16)
was somewhat higher than in the first experiment (23%), due mainly to four embryos that developed in the third replicate. Nevertheless, the cell means shown in Figure 2a demonstrate that a variety of changes in the composition of medium M16 produced by adjusting the concentrations of NaCl and pyruvate overcame the block to a lesser greater degree. Figure 2a (solid circles) also shows that ducing the pyruvate fect to that of reducing ing the block.
concentration the NaCI
had a clear concentration
or re-
additive efin overcom-
248
LAWITfS
AND
when
the
100
rather
than
90
In experiment three concentrations regression lines.
10 (a)
z
9
#{149} 0.13 0.23
mM P’YR mM PYR
8
o
mM
0
a:
0.33
#{149} MEAN
0
(c)
(b)
PYR -
RESPONSE
80
7
3-
#{149}
0
a: a-
.1 5
0
#{149} I
1-±
4
-f
M16-
0
o
3
U-
50
N
Ui
a:
60
-
z
a:
m
10
5.8
NoCI
11.6
23.3
0.13
LACTATE
0.23
PYRUVATE
CONCENTRATION
of NaCI, were
centrations periment
KC1, and
done
(mM)
not
Two all combinations
of NaCl, KCI, and A, the concentrations
summarized in Figure effect for NaHCO3 was
NaCI
NaHCO3.
in which
95 mM; KCI: 0, 2.4, and and 25.0 mM. The results
KCI was was not
marginally significant.
x NaJ-ICO3 significant.
factorial experof three con-
NaHCO3 were tested. In exused were NaC1: 75, 85, and
4.8 mM; and NaHCO3: 6.25, and the analysis of variance
3, a and highly
b, and
Table
significant,
significant, However,
the
3. The main
12.5 are main
effect
of
and the main effect for NaC1 there was a very significant
interaction. In experiment
All the other interactions B, the concentrations
were of NaC1
and KCI remained the same as in experiment A; however, the two lower concentrations of NaHCO3 were raised to 10 and 17.5 mM. The results and the analysis of variance are summarized
in Figure
to experiment
The
3, c and
A, where
of all three components nificant. Furthermore, NaHCO3 interactions
teractions
were two-way
KCI X NaHCO3
NaCI
had
d, and
Table
no effect,
3. In contrast the
main
effects
in experiment B were highly sigthe NaCl X NaHCO3 and KCI X were highly significant. All other in-
not significant. tables
showing
interactions
lustrated in Figure 3, a-d. cations and slopes of the NaCl and KCI concentrations of NaHCO3 in the ments reveals that
5b
medium. the overall
the from
NaCI
both
X
the
NaHCO3
experiments
and are
mM
df the
df
52),
=
80%
indicating
that
of NaC1 did mM NaHCO3,
stage. The data corresponding of KCI is also represented The the
of the
=
the
under
not the
embryos
regression NaHCO3
0.263, df 6.25 mM,
=
regression
nificantly
coefficient
different
=
developing
from
zero
(b
mM
the
was
0.49,
signifi-
was 25 mM concentration coefficient was 5b
Sb
=
the
0.263, con-
Similarly,
was not sig0.263, df =
NaHCO3
=
be-
to the three by linear regres-
coefficient concentration
52). When regression
at 12.5
these
affect the reresponse was
not significantly different from zero (b = 0.02, df = 52), indicating that under these conditions centration of KCI did not affect the response. the
df
NaHCO3 (b = 1.07, 5b = 0.26, negative at 25 mM NaHCO3 (b
about
Sb
mM,
data, corresponding to the is represented by linear coefficient was signifi-
with
was
17.5
52). Ax a concentration of 12.5 mM regression did not differ from zero
=
0.263,
=
10 to
=
52). In experiment B, the NaC1 data corresponding and 17.5 mM NaHCO3 can be adequately
by linear regression cient at the lower nificantly different 52), again indicating centration concentration
coefficient df = 52).
lines (Fig. concentration
3c). The regression coeffiof NaHCO3 was not sigfrom zero (b = 0.28, 5b = 0.24, df = that under these conditions the con-
of NaCl did not of NaHCO3
affect the was 17.5
was significantly negative At 25 mM NaHCO3, the
but significantly was very similar
to 10.0 represented
quadratic. to those
response. mM, the (b
When the regression
-1.03,
=
regression
s
Overall, the pattern found in the previous
of responses experiment.
The data corresponding to the three concentrations can also be adequately represented by linear regression (Fig. 3d). The regression coefficient was significantly ative
when
-1.45, NaHCO3
5b
the
NaHCO3
0.242, df 10,0 mM,
=
was
concentration =
was
52). When the regression
significantly different from zero (b = 52), indicating that under these
0.24,
=
negative
was
25
of KC1 lines neg-
mM
(b
the concentration coefficient was -0.12, conditions
5b
=
=
the
=
of not
0.242, df concen-
tration of KC1 did not affect the response. Similarly, the regression coefficient when the NaHCO3 concentration was 17.5 mM was not significantly different from zero (b = -0.78, =
0.242,
df
=
52).
il-
The main result was that the loregressions of the response on were dependent on the amount A comparison of both level of response was
The
3a) the of NaCI, regression
2-cell
= -0.81, NaHCO3
(b of
mM
Effect
A (Fig.
sion lines (Fig. 3b). cantly negative when
0.33
FIG. 2. The proportion (expressed either as the transformed proportion or a percentage) of CF1 mouse zygotes that develop to at least the 4cell stage in media containing combinations of different concentrations of NaCl, lactate, and pyruvate. (a) The main effect of NaCI (the symbols show each cell Itreatmenti mean In = 3) for the transformed proportions of each of the 27 media tested). (b) The main effect of lactate. (c) The main effect of pyruvate.
iments
0.10,
from
ranged
25 mM.
=
concentrations
Ho 95
NaHCO3
or
0.263, however,
5b
maximal,
F-
85
=
yond
0
of
6.5
conditions the concentration sponse. Furthermore, at 12.5
20
a
75
-1.15, NaHCO3, (b
L3Q
0
2
;t C)
,1f
(I)
level
cantly positive at 6.25 = 52) and significantly
70
6
BIGGERS
Blastocyst
Development
In all experiments
experi-
to blastocysts was fore not described
greater
cysts)
were
obtained
the
proportion
of zygotes
developing
generally very low. The results are therein detail. The best yields (43% blastowith
a modification
of M16
in which
OVERCOMING
109-
9
H3-
0 ci_
f
a)
‘
\
6.25 12.5
mM mM
NoHCO NoHCO
9
-
25.0
mU
NoHCO,
H-
I
6-
