/ . Embryo!, exp. Morph. Vol. 56, pp. 1-22, 1980
Printed in Great Britain © Company of Biologists Limited 1980
Effects of culture conditions on the developmental
programme of mouse blastocysts
By MARTIN H. SELLENS1 AND MICHAEL I. SHERMAN 2
From the Roche Institute of Molecular Biology
Nutley, New Jersey
SUMMARY
We have investigated the developmental potential of mouse blastocysts cultured under a
variety of conditions. A number of parameters were used as criteria for development and
differentiation, namely hatching of blastocysts from the zona pellucida and their adhesion to
the substratum, outgrowth and polyploidization of trophoblast cells, increase in cell number,
protein content, /?-glucuronidase activity, appearance of lactate dehydrogenase A subunits,
plasminogen activator production, and A5,3/?hydroxysteroid dehydrogenase activity.
Under optimal culture conditions, embryos grew relatively rapidly and expressed all the
differentiative markers for which they were tested. Under less supportive conditions, the
production of the markers was usually reduced quantitatively; the expression of some markers
could also be considerably delayed or even totally prevented. In fact, embryos cultured in the
least nutritive medium (one designed to support development only through pre-implantation
stages) appeared to be in a state of metabolic quiescence closely resembling that of blastocysts in ovariectomy-induced delay. Overall, the results of our investigations lead us to
propose that the expression of each of the aforementioned markers is probably under independent control and subject to intrinsic programming. Finally, the observation that some
markers are produced by embryos in suboptimal media whereas others are not, suggests that
the minimum metabolic level necessary for expression varies from one marker to another.
INTRODUCTION
Mouse blastocysts cultured in appropriate serum-containing media acquire
a number of properties analogous to those observed during the peri-implantation
period (the time just prior to, during, and shortly after, implantation) in utero
(for reviews, see Sherman & Wudl, 1976; Jenkinson, 1977). Among these are the
ability of the blastocyst to hatch from the zona pellucida and the acquisition of
an adhesive surface and subsequently of migratory properties by the outer,
trophoblast layer. Biochemical analyses indicate that blastocysts also express
several gene products for the first time during the peri-implantation period
both in utero and in culture. These include lactate dehydrogenase (LDH) A
subunits (Auerbach & Brinster, 1967, 1968; Monk & Ansell, 1976; Monk &
1
Author's present address: Department of Biology, University of Essex, Wivenhoe Park,
Colchester CO4 3SQ, U.K.
2
Author's address for reprints: Roche Institute of Molecular Biology, Nutley, New Jersey
071.10, U.S.A.
2
M. H. SELLENS AND M. I. SHERMAN
Petzoldt, 1977; Spielmann, Eibs, Jacob-Miller & Bischoff, 1978), plasminogen
activator (Strickland, Reich & Sherman, 1976; Sherman, 1980) and A5,3/?hydroxysteroid dehydrogenase (3/?-HSD) (Chew & Sherman, 1975; Sherman &
Atienza, 1975). The fact that blastocysts not only express these properties
in vitro but do so in proper sequence and close to the temporal schedule observed
in utero suggests that these events are either intrinsically programmed or triggered in response to activators present in serum-containing culture medium
as well as in the uterine milieu. The recent development of a partially defined
medium, EM2, which is serum-free but nevertheless supports blastocyst development and differentiation through peri-implantation stages (Rizzino & Sherman,
1979), tends to favour the former view.
Blastocysts cultured in EM2 medium hatch, attach and grow out along the
culture dish with the same high frequencies observed in serum-containing media,
albeit with some delay. However, in several other serum-free media tested, these
events were delayed even further or prevented totally (Rizzino & Sherman, 1979;
Sherman et al. 1979). We and others had found previously that hatching,
attachment and outgrowth were not necessary for the expression of some
differentiative markers by cultured blastocysts (Sherman, 1972 a, b; Barlow &
Sherman, 1972; Spielmann et al. 1978). In the present study, we have attempted
a more comprehensive comparison of the developmental properties of blastocysts in optimal medium and under culture conditions which are either suboptimal for, or preclusive of, the attachment and outgrowth of blastocysts.
Accordingly, in addition to assaying blastocysts under the various culture
conditions for the expression of the 'new' gene products mentioned above,
we have also determined their cell numbers and mitotic indices, total protein
contents, /?-glucuronidase activities [since levels of this enzyme rise rapidly
under optimal post-blastocyst culture conditions (Wudl & Sherman, 1976)]
and extents of polyploidization. Furthermore, we have compared the properties
of blastocysts cultured in media which do not permit attachment and outgrowth
with blastocysts under similar constraints in the uteri of ovariectomized
females.
Our results indicate that under conditions of metabolic insufficiency, certain
differentiative gene products are expressed, whereas the appearance of others
is either prevented or substantially delayed. Reasons for this apparent dichotomy
are considered.
MATERIALS AND METHODS
Embryos
Embryos were obtained from SWR/J (Jackson Laboratory, Bar Harbor,
ME) females, superovulated (Runner & Palm, 1953) and mated with SJL/J
(Jackson Laboratory) males. Blastocysts were collected on the afternoon of
the 4th day of pregnancy (the day of observation of the sperm plug is considered
the first day) by flushing the dissected uterine horns with phosphate-buffered
Mouse blastocyst development in vitro
3
saline (PBS - solution A of Dulbecco & Vogt). Prior to culture, all embryos
were washed once with PBS. Delayed blastocysts were obtained by ovariectomising pregnant females early on the 3rd day of pregnancy (Yoshinaga &
Adams, 1966). Progesterone therapy during delay was not used. Embryos were
collected 7 days after ovariectomy in the same way as for normal blastocysts
except that they were flushed into a culture dish containing a pad of 2 % agarose
(SeaKem, MCI Biomedical, Rockland, ME) to prevent attachment (Shalgi &
Sherman, 1979). Time in culture is referred to in terms of equivalent gestation
days (EGD), the age of the embryos from the time of conception. (When assays
were carried out for 24 h periods the EGD is expressed in half days; for example,
EGD 5-5 refers to an assay begun on EGD 5 and terminated on EGD 6.)
Media and culture conditions
Bovine serum albumin (BSA; Pentex/Miles Laboratories, Elkhart, IN) was
made up as a 6 % solution in distilled water, dialysed extensively at 4 °C against
distilled water, adjusted to a concentration of 3 % and stored in aliquots at
- 2 0 °C. Fetuin (from bovine serum, Pedersen method, Calbiochem, La Jolla,
CA) was solubilized as described by Rizzino & Sato (1978), dialysed extensively
at 4 °C against acidified distilled water (adjusted to pH 2-5 with 5 N-HC1), and
stored in aliquots at - 20 °C. Aliquots were thawed only once and during use
were stored for up to two weeks at 4 °C. In all media the following antibiotics
were used: penicillin (lOOu/ml), streptomycin (100/ig/ml) and kanamycin
(100 u/ml) (Gibco, Grand Island, NY).
PCM, the preimplantation culture medium of Goldstein, Spindle & Pedersen
(1975), was variously supplemented with 0-05 % fetuin (PCM + FET), with amino acids and vitamins (MEM vitamins, Gibco) at the concentrations used by
Spindle & Pedersen (1973) (PCM+AA+VIT), or with all of these components
(PCM+AA+VIT + FET).
EM2 is a serum-free medium which supports post-implantation development
(Rizzino & Sherman, 1979). It consists of equal parts of (i) NCTC 109 medium
(Microbiological Associates, Bethesda, MD) supplemented with 0-3% BSA
and antibiotics, and (ii) PCM + AA + VIT. This mixture is further supplemented
with 0-05 % fetuin. EM2 with fetuin omitted (EM2-FET) was also used in
these experiments.
NCTC 109 medium supplemented with antibiotics and with 10 % heat inactivated (56 °C, 20 min) fetal calf serum (FCS; selected lots from Microbiological
Associates) was the control medium for these experiments because it has been
found to be optimal in this laboratory for blastocyst development (Sherman,
1975).x This complete medium (abbreviated hereafter as cNCTC) was also
used in culture dishes coated with a thin pad of 2 % agarose (Sherman, 1978)
(cNCTC/agarose).
1
For footnote see following page.
4
M. H. SELLENS AND M. I. SHERMAN
Unless otherwise noted, all cultures were carried out in 3 ml medium in 35mm
tissue-culture-grade plastic dishes (Falcon, Oxnard, CA). Twenty to thirty
embryos were cultured per dish. Two milliliters of the medium were changed
every second day except where indicated. Cultures were maintained at 37 °C
in an atmosphere of 5 % CO2 in air and at high humidity.
Hatching, attachment and outgrowth
Embryos were inspected daily with a dissecting microscope to determine
times of hatching from the zona pellucida, attachment to the substratum and
outgrowth of trophoblast cells. T50 refers to the time at which 50% of the embryos had reached a given developmental stage.
Cell counts, mitotic indices and nuclear DNA measurements
The method of Tarkowski (1966), as modified by Pedersen & Cleaver (1975),
was employed to produce cellular spreads from whole embryos. The procedures
for determination of nuclear DNA content by microfluorometry of Feulgenstained spreads have been described elsewhere (Barlow & Sherman, 1972;
Wudl & Sherman, 1978). Liver nuclei DNA values were used to standardize
the microfluorometer. Cell numbers and mitotic indices (MI) were determined
from the same cell spreads by observation with phase contrast optics.
Protein content
Embryos were washed through three changes of PBS. The zona pellucida,
where present, was removed after the first wash by a brief exposure to PBS
adjusted to pH 2-5 with 5 N-HC1 and containing 0-4% (w/v) polyvinyl pyrrolidone (MW 40000) (Handyside & Barton, 1977). In this and other studies, embryos attached to, and/or outgrowing along, the substratum were carefully
detached and collected with a drawn-out and flame-polished capillary tube.
Thereafter these embryos were processed in the same way as non-adherent
embryos. The washed embryos were transferred in groups of three to five into
10 /d of 0-1 M lithium borate buffer, pH 8-7, containing 0-05 % sodium dodecyl
sulphate. Samples were frozen and thawed three times and the protein separated
from amino acids and peptides (MW ^ 6000 daltons) on a Bio-Gel P6 (BioRad Laboratories, Richmond, CA) column. Protein content was measured
using fluorescamine reagent (Bohlen, Stein, Dairman & Udenfriend, 1973) and
a preparative analytical peptide analyzer (Hoffman-LaRoche Inc., Nutley, NJ)
standardized with BSA (manuscript in preparation).
1
It has been reported (McLaren & Hensleigh, 1975) that postblastocyst development was
very poor in cNCTC. We and others (H. Spielmann, personal communication; C. Hammerberg, personal communication) have observed enormous variation in the effectiveness of
NCTC-109 medium from different suppliers. We have found postblastocyst development in
supplemented NCTC-109 medium to be highly reproducible provided that we use pretested
lots of serum and medium which is less than six months old and which has been purchased
from Microbiological Associates.
Mouse blastocyst development in vitro
5
Enzyme analyses
/?-Glucuronidase assays were carried out by microflurometric determinations using 4-methylumbelliferyl-/?-D-glucuronide (Sigma, St Louis, MO;
3-10-4 M in 0-1 M acetate buffer, pH 5-6) as substrate (Wudl & Sherman, 1976).
Each assay sample contained a sufficient number of embryos (from 2 to 30,
depending upon EGD and culture medium used) to give an aggregate enzyme
activity between 10~7 and 10~6 moles 4-methylumbelliferone produced per hour
at 37 °C. The production of plasminogen activator was assayed by the fibrinagar overlay procedure (Beers, Strickland & Reich, 1975; Sherman, 1980).
The production of plasminogen activator was measured semi-quantitatively
by determining the size of the lysis zones, after overnight incubation in the
reaction mixture, under a dissecting microscope. Scoring was in arbitrary units
as follows: no zone of lysis, 0; faint zone of lysis, 0-5; distinct lysis zone, 0 1 1-0 mm diameter, 1-0; 1-1 to 2 0 mm diameter, 2-0; greater than 2 mm diameter,
3-0. A5,3/?-Hydroxysteroid dehydrogenase (3/?-HSD) activity was determined by
radioimmunoassay measuring the conversion of pregnenolone to progesterone
by groups of 10-15 embryos (Marcal, Chew, Salomon & Sherman, 1975;
Salomon & Sherman, 1975). For LDH analyses, embryos were washed three
times with PBS, placed in groups of 50 to 100 into distilled water, and frozen
and thawed three times. The samples were then mixed with ethylene glycol
(17% final concentration) and bromphenol blue (Sigma), electrophoresed on
0-5 mm thick 8 % polyacrylamide slab gels (Dietz & Lubrano, 1967) until the
bromphenol blue marker reached the bottom of the gel (approx. 4 h), and
stained for LDH activity by the procedure of Shaw & Koen (1968).
RESULTS
Hatching, attachment and outgrowth
Hatching occurred under all culture conditions and in most cases attained
levels which were similar to those observed in optimal (cNCTC) medium
(Fig. 1). However, both the extent and rate of hatching were substantially
reduced in PCM + AA+VIT (Fig. ID). Although hatching was normal when
this medium was further supplemented with fetuin (Fig. 1C), it was noted that
during the initial several hours of culture, blastocysts in both of these media
did not appear to be as fully expanded as those under other culture conditions.
Hatching was delayed slightly, if at all, in EM2 media. Although all blastocysts
hatched in PCM media lacking amino acids (Figs. 1A and B), there was evidence
of a delay.
Attachment of blastocysts occurred soon after hatching in cNCTC medium
(Fig. 1G). Embryos attached to the substratum, although after longer periods
of culture, in the serum-free media, provided that fetuin was present (Figs. 1 A,
M. H. SELLENS AND M. I. SHERMAN
100 -
20 40 60 80 100 160
20 40 60 80 100160
Time in culture (h)
Fig. 1. Hatching, attachment and outgrowth of blastocysts in different culture
media. Embryos were collected on the fourth day of pregnancy and cultured in
groups of 20-30 in 35 mm dishes containing 3 ml of the following media (for full
description of culture media see Materials and Methods): (A) PCM+FET; (B)
PCM; (C) PCM+ AA+VIT+FET; (D) PCM+AA+VIT; (E) EM2; (F) EM2FET; (G) cNCTC; (H) cNCTC/agarose. Each point represents observations on a
total of 50 to 300 embryos from three separate experiments. Embryos were scored
by examination under a dissecting microscope for hatching from the zona pellucida
(O), attachment to the substratum (A), and trophoblast outgrowth ( • ) .
C and E). Attachment was completely prevented by coating the dishes with an
agarose pad (Fig. 1H) and was only transient in serum-free media lacking
fetuin (Figs. 1B, D and F).
Outgrowths of trophoblast cells were produced by all blastocysts in cNCTC
medium. Outgrowths were also observed in EM2 medium and in PCM+AA +
VIT + FET but their appearance was delayed and the extent of outgrowth
was smaller, particularly in the latter medium. Blastocysts cultured in PCM +
FET rarely gave rise to trophoblast outgrowths (Fig. 1 A) and on such occasions
Mouse blastocyst development in vitro
2
o
I
D
B
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50 J_l
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8
10
4 6 8
10
Equivalent gestation day
1
7
i
4
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6
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8 10
Fig. 2. Cell number and mitotic index of cultured blastocysts. Culture conditions
were as follows: (A) PCM (O), PCM+FET (A); (B) PCM + AA+VIT (O), PCM+
AA + VIT + FET (A); (C) EM2-FET (O), EM2 (A); (D) cNCTC/agarose (O),
cNCTC (A). Fourth day values ( • ) are from blastocysts collected without culture.
Embryos were collected from culture at the times indicated and cell spreads were
made on microscope slides as described in Materials and Methods. The cells were
counted and examined for mitotic figures by phase contrast microscopy. Between 5
and 12 embryos were examined and a mean cell number calculated. Mitotic index
(MI) values reflect the percent of cells in mitosis (each telophase pair was counted
as a single mitotic figure).
only a few outgrowing cells were in evidence. Outgrowths were never observed
under the other culture conditions.
Cell number and mitotic index
Under optimal conditions (Fig. 2D), cell number increased almost exponentially throughout the culture period. Embryos prevented from attachment and
outgrowth by the presence of an agarose pad contained considerably fewer
cells (Fig. 2D). By EGD 11, many of these embryos appeared disorganized and
showed signs of necrosis. Cell numbers observed in embryos cultured in EM2
medium initially increased exponentially, although with a prolonged doubling
time compared with embryos in cNCTC medium (Fig. 2C). Omission of fetuin
8
M. H. SELLENS AND M. I. SHERMAN
from EM2 medium did not affect the initial rise in cell number, but did influence
the final numbers reached (Fig. 2C). The gross morphology of the embryos
was similar to those cultured in cNCTC medium on agarose pads. In PCM 4AA+VIT + FET, embryos averaged two cell doublings by EGD 9 with no
subsequent increase in cell number (Fig. 2B). In the other suboptimal media,
maximum cell numbers reached only about 120 to 130 and this number declined
after EGD 8 (Figs. 2 A, B).
Whereas the MI in cNCTC medium declined gradually over the culture
period, consistent with the observed tailing off of the rate of increase of cell
number, the MI of embryos in most of the suboptimal media fell more precipitously (Fig. 2). The observation that the MI of embryos were similar in cNCTC
medium in the absence or presence of an agarose pad was interesting in view
of the differences in cell number (Fig. 2D). In the serum-free media, the MI
values generally correlated with the observed cell numbers. There was often a
transient increase in the MI of embryos on EGD 7 and again on EGD 9, in
each case on the day following renewal of the culture medium.
Protein content
The total protein content of embryos cultured in cNCTC medium increased
approximately 100-fold over the period of culture (Fig. 3 D), reflecting the increase
in cell number. Embryos cultured on agarose had a proportionately greater
increase in protein content than in cell number (see Table 1). Similar relationships were observed for embryos cultured in EM2 media (Fig. 3C and Table 1),
that is, the absence of fetuin resulted in an increase in the ratio of protein content
to cell number. The increase in protein content in these media over the period
of culture was approximately 25-fold. Embryos cultured in PCM supplemented
with amino acids and vitamins showed an increase in protein content of only
three- to fourfold either in the presence or absence of fetuin (Fig. 3 B). Once again,
although the increase in protein content was small compared with that of embryos in optimal medium, it was continuous, unlike the increase in cell number.
Finally, embryos cultured in PCM showed a pattern of protein content mirroring the cell number data: there was an increase until EGD 7 followed by a
gradual decrease over the remaining culture period (Fig. 3 A). However, in
PCM + FET, this decrease was not observed.
fi-Glucuronidase activity
Under all culture conditions, the /?-glucuronidase activity of the embryos
rose continuously through EGD 10 (Fig. 4). There were, however, variations
in the ratios of enzyme activity per cell and per unit protein (Table 1). As with
total protein content, the enzyme activity per cell was generally less in media
permitting outgrowth than in the equivalent media, lacking fetuin (or with an
agarose pad), which precluded outgrowth. The data in Table 1 also indicate that
Mouse blastocyst development in vitro
2000
1000
500
200
100
50
20
10
4
6
8
10
4
6
8 10
4 6 8 10
Equivalent gestation day
4
6
8
10
Fig. 3. Protein content of cultured blastocysts. Culture conditions and symbols
are the same as for Fig. 2. Embryos were prepared for protein analysis as described
in Materials and Methods. Samples contained three tofiveembryos and values were
averaged from three separate experiments.
there is a large increase in the amount of /?-glucuronidase activity relative to
protein content during the period of culture under all conditions used.
DNA content
The DNA content of the largest trophoblast cells of blastocysts cultured in
cNCTC medium underwent five to six cycles of endoreduplication by EGD 11
(Fig. 5D)5 about one cycle more than the largest trophoblast cells of embryos
cultured in cNCTC medium on an agarose pad. The largest trophoblast cells
from embryos cultured in EM2 media (Fig. 5C) or in supplemented PCM
(Fig. 5B) underwent fewer cycles and the time of onset of polyploidization was
delayed. The presence or absence of fetuin did not markedly affect the ploidy
levels reached in these media. Fetuin was, however, essential for the onset of
polyploidization of trophoblast cells in otherwise unsupplemented PCM (Fig.
5 A): in PCM alone there was no evidence of polyploidization during seven days
of cultnre, whereas polyploidization was observed in trophoblast cells cultured
10
M. H. SELLENS AND M. I. SHERMAN
Table 1. Protein contents andft-glucuronidaseactivities of uncultured fourth day
or delayed blastocysts and of biastocysts cultured in various media
Conditions
Protein
content
per cell
(ng)
/?-Glucuronidase
activity
per cell
/?-Glucuronidase
activity per
ng protein
Relative
/?-glucuronidase
activity*
Fourth day blastocysts
In utero delayed blastocysts
PCM
PCM + FET
PCM + AA+VIT
PCM + A A + VIT+FET
EM2-FET
EM2
cNCTC/agarose
cNCTC
0-39
018
017
0-43
0-84
0-31
119
0-48
1-28
0-72
3-2
26-7
17-3
36-3
360
18-8
42-4
17-8
50-6
22-7
8-2
1480
1000
85-3
42-7
60-3
35-7
37-3
39-6
31-8
180
12-2
10-4
5-2
7-4
4-3
4-5
4-8
3-9
Values for 4th day blastocysts are taken from data in Figs. 2 to 4. Values for cultured embryos on EGD 10 and for blastocysts delayed in utero for an equivalent period of time are
taken from Figs. 2 to 4 and Table 4, respectively.
/?-Glucuronidase activity is expressed as fmoles 4-methylumbelliferone produced/embryo/hr.
* Relative increase in /?-glucuronidase specific activity is calculated as the /?-glucuronidase
activity per ng protein under each condition divided by 8-2, the value for 4th day (uncultured)
blastocysts.
in PCM + FET, although only a single endoreduplicative cycle had taken place
by EGD 11.
Plasminogen activator production
Under all culture conditions, lysis zones indicating secretion of plasminogen
activator first became detectable in embryos assayed between EGD 5 and 6
(Fig. 6). In fact, there was little difference in the size the lysis zones prior to
EGD 7. Although embryos continued to secrete plasminogen activator for the
Fig. 4. /?-Glucuronidase activity of cultured blastocysts. Culture conditions and
symbols were the same as for Fig. 2 and enzyme activity was determined as described
in Materials and Methods. Between four and six samples from two separate experiments, each sample containing several embryos, were evaluated and mean values
calculated.
Fig. 5. Nuclear DNA content of cultured blastocysts. Culture conditions and
symbols were the same as for Fig. 2. Cell spreads of embryos collected in two separate experiments were made as for Fig. 2 and the nuclei were stained with Schiff's
reagent. DNA measurements were made by microfluorometry and expressed as
multiples of the haploid amount (C) by reference to values obtained for liver
nuclei. Each point represents the average of measurements on a total of 18 to 30
nuclei, taking the largest three to five nuclei from each of at least five separate
embryos.
Mouse blastocyst development in vitro
10 4
6
8 10 4
6
8
Equivalent gestation day
11
10 4
Fig. 4
256
128
64
32
1.6
Q
A
I
8
1 I
10
I
4
I
I
I
6
8 10
4 6
8
Equivalent gestation day
I
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I I
Fig. 5
I I
I I
I I
10
I
I
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8
I
I I
10
12
M. H. SELLENS AND M. I. SHERMAN
2-5 -
A
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B
-- c
-- D
£ 2-0
1-5
10
0-5
8-5 10-5
4-5
6-5 8-5 10-5 4-5 6-5
Equivalent gestation day
5 10-5
4-5 6-5 8-5 10-5
Fig. 6. Plasminogen activator activity in cultured blastocysts. Culture conditions
and symbols were the same as for Fig. 2. Enzyme activity was determined by the
fibrin-agar overlay procedure and lysis zones were inspected after overnight
incubation. The sizes of the lysis zones are expressed in terms of arbitrary units
as explained in Materials and Methods. Each point represents the analysis of at least
20 embryos.
duration of the culture period, the average size of the lysis zone differed among
culture conditions as would be expected from the other parameters already
considered. It should be stressed that lysis zone size is only a semi-quantitative
estimation of plasminogen activator production and secretion. For example,
outgrowing blastocysts might have larger lysis zones than those failing to outgrow
partly because lysis zone size includes the extent of outgrowth in the former
case. Another consideration is that the arbitrary units used to represent activity
are based on linear measurements, i.e. the diameter of the lysis zones, whereas a
more realistic estimate of the amount of enzyme secreted might be in terms of
the volume of the lysis zone (which is difficult to measure because of variability
in the thickness of the agar overlay). It is, nevertheless, clear from these experiments that embryos under all culture conditions secreted plasminogen activator
continuously and without an initial delay and that the amount of plasminogen
activator produced per embryo was similar in EM2 and cNCTC medium,
substantially less in PCM + AA + VIT, and even less in unsupplemented PCM.
A5,3(3-Hydroxysteroid dehydrogenase activity
There were dramatic effects of culture medium on the ability of embryos to
convert pregnenolone to progesterone via 3/?-HSD activity (Fig. 7). Three
Mouse blastocyst development in vitro
4-5
6-5
8-5
10 5 4-5
6-5
8-5 10-5 4-5 6-5 8-5 1 0 5
Equivalent gestation day
4-5
13
6-5
8-5 10-5
Fig. 7. Conversion of pregnenolone to progesterone by blastocysts cultured in
various media. Culture conditions and symbols were the same as for Fig. 2 except
that all media were supplemented with 1 /*g/ml pregnenolone and that FCS, where
used, was dextran-norit treated. 3/?-HSD activity was measured for groups of .10 to
15 embryos by determining the amount of progesterone secreted into the medium
[Chew & Sherman (1975) have demonstrated that 95 % or more of the progesterone
formed by blastocysts can be detected in the culture medium at any time during
the assay period]. Each point represents the average of determinations on samples
of medium from at least six blastocyst cultures from three separate experiments.
points are noteworthy: (a) embryos in PCM (with or without fetuin) failed
to produce detectable amounts of progesterone (Fig. 7 A); (b) in PCM + AA +
VIT small amounts of 3/?-HSD activity were observed but, as with polyploidization, the time of appearance of measurable levels of progesterone was
delayed substantially; and (c) embryos in EM2 medium (with or without fetuin)
appeared to produce much more progesterone than those in cNCTC medium.
The last observation is expected on the basis of previous findings and is related
to the fact that the inner cell mass (ICM) of the blastocyst develops more
extensively in cNCTC medium than in EM2 medium (see Sherman, Atienza,
Salomon & Wudl, 1977; Rizzino & Sherman, 1979).
Lactate dehydrogenase activity
Prior to implantation, mouse embryos contain LDH-1 (four B subunits)
as the predominant isozyme, whereas following implantation electrophoretic
analysis reveals the presence of enzyme containing A subunits, namely LDH-5
(4A), LDH-4 (3A, IB) and LDH-3 (2A, 2B) (Auerbach & Brinster, 1967,
2
EMB 56
LDH-1
LDH-2 LDH-3 LDH-4 LDH-5
LDH-1 LDH-2 LDH-3
LDH-4
Equivalent gestation day 10
LDH-5
W
f
+
±
±
±
+
+
+
+
+
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cNCTC
cNCTC/agarose
++
±
+
+
+
++
+
±
++
++
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EM2
+
±
±
++
++
™
EM2
+
±*
±*
EM2-FET
+
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±
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+
^
±
PCM + AA + V1T + FET
+
±
*
±
±
±
2
PCM + AA + YIT
+
± * ± * ± * ±*
±
±
°
PCM + FET
+
_
_
_
_
_
_
_
_
_
g
_
_
_
^
PCM
+
_
_
_
_
_
_
Disrupted cells were analysed for LDH content by electrophoresis on polyacrylamide slab gels as described in Materials and Methods. Fifty to c/i
100 embryos were used in each analysis. Gels were scored for LDH activity independently by both investigators. Scoring was the same except for E
some cases in which only one investigator noted the presence of a faint band of activity. Scoring was as follows: + + , strong band of LDH activity ^
+ , distinct band; ± , faint band; ± *, scored ± by one investigator and — by the other; —, no band visible. Unlike the observations of Monk and g
Petzholdt (1977), extra bands, i.e. those not corresponding to LDH 1, 2, 3, 4 or 5, were not noted. We have also confirmed previous observations >
(Spielmann et al. 1978) that LDH activity in the FCS component of cNCTC does not comigrate with any of the murine LDH bands.
2
Culture medium
Equivalent gestation day 8
Table 2. Lactate dehydrogenase electrophoretic profiles from embryos cultured in various media
Mouse blastocyst development in vitro
15
1968; Monk & Ansell, 1976; Monk & Petzoldt, 1977; Spielmann et ah 1978).
Consistent with these reports, blastocysts cultured in cNCTC medium produced
A subunits within four days. By EGD 10, A subunits were predominant, since
only LDH-4 and LDH-5 bands were observed (Table 2). Similar patterns were
observed in EM2 media and in PCM+AA+VIT, both with and without
fetuin. However, in PCM with or without fetuin, no LDH activity was detectable by EGD 10 under our conditions. Conversely, blastocysts cultured in
cNCTC on an agarose pad showed the normal appearance of A subunits, but
the persistence of a strong LDH-1 band on EGD 10 suggested that there had
been no obvious diminution of B subunits.
In cNCTC/agarose, some blastocysts form aggregates whereas others do not.
Accordingly, we analyzed groups of aggregated and nonaggregated blastocysts
for LDH activity. We observed that on EGD 6 the aggregated group of embryos
contained bands of LDH-2 and LDH-3 as well as LDH-1, whereas non-aggregated embryos contained only LDH-1. However, by EGD 10, the profiles for
the two groups contained all five LDH bands.
Blastocysts delayed from implanting in vivo
In order to determine whether embryos prevented from attachment and/or
outgrowth in vitro resembled those prevented from implanting in vivo in other
respects, we compared the properties of blastocysts maintained in ovariectomised
mothers for one week or cultured for a similar period of time, either in PCM,
PCM + FET, PCM+AA+VIT or EM2-FET (Table 3).
In general, the results indicated that the properties of implantation-delayed
blastocysts most closely resembled those of embryos cultured in PCM, with or
without fetuin, and suggested that the development of blastocysts cultured in
PCM+AA+VIT or EM2-FET was more advanced than in blastocysts delayed
in vivo. It should be noted in particular that in v/vodelayed blastocysts resemble
embryos cultured in PCM in that they fail to polyploidize, secrete relatively
small amounts of plasminogen activator and do not possess detectable 3/?HSD and LDH-5 activities.
DISCUSSION
In this study, we have for the first time attempted to quantitate a spectrum
of developmental parameters as well as markers of differentiation for blastocysts developing under a variety of culture conditions. In cNCTC, a medium
which has been demonstrated previously to support substantial post-blastocyst
development in vitro (Sherman, 1975), embryos resemble qualitatively those
developing in vivo with respect to the timing and sequence of appearance of the
markers under investigation. However, even these in vitro conditions, which are
the most favorable that we have used, do not support embryonic growth at a
rate similar to that which occurs in vivo. For example, an 8th day conceptus
2-38
±
±
10
392
140
45
2-2
331±88
EM2-FET
014
11
75
3-2
14
11
89 ±52
PCM + AA + VIT
9 n n t a fnr in ,itt>r<
< 010*
0
34
2-9
6
0-9
80±ll-4
PCM+FET
Culture media
a.
y
g
y p
following total numbers of embryos (number of separate determinations in parentheses): cell number and MI, 9 (the MI value of 01 reflects, in fact,
only a single mitotic figure in approximately 1000 cells); protein content, 35 (5); /?-glucuronidase activity, 66 (6); ploidy, 9 (the 30 largest nuclei
were measured); plasminogen activator activity, 32 (3); 3/?-HSD, 75 (5); LDH, 50 (1).
* Becuase of weak cross-reactivity of the substrate (pregnenolone) with the antibody, 0 1 pmole/embryo/day is the lowest meaningful level of
progesterone production measurable by the radioimmunoassay.
1—1 a n H TS»V»1F\
< 010*
< 010*
Fioc
0
18
1-8
4
0-8
105 ±32
PCM
01
21
3-1
4
0-5
delay
116 + 22
"Hflta fnr rnitiirpH emhrvns are FfrD 10 or 10-5 v;}1np<i taVpn from
Cell numbers ±SD
Mitotic index
Protein content (ng/embryo)
/?-Glucuronidase activity (pm/embryo/ hr)
Ploidy (C)
Plasminogen activator activity
(arbitrary units)
3/?-HSD
(pm progesterone/embryo/day)
LDH-5
Parameter
Tn u tt> f*/i
jri ttitzru
Table 3. Comparison of properties of blastocysts delayed from implantation in utero with those prevented from attachment and/
or outgrowth in culture for an equivalent period of time
0
•>>.
> ^
r
w1
r
W
l-M
SHE
Mouse blastocyst development in vitro
17
in utero contains approximately 20/*g total protein (Sherman, unpublished
observations) and the egg cylinder alone contains about 16000 cells (Snow,
1976); in cNCTC on EGD 8, after the most rapid period of growth in vitro,
embryos contain only 600 ng protein and about 900 cells, including trophoblast.
On the basis of the protein values, we can estimate that the approximate
protein do.ubling time in cNCTC is about 19 h from the 4th day to EGD 8
(approximately five doublings in 96 h), whereas it is twice as fast (about ten
doublings in 96 h, giving a doubling time of 9-6 h) in utero. The calculation of
the difference in doubling time with respect to cell number is complicated by the
fact that all cells of the blastocyst appear capable of division from the 4th to the
5th day, both in vitro (Fig. 2) and in utero (Barlow, Owen & Graham, 1972),
but thereafter some of the cells become non-dividing as they polyploidize to form
giant trophoblast cells. If we use 42 cells as an estimate of the average number of
the latter cells on the 5th day (see Table 2 of Sherman & Atienza, 1975), then
the doubling time of the remainder of the population is approximately 18 h
in cNCTC (56 cells to 920 cells in 72 h) and approximately 9 h in utero (56 cells
to 16 000 cells in 72 h). Although these estimates do not take into account other
possible influential factors such as cell death, they nevertheless suggest that (a)
protein and cell doubling times are about twice as rapid in vivo as in vitro and
(b) since the protein doubling time (of both ICM and trophectoderm derivatives)
is very similar to the cell doubling time (ICM derivatives only), protein content
most likely increases at the same rate in the total ICM-derived cell population
as in the total trophectoderm-derived cell population.
Several previous studies have focussed upon the molecular requirements for
hatching, attachment and outgrowth in vitro. Contrary to the observations
of Spindle & Pedersen (1973), we have not noted that hatching is inhibited by the
absence of amino acids (Fig. 1). This discrepancy might be due to the different
protein concentrations (and thus the potential source of exogenous amino
acids) in the media: in the study by Spindle & Pedersen, the medium contained
1 % FCS, that is, approximately 0-5 mg/ml total protein, whereas the concentration of BSA in PCM amounts to 3 mg/ml. Nevertheless, aside from a requirement for glucose (Wordinger & Brinster, 1976), presumably as an energy
source for blastocyst expansion and contraction during hatching, the blastocyst appears to be undemanding in terms of nutrient requirements for shedding
of the zona pellucida.
Blastocysts will attach permanently to the culture dish and trophoblast cells
will outgrow along it only in the presence of an appropriate substratum, an
adequate source of energy, and an exogenous supply of amino acids (Gwatkin,
1966tf, b; Spindle & Pedersen, 1973; Wordinger & Brinster, 1976; Van Blerkom,
Chavez & Bell, 1979; Rizzino & Sherman, 1979). Both serum and fetuin preparations contain a component which promotes blastocyst adherence, though not to
an agarose pad (Fig. 1; Sherman, 1978). Fetuin does not appear to be required
18
M. H. SELLENS AND M. I. SHERMAN
for the onset of blastocyst adhesiveness; however, attachment is only transient
in the absence of fetuin preparations or serum (Fig. 1B, D, F). We should stress
that we have not yet established fetuin itself to be the agent which facilitates
adhesion since our previous studies indicate that the fetuin preparations used to
contain contaminating proteins, albeit at relatively low levels (Rizzino & Sherman, 1979).
Unlike Gwatkin (1966a, b) and Spindle & Pedersen (1973), we have not found
that arginine and leucine are required for blastocyst attachment in our media
(e.g. Fig. 1 A). We can confirm the requirement for amino acids for trophoblast
outgrowth (Gwatkin, 1966a, b; Spindle & Pedersen, 1975) in our serum-free
media provided that albumin and fetuin preparations are dialyzed prior to use.
However, the observed delay of trophoblast outgrowth in serum-free media
containing fetuin and amino acids (Figs. 1C, E) suggests that other factors, as
yet undefined, are also involved.
In general, blastocysts cultured under conditions which prohibit trophoblast
outgrowth (that is, in serum-free media lacking fetuin or in serum-containing
medium with an agarose pad) develop almost as well as those growing in a
permissive medium. There are, however, some notable exceptions. Embryos
prevented from outgrowth contain fewer cells beyond three days of culture than
those capable of outgrowth. We believe this difference to be due primarily to an
increase in the death rate of ICM-derived cells which become internalized in
greater numbers by the lack of outgrowth and which, therefore, have impaired
access to nutrients. This view is supported by the observed differences
in protein content relative to cell number in outgrowing vs. non-outgrowing embryos: the former differences are generally much smaller than the
latter (cf. Figs. 2, 3), suggesting that giant trophoblast cells, which undoubtedly
contain more protein than individual ICM cells, constitute a greater proportion
of the cell population in the absence of outgrowth. Figure 5 confirms that
trophoblast cells polyploidize to similar extents in the presence or absence of
outgrowth.
During development in utero and under optimal culture conditions, periimplantation embryos show a progressive decrease in LDH 1 activity and the
appearance for the first time of LDH A subunits (Auerbach & Brinster, 1967,
1968; Monk & Ansell, 1976; Monk & Petzoldt, 1977; Spielmann et al. 1978).
Monk & Petzoldt (1977) proposed from studies on embryos cultured in hanging
drops that cell adhesion (either to the substratum or to other cells) was the
stimulus for the production of A subunits of LDH. Spielmann et al. (1978),
however, failed to observe suppression of A-subunit production in cultured
blastocysts which remained viable but failed to hatch from their zona pellucidae.
Our results are intermediate between those of Monk & Petzoldt and of Spielmann
and his colleagues: individual embryos cultured on agarose do produce detectable levels of A subunits, but at a later stage than aggregated embryos under the
same culture conditions. It is, therefore, likely, as considered by Monk &
Mouse blastocyst development in vitro
19
Table 4. Threshold levels for expression of developmental and differentiative
markers during peri-implantation stages of mouse embryogenesis
Marker
Threshold level
Acquisition of surface adhesiveness
Secretion of plasminogen activator
Increase in /?-glucuronidase activity
Polyploidization
Increase in total protein content
Outgrowth of trophoblast cells
Appearance of A6,3/?-hydroxysteroid dehydrogenase activity
Appearance of lactate dehydrogenase A subunits
Increase in cell number
Low
Low
Low
Intermediate
Intermediate
High
High
High
High
Markers are considered to be in the low threshold category if they were observed under all
culture conditions. Intermediate threshold markers are not observed in PCM. High-threshold
markers are not observed in PCM or PCM+FET.
Petzoldt (1977), that even contact of blastomeres with each other following
collapse or occlusion of the blastocoel, as eventually occurs when individual
blastocysts are cultured on agarose or when they fail to hatch under other
culture conditions, is adequate to stimulate production of LDH A subunits. Our
LDH studies indicate that as well as producing LDH A subunits, blastocysts
cultured on agarose uniquely maintain high levels of LDH B subunits. Whether
this reflects the continuing synthesis of these subunits or a decrease in their
degradation rate is not clear. Nevertheless, the pattern of enzyme activity (LDH
5 and LDH 1 greater than LDH 3) indicates that the A and B subunits do not
interact freely, presumably because they are produced at different times and/or
in different cells.
The observation that blastocysts initiate polyploidization in PCM supplemented with fetuin, but not in PCM alone (Fig. 5 A), was unexpected and is not
readily explained. We have previously proposed that the role of the fetuin
preparation in blastocyst development in vitro is to provide a substratum suitable for attachment and outgrowth (Rizzino & Sherman, 1979). However,
outgrowth does not occur in PCM + FET due to the lack of added amino acids;
therefore, fetuin, or a contaminating factor in the fetuin preparation, presumably
facilitates the onset of polyploidization in this medium at some other level.
Because the various parameters under study do not necessarily respond in the
same way to alteration of the culture conditions, we propose that there are
variable thresholds for gene expression during the peri-implantation period. We
have separated the parameters measured into three classes based upon the
nutrient requirements for their expression (Table 4). We should stress, however,
that this compartmentalization is not meant to be rigid; indeed, there might be a
contiguous spectrum of thresholds for expression of genes during the periimplantation period, and this might have been apparent if we measured many
20
M. H. SELLENS AND M. I. SHERMAN
more parameters under a larger array of culture conditions. Nevertheless, it is
clear that by alteration of culture conditions, we have been able to suppress the
appearance of some gene products but not of others.
A variety of factors might account for the differential effects that we have
observed. For instance, differences in the complexity of the events might explain
why there is a higher threshold for division of cells in the ICM (involving DNA
replication and mitosis) than for polyploidization of trophoblast cells (which
involves only replication). The same might apply to trophoblast outgrowth
vs adhesion. Also, exceptional enzyme stability might result in increasing
/?-glucuronidase activities, even under the least favourable culture conditions
[Wudl (1974) has found enzyme activity to be linear for at least 168 h at 37 °C
and at least 60 h at 56 °C]. Differential gene expression during the peri-implantation period might also reflect differential times of mRNA transcription, mRNA
stability and/or preferential mRNA translation. It appears that all of these
factors are involved in gene expression during preimplantation stages of
mammalian embryogenesis (reviewed by Sherman, 1979; see also Van Blerkom
et ah 1979, for a consideration of the control of gene expression during implantation delay and during release from delay). Experiments in progress are consistent
with the view that mRNAs for some of the low threshold events in Table 5 are
produced earlier than those for some of the high threshold events (J. Schindler,
unpublished observations). It is anticipated that with further study, evidence
will also be found for translational control of gene expression during the periimplantation period.
We are grateful to Ms Jill Lunn for computation of DNA values and to Drs J. Schindler,
J. Monahan and H. Ennis for comments on the manuscript.
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Note added in proof
The fluorescamine assay as used by us to measure protein contents involves reaction
primarily with lysine residues (Bohlen et al. 1973). We had assumed that proteins in cultured
blastocysts contained, on average, an equal amount of all amino acids, so that the lysine
content would be 5%. From actual amino acid analyses of proteins from cultured blastocysts (Sellens, M. H., Stein, S., and Sherman, M. I., manuscript in preparation), we have
now learned that the average lysine content is approximately 7%. Therefore, protein values
given in Fig. 3 are uniformly overestimated by approximately 30%.