/. Embryol. exp. Morph. Vol. 34, 2, pp. 467-484, 1975
Printed in Great Britain
467
Effects of bromodeoxyuridine,
cytosine arabinoside and Colcemid upon in vitro
development of mouse blastocysts
By MICHAEL I. SHERMAN 1 AND SUI BI ATIENZA 1
From the Roche Institute of Molecular Biology, New Jersey
SUMMARY
Mouse blastocysts in culture have been treated with increasing concentrations of cytosine
arabinoside, bromodeoxyuridine or Colcemid. Concentrations of all three antimetabolites
have been found which interfere with neither hatching of the blastocysts from their zona
pellucidae nor subsequent attachment of the blastocysts to the culture dish, but which
eventually result in death of the inner cell mass (ICM) and its derivatives. The effect upon the
ICM is selective at these antimetabolite concentrations since many or, in some cases, all
trophoblast cells continue to survive, and by a number of criteria, undergo normal patterns
of differentiation and development.
INTRODUCTION
In recent years, conditions for post-blastocyst culture have improved considerably. In our laboratory we have demonstrated biochemically that both
inner cell mass (ICM) and trophectoderm derivatives undergo differentiation
in vitro (see Sherman, 1974, 1975 c for reviews), while Hsu and co-workers
(see Hsu, Baskar, Stevens & Rash, 1974) have reported that a fraction of
blastocysts could give rise to somite-stage embryos under their culture conditions. The conditions used by Hsu and by us, although not dramatically
different, do not yield the same results: while the inclusion of a collagen substratum and human cord serum can lead to development of morphologically
recognizable yolk sac and embryo proper (Hsu, 1973), a plastic substratum
and fetal calf serum favour characteristic differentiation of trophoblast and
yolk sac cells (see Sherman, 1975 c). Although some tissues of the embryo
proper might develop in the latter case, structures such as heart and somites
are absent.
Various patterns of post-blastocyst development might thus be achieved at
will by the inclusion or omission of substances in the culture medium. This
would facilitate understanding developmental problems involving cell lineages
as well as the nature of cell-cell interactions. To investigate the feasibility of
1
Authors' address: Roche Institute of Molecular Biology, Nutley, New Jersey, 07110,
U.S.A.
468
M.I.SHERMAN AND S.B.ATIENZA
developing selective culture conditions, we have begun a study of the effects
of antimetabolites on post-blastocyst development. The studies described here
utilize antimetabolites whose primary action is to interfere with replication
and the cell cycle. The rationale was to take advantage of the difference between ICM cells, which divide rapidly, and polyploid trophoblast cells which,
although they replicate, are amitotic and nondividing (see Graham, 1973, for
a review). We have observed that dosages of cytosine arabinonucleoside (Ara C),
5-bromo-2'-deoxyuridine (BUdR) and Colcemid can be found which are lethal
to ICM cells while they fail to hinder development and differentiation of
trophoblast.
METHODS AND MATERIALS
Materials
[3H]thymidine and [3H]progesterone were purchased from New England
Nuclear Inc., Boston, Massachusetts, and [3H]BUdR was obtained from
Schwarz-Mann, Orangeburg, New York. BUdR and Ara C were purchased
from Sigma Chemicals, St Louis, Missouri. Dulbecco-modified Eagle's medium,
Colcemid, penicillin, streptomycin and kanamycin were obtained from Gibco,
Grand Island, New York. NCTC-109 medium and fetal calf serum were
purchased from Microbiological Associates, Bethesda, Maryland. Progesterone
and pregnenolone were purchased from Steraloids, Inc., Pawling, New York,
and were recrystallized before use. Antiprogesterone antisera (lots S49, no. 6,
and S257, no. 2) were purchased from Dr Guy Abraham, Harbor General
Hospital, Torrance, California. Kodak NTB-2 Nuclear Track Emulsion was
obtained from Eastman Kodak Company, Rochester, New York.
Collection and culture of blastocysts
SWR/J females and SJL/J males, both obtained from Jackson Laboratories,
Bar Harbor, Maine, were mated after the former had been induced to superovulate (Runner & Palm, 1953). The day of observation of the sperm plug is
considered the first day of pregnancy. Fourth-day blastocysts were flushed
from uteri with phosphate-buffered saline (PBS). Blastocyst cultures were
carried out as described previously (Sherman, 1975o), utilizing NCTC-109
medium (Evans, Bryant, Kerr & Schilling, 1964), supplemented with 10%
heat-inactivated fetal calf serum and penicillin (100 i.u./ml), streptomycin
(lOOyMg/ml) and kanamycin (lOO^g/ml). Where indicated, Dulbecco-modified
Eagle's medium (DME; Vogt & Dulbecco, 1963) was used instead of NCTC109. Antimetabolites were added in small volumes of PBS or water. Blastocyst
hatching and attachment were monitored as described elsewhere (Salomon &
Sherman, 1975; Sherman & Salomon, 1975). Embryonic ages will be described
in terms of equivalent gestation days (e.g.d.), i.e. the age of the embryos had
they been left in utero.
Antimetabolite treatment of blastocysts
469
Measurement of nuclear diameters, areas and DNA contents
Blastocyst cultures were visualized with phase optics of a Wild M40 microscope and photographed with a Polaroid camera attachment. The largest
and smallest diameters were measured and averaged. Corrections were made
for the 165-fold magnification.
For microspectrophotometric determinations, cells were stained with Feulgen
(Barlow & Sherman, 1972). The largest {dt) and smallest (ds) diameters of each
trophoblast nucleus were measured, and nuclear areas were calculated according
to the formula A = n. ^dt.^ds. Nuclear absorbances at 565 nm were measured
with a Leitz MPV11 microscope photometer.
Autoradiography
Blastocysts were cultured in NCTC-109 medium on coverslips in 35 x 10 mm
culture dishes. After the appropriate time in culture, thymidine-methyPH
(20-5 Ci/mmole) was added to the control, Ara C or Colcemid-treated cultures,
so that the final concentration was 1 /^Ci/ml and the final specific activity was
24-2 mCi/mmole, taking into account the unlabeled thymidine already in the
NCTC-109 medium. Similarly, 5-bromo-2'-deoxyuridine-6-3H (13 Ci/mmole)
was added to BUdR-treated cultures so that the final concentration and specific
activity were 1 /tCi/ml and 10 mCi/mmole, respectively. After a 24 h incubation period, coverslips containing attached blastocysts were washed in PBS,
placed in ethanol: glacial acetic acid (3:1) for lOmin and then transferred to
ethanol for 20 min. After air drying, coverslips were mounted on slides, and
washed in cold PBS (10 min), cold 5% trichloroacetic acid (10 min) and two
changes of cold 70 % ethanol (5 min each); slides were subjected to autoradiographic analysis as described by Kopriwa & LeBlond (1962), using
Kodak NTB2 Nuclear Track Emulsion. Slides were exposed at 4 °C for 14
days, developed and stained with Giemsa.
Assays for progesterone production
Blastocysts were cultured in NCTC-109 medium supplemented with dextrannorit-treated serum (Salomon & Sherman, 1975). Pregnenolone was added to
a final concentration of 1-2 ^g/ml. After 24 h incubation, medium was collected and assayed for progesterone content by the radioimmunoassay procedure described previously (Chew & Sherman, 1975; Sherman, \915b).
Medium with pregnenolone but without cells was incubated for 24 h and
assayed as a background control.
RESULTS
Effects of antimetabolites on hatching and attaching
A range of concentrations of Ara C, BUdR and Colcemid was tested for
effects upon hatching and attachment of blastocysts in NCTC-109 medium.
470
M.I.SHERMAN AND S.B.ATIENZA
10-3
Ara C (M)
Fig. 1. Effects of Ara C on blastocyst hatching and attachment. Blastocysts and
some late morulae were obtained on the 4th day of gestation and placed in lots
of 25 directly into NCTC-109 medium. Ara C at the indicated concentrations was
added either immediately or after 24 h. The final percentage of blastocysts that
had both hatched and attached to the culture dish was determined after 70 or more
hours in culture. O, Ara C added on first day of culture (i.e. fourth e.g.d.); • ,
Ara C added after about 24 h of culture (i.e. fifth e.g.d.).
Fig. 1 illustrates that Ara C added at the start of the culture period or after
24 h does not have a marked effect upon hatching and attachment, even at
concentrations as high as 10"3 M. BUdR at concentrations greater than 10~4 M
has a deleterious effect on blastocyst hatching and attachment, but only when
added at the beginning of the culture period; when added after 24 h of culture,
little effect is seen up to 10~3 M (Fig. 2). Even at the highest doses used here'
only about 50 % of the blastocysts are prevented from further development.
The bulk of the cultures to be described in this study were carried out in
NCTC-109, since in our hands trophoblast and ICM develop best in this
medium. However, NCTC-109 contains deoxycytidine and thymidine (ca.
3 x 10~5 M each), which can protect at least in part against the effects of Ara C
and BUdR (Chu & Fischer, 1962; Coleman, Coleman, Kankel & Werner,
1970; Meuth & Green, 1974). In DME medium, which lacks deoxynucleosides^
BUdR and Ara C block hatching and attaching completely at 10~5 M.
The effects of Colcemid are much more drastic than those of BUdR (Fig. 3).
Concentrations greater than 3 x 10~8 M completely block blastocyst hatching
and attachment. When Colcemid is added 24 h, or even 48 h, after the beginning of culture, a substantial fraction of the blastocysts fail to develop. The
48 h values do not reflect the full effectiveness of the antimetabolite, since
some of the blastocysts have already attached to the culture dish by this time.
Antimetabolite treatment of blastocysts
II
100
"8
80 --
^
60 --
i
i
1
a
o
•
o
i
i
i
i
•
9
i
—o—
471
1
-
•
•
\
O \
\° ~
o
40 _
20 -
1
-H10
i
i
10"5
BUdR (M)
1
10
1
1
lO
Fig. 2. Effects of BUdR on blastocyst hatching and attaching. Experiments
were carried out as described in the legend to Fig. 1, except that BUdR was used.
O, BUdR added on first day of culture (fourth e.g.d.); • , BUdR added after
about 24 h of culture (fifth e.g.d.).
"" 1
100.
i
1
i
I
i
1
I
I
-
ft
80
60
40
\
20
-0-L-
\
•
\ V—_ 1
o
' o '
'
'
12
18
Colcemid(Mxl0-8)
' 6
24
30
Fig. 3. Effects of Colcemid on blastocyst hatching and attachment. Experiments
were carried out as described in Fig. 1, except that Colcemid was used. O, Colcemid
added on first day of culture (fourth e.g.d.); A, Colcemid added after about 24 h
of culture (fifth e.g.d.); D, Colcemid added after about 48 h of culture (sixth e.g.d.).
To determine whether those blastocysts which had been able to develop in
the presence of antimetabolites had nevertheless been delayed in their rates of
hatching and attaching, time studies were carried out as described by Salomon
30
E M B 34
472
M. I. SHERMAN AND S. B. ATIENZA
FIGURE 4
Morphology of control and antimetabolite-treated blastocyst cultures. Photos
were taken at the same magnification, with phase contrast optics, after seven days
in culture (eleventh e.g.d.). Antimetabolite treatments were continuous, and
NCTC-109 medium was used. (A) Control; (B) 2 x 10"5 M Ara C added on the
first day of culture (fourth e.g.d.); (C) 10~4 M BUdR added on the second day
of culture (fifth e.g.d.); (D) 3 x 10 8 M Colcemid added on the third day of culture
(sixth e.g.d.). Scale = 100 fim.
Antimetabolite
473
treatment of blastocysts
Table 1. Effects of antimetabolite upon post-blastocyst development
Time of
Anti-
r\ 11ii
metabolite
AraC
Medium
NCTC-109
DME
BUdR
NCTC-109
J-/LHdlILMl KJl
(h)
treatment
0
24 h
Continuous
Continuous
Continuous
Continuous
Continuous
24 h
Continuous
24 h
Continuous
Continuous
Continuous
Continuous
Continuous
24 h
Continuous
24 h
Continuous
24
48
0
24
0
24
DME
Colcemid
NCTC-109
Lethal dose for:
oflfl it ion
diiiil HUH
48
0
24
0
24
48
ICM
Trophoblast
^ 3x 10-4M
2 to 5x 1 0 - 5 M
5 x 10-5 M
10"3M
7
5X10- M
5 to7-5xl0- 7 M
7
5xl0" M
5 to7-5x!0- 7 M
5
1 to 3 x 10-* M
5xlO~ to 10-* M
10-* M
5X10-5M
1 to 3x 10-* M
> 3xl0-*M
5x 1 0 - 5 M to 10-* M 3xl0-*M
10-* M
5xJ0-*M
5xlO~ 7 M
3X10-6M
7-5 xlO- 7 to 10-6 M ^ 3X 10- 6 M
3 to 9x 10-8M
9 x 10~8 M
10-* M
10-5M
10-5M
5xlO- 5 M
2-7X10~7M
> 1-5X10-6M
3 to9xlO-8M
J-8x 1 0 - 7 M
> 1-5X10-6M
> 1-5X10-6M
> 2-7 x 10-7 M
9xlO- 8 to
1-8X10-7M
Blastocyst cultures were begun on the fourth day of gestation. In general, lots of 25 blastocysts were cultured in 3 ml of medium. Antimetabolites were added either at the beginning
of the culture period, 0 h, or after 24 or 48 h of culture, as indicated. Lethality was monitored 7-8 days after the beginning of culture, i.e. on the 11 th—12th e.g.d. In some cases, a
range of lethal doses are given, indicating that there was slight variability from one experiment to the next.
& Sherman (1975). Ara C- and BUdR-treated blastocysts showed no significant
delays in the rates of hatching and attaching when compared with controls.
The only evidence of substantially delayed hatching and attachment rates were
observed when blastocysts were pulsed with Colcemid for one day after 24 h
of culture. In these cultures, the average rates of both hatching and attachment
were retarded by as much as 24 h compared to controls. With this exception,
our observations indicate that blastocysts either fail to develop further in
response to antimetabolites, or they hatch and attach along a normal, or close
to normal, schedule.
Effects of antimetabolites upon trophoblast and inner cell mass viability
Ara C, BUdR and Colcemid were tested for their effects upon ICM and
trophoblast development. Both continuous exposures and 24 h pulse treatments
were utilized. Trophoblast cells are more resistant to all three antimetabolites
than ICM cells (Table 1). At appropriate concentrations, the ICM can often
be seen to undergo degeneration as soon as blastocyst outgrowth along the
30-2
474
M. I. SHERMAN AND S. B. ATIENZA
Table 2. Effect of antimetabolites upon number of trophoblast cells
per blastocyst
Antimetabolite
None
Ara C, 2 x 10"5
BUdR, 10" 4 M
Number of
blastocysts
counted
Average number
of trophoblast
cells ±S.D.
Range
21-80
41-8 ±14-7
9-30
20-4 ± 6-3
39-75
55-3 ±10-3
9-69
Colcemid, 9 X 1 Q - 8 M
42-8 ±19-1
All antimetabolites were added 24 h after the beginning of blastocyst culture in NCTC-109
medium, and were maintained continuously. Numbers of trophoblast cells were measured
on the fifth day of culture (ninth e.g.d.), after most of the 1CM cells had disappeared.
M
26
19
19
9
culture dish has begun, and by 72 h after antimetabolite addition, few unhealthy
ICM cells, if any, are present. The result is a pure monolayer of trophoblast
cells which maintains a healthy appearance for a number of days (Fig. 4).
Although there were slight variations from one experiment to another, the
differences between the lethal concentrations of Ara C, BUdR and Colcemid
for ICM and trophoblast were, in most cases, at least twofold and usually fiveto sixfold. Particularly with Ara C, antimetabolite concentrations having little
effect upon blastocyst hatching and attachment subsequently proved to be
lethal to ICM cells.
Just as with blastocyst hatching and attachment, ICM and trophoblast cells
are both much more resistant to the effects of BUdR and Ara C when cultured
in NCTC-109, compared with DME, medium. ICM and trophoblast cells are
20- to 40-fold more sensitive to Ara C in DME medium, while both cell types
are 30-100 times as sensitive to BUdR in DME compared with NCTC-109
medium (Table 1).
Table 1 illustrates that concentrations of Ara C, BUdR and Colcemid which
are lethal to ICM when added at the beginning of the culture period are
generally equally deleterious when added after 24 h of culture. On the other
hand, resistance of trophoblast cells to the antimetabolites usually increases
when the time of addition is delayed by a day. Twenty-four-hour treatments
with Ara C, BUdR and Colcemid are not as effective as the continuous presence
of the antimetabolites in killing ICM and trophoblast cells (Table 1). However,
lethal concentrations of BUdR for both ICM and trophoblast are only twoto threefold greater in 24 h pulses than in continuous treatment, while the
differences with Ara C and Colcemid are tenfold or greater.
The data for lethality of trophoblast cells in response to antimetabolites as
described in Table 1 are all-or-none in so far as cultures were scored for the
presence or absence of trophoblast. This data does not deal with the question
of whether all trophoblast cells of a treated blastocyst survive at antimetabolite
Antimetabolite treatment of blastocysts
475
A
18
36
54
72
90
108 126
Nuclear diameter (/.im)
Fig. 5. Diameters of trophoblast cell nuclei from control and antimetabolitetreated blastocyst cultures. All antimetabolite treatments were continuous and
NCTC-109 medium was used. Measurements were made from photos of cultures
taken on the 11th e.g.d., except in (A), wherein a long-term diploid blastocystderived culture was photographed and nuclei were measured. (B) Control blastocyst
cultures; (C) cultures treated with 2x 10~5 M Ara C on the first (fourth e.g.d.) or
second (fifth e.g.d.) days of culture; (D) cultures treated with 10~4 M BUdR on the
first day (fourth e.g.d.); (E) cultures treated with 9x 10~8 M Colcemid on the third
day (sixth e.g.d.). The numbers of nuclei measured are as follows: (A) 110, (B) 307,
(C) 102, (D) 201, (E) 183.
concentrations which are lethal to ICM cells. This was investigated by counting
the number of trophoblast cells in control blastocysts and blastocysts treated
with antimetabolites at levels that eliminated ICM cells (Table 2). Counts of
cell numbers are derived from the numbers of nuclei. Care was taken to distinguish between binucleate trophoblast cells (Sherman, 1975 b) and two cells
in close apposition to each other. Also, control culture values are underestimates
in many cases because the large clumps of ICM cells cover a portion of the
trophoblast monolayer. The data in Table 2 suggest that Ara C causes a marked
reduction in trophoblast cell number, while BUdR and low concentrations of
476
M. I. SHERMAN AND S. B. ATIENZA
A
1
1
I 1
4 -
D
B
4 1
i
4
I
i
8
12
16
Nuclear area (//m2 x 10~3)
n
-
i
hn
n n i
n
8
16
24
32
Absorbance (arbitrary units)
Fig. 6. Microspectrophotometry of trophoblast nuclei from control and BUdRtreated cultures. BUdR (10~4 M) was added on the second day (fifth e.g.d.) of
culture in NCTC-109 medium. All measurements were made as described in
Methods after 7 days of culture (11th e.g.d.). A and C are measurements on
trophoblast nuclei from control cultures. B and D are measurements on trophoblast
nuclei from BUdR-treated cultures.
Colcemid do not. We did not observe large increases in the number of trophoblast cells as Garner (1974) reported when she added very low concentrations
of BUdR to preimplantation-stage embryos.
Since Barlow & Sherman (1972) reported a positive correlation between the
extent of polyploidization and trophoblast nuclear diameters, nuclear diameters
of antimetabolite-treated and untreated trophoblast cells were compared (Fig.
5). Measurements were made on the 11th e.g.d., at which time all JCM derivatives had been killed in treated cultures. Only trophoblast nuclei were measured
in the control cultures. To provide an indication of the range of diameters of
diploid cell nuclei, measurements were performed on a blastocyst-derived cell
line at a time when the chromosome number was known to be diploid (Sherman,
1975a). In this case, nuclear diameters ranged from 7-24 /mi (Fig. 5A). About
30 % of control trophoblast nuclei have diameters within this range (Fig. 5 B);
these are most likely ectoplacental cone cells, since they appear to undergo
polyploidization after several days of culture (Sherman, 1915a). The remainder
of the nuclei are presumably polyploid, and have a range of diameters between
25-90 jam. Fig. 5C shows that Ara C-treated trophoblast cells also have nuclei
which are in the polyploid region. Although the doses of Ara C used appear
not to have completely blocked DNA synthesis in trophoblast cells (see also
Table 3), the range of nuclear diameters is smaller than that of the controls.
Furthermore, although the percentage of nuclei in the diploid range in the Ara C
sample is about the same as in the controls, these are probably not due to
actively dividing ectoplacental cone cells, but to trophoblast cells that have
failed to undergo polyploidization.
Antimetabolite treatment of blastocysts
All
Table 3. Thymidine and bromodeoxyuridine incorporation into acid-insoluble
material in trophoblast nuclei from control and antimetabolite-treated blastocyst
cultures
Time of addition of radioisotopes-equivalent gestation age
10-11
8-9
Antimetabolite
No. nuclei
labeled
Time of
addition No. nuclei
(h)
studied
None
AraC(2xl0~ 5 M)
(10-5M)
BudR ( 1 0 - 4 M )
—
0
24
24
1352/1372
167/190
168/178
595/600
Colcemid(3x 10- 8 M)
24
—
(9X10-8M)
(1-8X10-7M)
(2-7X10~7M)
/o
98-5
87-9
94-4
99-2
12-13
No. nuclei
labeled
No. nuclei
studied
No. nuclei
labeled
No. nuclei
studied
126/213
59-2
535/943
56-7
—
63/150
—
42-0
113/312
—
63-8
—
268/304
88-2
—
—
—
—
229/269
85 1
—
—
—
—
269/314
85-7
—
—
—
—
Blastocyst cultures in NCTC-109 medium were begun on the fourth day of gestation.
Antimetabolite treatments were continuous. [3H]thymidine was added at the indicated time
except for the BUdR experiments, in which [3H]BUdR was used.
48
48
48
The distribution of nuclear diameters in Colcemid-treated trophoblast cells
(Fig. 5E) does not differ markedly from that of controls. The smaller percentage
of nuclei in the diploid range is consistent with the failure of ectoplacental
cone cells to proliferate in the presence of Colcemid, perhaps due both to a
direct mitotic block, and, indirectly, to the removal of the ICM (see Sherman,
19756).
The distribution of nuclear diameters in BUdR-treated cells (Fig. 5D) was
unexpected. Instead of having a range of nuclear diameters equal to, or somewhat less than, the controls, the great proportion of the nuclei are larger than
the majority of the controls (see, for example, Fig. 4). These large nuclei might
have arisen by accelerated cycles of endoreduplication or by flattening of the
trophoblast cells against the culture dish in response to BUdR. To distinguish
between these two alternatives, control and BUdR-treated blastocyst cultures
were stained with Feulgen, and the absorbance of the 25 largest trophoblast
nuclei in each case was measured by microspectrophotometry. Fig. 6 reveals
that although the largest BUdR-treated nuclei generally occupy a greater area
than the largest of the control nuclei, the absorbances are within the same
range. Since DNA is specifically stained by this procedure, it is concluded that
the BUdR-treated trophoblast nuclei do not endoreduplicate more quickly than
the controls, but that they are merely more flattened. BUdR also causes flattening of other cell types due to a tighter adherence to the substratum (see
Rutter, Pictet & Morris, 1973).
478
M. I. SHERMAN AND S. B. AT1ENZA
0
6-5
8-5
10 5
12 5
Equivalent gestation age (days)
Fig. 7. Progesterone production by control and antimetabolite-treated blastocyst
cultures. The number of blastocysts used per culture and the treatments are as
follows: (A) 300 blastocysts, Ara C (2x 10~5 M) added on the first day of culture
(fourth e.g.d.); (B) 50 blastocysts, BUdR (3 x 10~4 M) added on the second day of
culture (fifth e.g.d.); (C) 30 blastocysts, Colcemid (9x 1O~8 M) added on the third
day of culture (sixth e.g.d.).
To complement the observations described above, blastocyst cultures were
exposed to radioactive thymidine or BUdR, and DNA synthesis in trophoblast
nuclei was monitored by autoradiography (Table 3). Radio-isotopes were
added on the fourth, sixth or eighth day of culture (equivalent gestation ages
of eight, ten and twelve days, respectively), and cells were incubated for a
further 24 h.
Almost every trophoblast nucleus in control cultures was labeled with [3H]thymidine over the 8th to 9th e.g.d. (Table 3); grain density was very high over
most of these nuclei following the 2-week exposure period. Clumps of 1CM
cells were also very heavily labeled.
The percentage of labeled trophoblast nuclei in control cultures drops sharply
when [3H]thymidine is added on the 10th e.g.d. There is only a very slight
further decrease, if any, in the percentage of labeled trophoblast nuclei when
[3H]thymidine is not added until the 12th e.g.d. The reason for the decrease in
labeling of trophoblast nuclei with time in culture is not readily apparent.
Antimetabolite treatment of blastocysts
479
Table 4. Progesterone production by control and antimetabolitetreated blastocyst cultures
Sample
Equivalent gestation day Progesterone produced
showing peak production (p-mole/blastocyst/24 h)
Control
A r a C treated
Control
BUdR treated
Control
8-5
1-18
11-5
2-98
8-5
014
10-5
0-37
8-5
108
11-5
1-38
Colcemid treated
10-5
1-61
Data for the antimetabolite-treated cultures are taken from the experiments described in
Fig. 7. Control cultures, containing approximately the same number of blastocysts as the
experimentals in each experiment, were simultaneously analyzed. The maximal progesterone
production over the period studied is given in each case. Two values are given for the
Colcemid control culture since two peaks of activity were observed.
A high proportion of trophoblast cells developing from blastocysts in the
presence of Ara C or Colcemid (at a variety of concentrations) incorporate
[3H]thymidine into acid-insoluble material on the eighth e.g.d. (Table 3).
However, the percentages of labeled nuclei are consistently lower than those of
controls. This is also true of Colcemid-treated cultures incubated with [3H]thymidine on the 10th e.g.d.
Blastocyst cultures treated with BUdR were incubated with [3H]BUdR instead
of [3H]thymidine. Table 3 indicates that as many trophoblast nuclei are labeled
in BUdR-treated as in control cultures, whether the radioactive nucleosides are
added on the eighth or twelfth e.g.d. In the eighth e.g.d.-BUdR-treated cultures, nuclei in ICM-derived cells which are still surviving at this time have a
grain density similar to that of trophoblast cells. It therefore appears as though
the differential sensitivity of ICM and trophoblast cells to BUdR is due neither
to differential uptake of the nucleoside analogue nor to differential incorporation into DNA.
Effects of antimetabolites upon trophoblast morphology
Morphological aspects of trophoblast development in blastocyst cultures
have been described in detail elsewhere (Sherman, 1975 a, b); briefly, trophoblast
cells flatten out against the culture dish shortly after attachment, the cytoplasm
is highly vacuolated, and cellular boundaries are unclear. During the period
of culture, the cells and their nuclei enlarge as polyploidization proceeds. The
only other marked changes observed are a disappearance of vacuolation after
about 5 days of culture and the subsequent appearance of perinuclear granules.
These morphological characteristics of trophoblast cells are unaffected by
antimetabolites present at concentrations lethal to ICM cells.
480
M. I. SHERMAN AND S. B. ATIENZA
Effects of antimetabolites on progesterone synthesis by trophoblast cells
We have shown elsewhere that cultured blastocysts are capable of synthesizing progesterone, and that the progesterone formed is secreted into the
culture medium (Chew & Sherman, 1973, 1975). Since cultures of trophoblast
cells dissected from the placenta at mid-gestation (Sherman & Salomon, 1975)
or developing from trophoblastic vesicles (Sherman, 1915b) are capable of
producing progesterone, at least part of the progesterone formed in blastocyst
cultures is most likely produced by trophoblast cells. In fact, since only trophoblast of all in vivo mid-gestation embryo-derived tissues has A5,3/?-hydroxysteroid dehydrogenase activity (Chew & Sherman, 1973, 1975), trophoblast
may well be the only cells in blastocyst cultures producing progesterone. We
therefore tested trophoblast cells in antimetabolite-treated blastocyst cultures
to determine whether they were capable of producing progesterone.
Fig. 7 illustrates that trophoblast cells developing from blastocyst cultures,
whether they are treated with Ara C, BUdR or Colcemid, are capable of synthesizing progesterone when pregnenolone is added to the culture medium.
Activity peaks between the 10th to the 12th e.g.d., the time at which homogenates of trophoblast cells dissected from the uterus show maximal progesterone-forming activity (Chew & Sherman, 1973,1975). The pattern of progesterone
production in control cultures is not predictable as it is in antimetabolitetreated cultures. Peak progesterone production in control cultures can occur
not only between the 10th and 12th e.g.d., but at other times as well; double
peaks are also observed (Table 4).
Although the patterns of progesterone production by trophoblast cells in
antimetabolite-treated cultures are similar to in utero trophoblast patterns, it
can be argued that the levels of progesterone synthesis are depressed. In fact,
Table 4 illustrates that although there is variation from one experiment to
another, peak levels of progesterone production by antimetabolite-treated
cultures are consistently higher than those of control cultures. We shall show
elsewhere that this is at least partly due to a depletion by ICM cells of some of
the progesterone synthesized by the trophoblast. It is likely that the time of
peak progesterone production in control cultures is unpredictable because a
variable population of ICM cells develops in different cultures, thereby metabolizing the progesterone formed to different degrees.
DISCUSSION
Although Ara C, BUdR and Colcemid probably all differ in their primary
antimetabolic effect, all three can be used to destroy the ICM moiety of blastocysts in culture, resulting in the production of pure trophoblastic monolayers.
A useful feature of the Ara C and BUdR treatments is the delayed effects of
the antimetabolites. They can be added to unhatched blastocysts, and will
Antimetabolite treatment of blastocysts
481
not interfere with either hatching or attaching at doses which will result in
elimination of all ICM cells within 72 h. In this way, cultures containing relatively large numbers of pure trophoblast cells can be obtained as early as the
eighth e.g.d. Although pure trophoblast monolayers can also be obtained by
culturing 'trophoblastic vesicles' derived from disaggregated blastomeres of
cleavage-stage embryos (Tarkowski & Wroblewska, 1967; Sherman, 1975 &),
antimetabolite treatments are much simpler and less time-consuming.
The reason for the differential sensitivities of ICM and trophoblast cells to
Ara C, BUdR and Colcemid is not clear. One problem is that all three antimetabolites can interfere with cell function in more than one way. Indeed, if
Colcemid blocked cell functions only by its best known effect, namely preventing
further development of cells in mitosis, giant trophoblast cells would be completely refractory, since they are amitotic. Yet, polyploid trophoblast cells are
killed following continuous exposure to Colcemid at concentrations greater
than 2 x 10~~7 M. Even lower concentrations of Colcemid appear to reduce the
extent of thymidine incorporation into DNA by trophoblast nuclei (Table 3).
Colcemid has in fact been found to affect cells in ways unrelated to mitosis
(e.g. Vasiliev et al. 1970; Rizzoni & Palitti, 1973).
Ara C might disrupt replication at a number of steps, as reviewed by RoyBur man (1970). While BUdR has not been reported to have an adverse effect
upon replication per se, it does inhibit ribonucleotide reductase (Meuth &
Green, 1974). BUdR is also incorporated into the DNA, where it can act as
a mutagen, at least in prokaryotes (Brockman & Anderson, 1963), and alter
the stability of the chromatin (David, Gordon & Rutter, 1974). All of the above
actions can be lethal to a cell.
Higher concentrations of Ara C, BUdR and Colcemid might be required to
kill trophoblast cells compared to those of the ICM because the former are
less permeable to the antimetabolites. However, the autoradiographic experiments indicating incorporation of BUdR into trophoblast DNA (Table 3)
would argue against the inability of that antimetabolite to enter trophoblast
cells. Furthermore, Rowinski, Solter & Koprowski (1975) have found a
differential sensitivity of trophoblast and ICM cells to actinomycin D, cordycepin and cycloheximide, similar to that reported here, and a differential
sensitivity to X-irradiation has also been observed (Goldstein, Spindle &
Pedersen, 1975). It is unlikely that permeability differences exist for such a
diverse array of antimetabolites. Similarly, while a relatively large pool of
deoxyribonucleotides in trophoblast, but not ICM, cells could counter the
adverse effects of Ara C and BUdR (Puck & Kao, 1967; Meuth & Green,
1974), this could not explain the observations with Colcemid or the antimetabolites used by the others.
Some other possibilities remain which may be more likely explanations of
the observed results. The first is that ICM cells may be relatively undifferentiated
compared to trophoblast between the fifth to seventh e.g.d., and that even a
482
M. I. SHERMAN AND S. B. ATIENZA
slight disruption of the critical events which are taking place during this time
period might have a drastic effect upon the ICM. Golbus & Epstein (1974) and
Garner (1974) have noted that preimplantation mouse embryos are very sensitive to BUdR; as they noted, sensitivity was much greater than that of several
in vitro differentiating systems that have been studied. ICM cells in our cultures
appear to share this marked sensitivity to BUdR, and to Colcemid as well,
compared to other cells in culture. Studies by Gontcharoff & Mazia (1967)
also indicated that early sea urchin embryos are markedly more sensitive to
BUdR than are embryos which have reached the 100-cell blastula stage. Consistent with all this is our preliminary observation that BUdR added after
72 h of culture does not have the devastating effect upon ICM cell derivatives
that it has when added at earlier times (unpublished observations).
Another characteristic of ICM cells that might render them more sensitive
to a wide spectrum of antimetabolites is their rapid rate of division; giant
trophoblast cells, which are non-dividing, might for this reason be able to
survive sharp reductions in the rates of DNA, RNA or protein synthesis.
Finally, the multigenomic nature of polyploid trophoblast cells may serve as
a protective device against antimetabolites: if some genes or gene functions are
made inoperative at any level by an antimetabolite, the presence of unusually
large numbers of alternate genes may compensate for the loss. Further studies
are necessary to test these proposals.
The polyploid nature of trophoblast cells may also explain why BUdR does
not block the expression of A5,3/?-hydroxysteroid dehydrogenase, as it does
with other so-called 'luxury' enzymes (see Rutter et al. 1973). BUdR may not be
effective in this case because trophoblast cells are non-dividing, or because the
antimetabolite is unable to inactivate all the A5,3/?-hydroxy steroid dehydrogenase
genes in these multigenomic cells.
The experiments described here and elsewhere (Sherman, 19756, c) indicate
that trophoblast development and differentiation can take place independently
of the ICM. Since it has also been demonstrated that post-blastocyst development and differentiation do not require uterine-specific factors (see Sherman,
1974; Sherman & Salomon, 1975), the information for programming trophoblast cell development appears to lie within that cell. On the other hand, our
abilities to monitor the biochemical properties of trophoblast cells without
interference by ICM cells may lead to information about biochemical cooperation between trophoblast and ICM cells. The utilization by ICM-derived
cells of progesterone produced by trophoblast is one such example.
Antimetabolite treatment of blastocysts
483
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(Received 14 March 1975, revised 6 May 1975)