/ . Embryol. exp. Morph. Vol. 36, 3, pp. 653-662, 1976
Printed in Great Britain
653
Patterns of lactic dehydrogenase
isozymes in mouse embryos over the implantation
period in vivo and in vitro
By MARILYN MONK 2 AND JOHN ANSELL 1
From the MRC Mammalian Development Unit, London
SUMMARY
Following blastocyst implantation, or outgrowth in vitro, the LDH isozyme pattern
changes from that of the maternally inherited B subunit isozyme form (LDH-1) to a pattern
dominated by A subunits (Auerbach & Brinster, 1967, 1968). In preimplantation embryos
we have also observed additional isozyme bands, as yet unidentified. An analysis of the
pattern of newly synthesized LDH isozymes and specific activity of LDH in different regions
of early post implantation embryos suggests that there is a sequential activation of A and B
subunits, and that activity first appears in ICM- (inner cell mass) derived tissues and then in
trophoblast-derived tissues. In vitro, in the absence of ICM cells, the transition of LDHisozyme pattern does not occur in outgrowing trophoblast giant cells. This suggests a possible
inductive interaction between ICM and trophoblast.
INTRODUCTION
Lactic dehydrogenase (LDH, E.C. 1.1.1.27) is a tetramer composed of
combinations of two subunits, A and B, coded for by separate genes (Shaw &
Barto, 1963). There are five isozymic forms; LDH-1 (4B), LDH-2 (1A3B),
LDH-3 (2A2B), LDH-4 (3A1B), and LDH-5 (4A), which vary in electrophoretic mobility. The relative proportion of these forms varies from tissue to
tissue in the adult animal presumably because of the differences in the rates
of expression of the two genes (Markert & Moller, 1959). A sixth LDH
isozyme, termed LDH-X, is synthesized by primary spermatocytes (Hawtrey &
Goldberg, 1968). It is a tetramer of C subunits, C being a third type of subunit
under independent genetic control (Zinkham, 1968).
In mouse embryos, the distribution of LDH amongst the five isozymic
forms, LDH-1 to LDH-5, varies with stage of development. The level of LDH
in the mouse egg is high (Brinster, 1965; Epstein, Wagienka & Smith, 1969)
and is thought to be exclusively in the B subunit form, or LDH-1 (Auerbach &
Brinster, 1967; Rapola & Koskimies, 1967). This maternally inherited LDH-1
1
Author's address: Department of Zoology, University of Edinburgh, Edinburgh 9,
Scotland.
2
Author's address: MRC Mammalian Development Unit, Wolfson House, 4 Stephenson
Way, London NW1 2HE, England.
654
M. MONK AND J. ANSELL
is progressively degraded during preimplantation development (Brinster, 1965;
Spielmann, Erickson & Epstein, 1974) and there is evidence that newly
synthesized enzyme does not appear before implantation (Epstein et al. 1969;
Erickson et al. 1975). At about the time of implantation (between 4 and
6 days gestation) the pattern changes, presumably due to the appearance of
LDH coded for by the embryonic genome, LDH-5 (the A subunit form)
predominating (Auerbach & Brinster, 1967; Wolf & En gel, 1972; Engel &
Petzoldt, 1973). Diapause blastocysts, where implantation has been prevented
by ovariectomy on the 3rd day of pregnancy, fail to show this transition,
suggesting that the implantation event itself is required for activation of embryocoded LDH expression (Auerbach & Brinster, 1967). Outgrowths obtained after
4 days culture of blastocysts in vitro also show a transition of LDH isozyme
activity to that of LDH-5 (Auerbach & Brinster, 1968).
The possibility that the appearance of LDH-5 is causally related to implantation suggests its use as a marker in the investigation of processes regulating
postimplantation development. In this paper we investigate postimplantation
isozyme patterns in different regions of the early embryo in vivo and in vitro.
MATERIALS AND METHODS
Mouse embryos were obtained from the randomly breeding stocks, CD1
(Charles River & Co.), MF1 (Olac, Bicester) or ' Q ' , bred in our laboratory.
Medium used for collection of embryos was Dulbecco's phosphate buffered
saline (PBS), or PBS supplemented with bovine serum albumin, sodium
pyruvate, glucose, penicillin and phenol red (PB1, Whittingham & Wales,
1969). On the day of the copulation plug, unfertilized eggs were teased from
oviducts of females mated with vasectomized males. Cumulus cells were
removed by treatment for 5 min with hyaluronidase (Sigma, 300 units/ml in PB1).
Eight-cell eggs were obtained by flushing oviducts dissected from females on
the 3rd day of pregnancy, and blastocysts by flushing uteri of females on the
4th day of pregnancy. Postimplantation embryos were dissected out of the
uterus into PB1. Samples for electrophoresis were washed through several
changes of PB1 or PBS, frozen and thawed twice, clarified by centrifugation,
and the supernatants stored in liquid nitrogen. Eight-cell embryos or blastocysts for culture were transferred to droplets of culture medium under oil.
Culture medium for 8-cell embryos was medium 16 (Whittingham, 1971).
Trophoblastic vesicles were prepared by the addition of tritiated thymidine at
a final concentration of 0-05 /tCi/ml to the medium for 24 h (Snow, 1973).
Outgrowths from trophoblastic vesicles or control blastocysts (produced by
culture from the 8-cell stage without tritiated thymidine) were harvested after
culture in medium 16 supplemented with 5 % foetal calf serum for a further
5 days. In some experiments blastocysts were cultured in Eagle's minimum
essential medium (MEM, Flow Laboratories) supplemented with sodium
Lactic dehydrogenase isozymes in mouse embryos
655
bicarbonate (2 g/J), glutamine (2 HIM), penicillin (100 units/ml), streptomycin
(50 /'g/ml) and 20 % foetal calf serum (Flow Laboratories) or human cord
serum (kindly supplied by University College Hospital). The media were
filtered and equilibrated to 37 °C and 10 % CO2 in air atmosphere before the
embryos were introduced.
Discontinuous polyacrylamide slab gel electrophoresis was carried out using
the solutions of Davis (1964) with a 2-5 % acrylamide stacking gel pH 6-7,
7 % separating gel pH 8-9, and a Tris-glycine tank buffer pH 8-3. Approximately
10 /d of sample was mixed with an equal volume of 40 % sucrose containing
bromophenol blue (0-01 %). Electrophoresis was carried out at 4 °C for
approximately 3 h at 100 V or until the bromophenol blue marker reached
the bottom of the gel. The gels were stained at 37 °C for LDH activity with
the staining solution of Engel & Kreutz (1973). When staining had reached
the desired intensity gels were fixed in 7 % acetic acid. Densitometer tracings
were made with a Joyce Loebl Densitometer.
LDH activity was assayed by the method of Brinster (1965) and protein
determinations were made by the Lowry technique using bovine serum albumin
as the standard (Lowry, Rosebrough, Farr & Randall, 1951).
RESULTS
In vivo development
Fig. 1 shows the distribution of LDH isozyme activities in an extract of
unfertilized eggs (Slot 4) as well as in extracts of adult skeletal muscle and
testis (Slots 1 and 2). The eggs, obtained from the oviducts of females mated
with vasectomized males, contain predominantly LDH-1. In addition there
are two faint bands, one running just behind LDH-1, and the other running
slightly more slowly than LDH-2. We have observed these additional
bands in all preimplantation embryos examined (1-cell, 8-cell, early and
late blastocysts) as well as in unfertilized eggs, whether spontaneously
ovulated or hormonally induced. They are present in all of the three strains
of mice examined (Q, CD1 and MF1). We have not identified these additional
bands. However, the band running behind the position of LDH-2 could be
a tetramer of 3B subunits and 1C subunit since it runs midway between that
of a 4B tetramer (LDH-1) and that of a 2B2C tetramer artificially created by
dissociating and reassociating the subunits of a whole testis extract (Slot 3).
Fig. 2 shows isozyme patterns in blastocysts and in postimplantation embryos
dissected from the uterus early and late on the 7th day of gestation. Blastocysts
contain LDH-1 together with the additional bands (Slot 1). Postimplantation
embryos (Slot 2) show the transition of LDH isozyme pattern to one dominated
by A subunits, LDH-5 (4A) and LDH-4 (3A1B). A faint residual band of
maternally inherited LDH-1 is visible. With further development, LDH-3
activity appears (Slot 3) owing to an increasing contribution to the pattern
656
M. MONK AND J. ANSELL
5
13 2
H S
LDH-X
LDH-5
1B3C
LDH-4
• 2B2C
LDH-3
•LDH-2
LDH-1
Slot
1
Fig. 1. LDH isozyme patterns in unfertilized eggs (Slot 4), and in adult mouse
testis before (Slot 2), and after (Slot 3), treatment to dissociate and reassociate
LDH subunits. Under the conditions of enzyme extraction and running of the
gel there is no dissociation and reassociation of subunits between independently
synthesized forms of the tetrameric enzyme. However, such dissociation and
reassociation can be artificially engineered by freezing and thawing mixtures of
isozymes in one molar sodium chloride in sodium phosphate buffer at pH 7
(Markert, 1963). Adult mouse skeletal muscle (Slot 1) is shown to give the position
of LDH-5 and LDH-4. An extract of a total of 48 unfertilized eggs, obtained
from oviducts of CD1 mice approximately 10 h after mating with vasectomized
males, was applied to the gel. The direction of migration is shown. Further details
are provided in Materials and Methods.
of de novo production of B subunits in the embryo. Measurements of specific
activity of total LDH in the extracts, have shown a progressive increase in
embryos from mice on the 7th, 8th and 9th day of pregnancy.
Fig. 3 shows the pattern of LDH isozymes, in different regions of embryos,
obtained on the 8th (primitive streak stage) and 9th days of gestation. Slots 1,
2 and 3 (Fig. 3) show LDH isozymes in extracts of ectoplacental cone, and
extra-embryonic and embryonic regions of the egg cylinder of 8th day embryos.
Lactic dehydrogenase isozymes in mouse embryos
657
LDH-5
LDH-4
LDH-3
LDH-2
LDH-1
Slot
1
Fig. 2. LDH isozyme patterns before and after implantation. Polyacrylamide slab
gel elect rophoresis of extracts of 33 CD1 blastocysts (Slot 1) and of approximately ten CD1 postimplantation embryos (Slots 2 and 3) dissected from the
uterus early and late on the 7th day of pregnancy. Adult mouse heart (Slot 4) and
skeletal (Slot 5) muscle serve to indicate the positions of the five isozyme bands.
Slots 4 and 5 (Fig. 3) show LDH isozyme activities in extracts of yolk sac and
embryonic tissues dissected from 9th day embryos. Densitometer tracings of
these gels are also shown in Fig. 3. The gels show that a greater degree of
B subunit synthesis, as reflected by the relative amounts of LDH-4 and 3,
has taken place in embryonic regions compared with extra-embryonic regions
of these early postimplantation embryos. In addition the total specific activity
of LDH was higher in embryonic than in extra-embryonic regions.
In vitro development
Outgrowths from blastocysts cultured for 4 days show some LDH-5 isozyme
expression (Auerbach & Brinster, 1968). Fig. 4 shows further development of
the LDH isozyme pattern in vitro after culture in the presence of human cord
serum which favours inner cell mass (ICM) development into egg cylinder-like
structures (Hensleigh, personal communication). After 6 days in culture the
in vitro pattern is very similar to that of embryos dissected from the uterus on
the 9th day of pregnancy.
Since the in vivo analyses suggest that expression of embryo-coded LDH
occurs earlier in ICM-derived tissue than in trophoblast-derived tissue we
investigated the possibility that an ICM is required for the appearance of
42
EMB 36
658
M. MONK AND J. ANSELL
8th
day
epc
9th
day
3
LDH
Fig. 3. Regional LDH isozyme patterns in postimplantation embryos. After
removal from their decidua in the uterus, Q embryos were dissected into component
parts as follows. From 8th day embryos ectoplacental cone and primary giant
cells {epc, Slot 1) were separated from the egg cylinder, and then extra-embryonic
(ee, Slot 2) and embryonic (e, Slot 3) regions were obtained by cutting the egg
cylinder in half at the level of the amnion; from 9th day embryos, yolk sac (ys,
Slot 4) was carefully cut away from embryonic tissue (e, Slot 5). Specific dissected
regions for each stage of development were pooled, extracts prepared as described
in Materials and Methods and assayed for LDH activity. Activity corresponding
to approximately 26 nmol NADH oxidized per minute at 37 °C by 10~3 molar
pyruvate at pH 7-5 was applied to each slot. Densitometer tracings were made
the same day. Equivalent results were obtained using CD1 embryos.
Lactic dehydrogenase isozymes in mouse embryos
659
LDH
Fig. 4. Development of LDH isozyme pattern in vitro. LDH isozymes in 9th day
embryos dissected from the uterus and in approximately 100 outgrowths from CD1
blastocysts cultured for 6 days in MEM supplemented with 20 % human cord
serum.
-ICM
•
LDH
1
Fig. 5. LDH in outgrowths from trophoblastic vesicles and blastocysts cultured
for a total of 6 days from the 8-cell stage. Extracts from approximately 50 outgrowths were applied to each slot. Trophoblastic vesicles were prepared as
described in Materials and Methods. The positions for isozymes LDH-5 to
LDH-1 were taken from a densitometer tracing of adult kidney extract run in
the same gel.
LDH-5 in trophoblast cells. Tritiated thymidine treatment (Snow, 1973) was
employed during culture from the 8-cell stage to produce trophoblastic vesicles
devoid of ICM. Outgrowths from such vesicles show giant cell transformation
but no proliferation of trophoblast cells (Ansell & Snow, 1975). Figure 5
shows that such outgrowths do not contain LDH-5.
42-2
660
M. MONK AND J. ANSELL
DISCUSSION
Our analysis of pre- and postimplantation LDH isozyme patterns confirms
the results of earlier workers that LDH-1 is the predominant form in preimplantation embryos. Early postimplantation embryos show an initial appearance of the A subunit form, or LDH-5, followed by the appearance of other
isozymes as the contribution of B subunits to the pattern increases (see also
Engel & Petzoldt, 1973). This suggests a sequential activation of A and B
subunit production. In addition, the pattern of appearance of these different
isozymes is regulated differently in different regions of postimplantation
embryos. This result, together with the observation that specific activity of
LDH is higher in embryonic regions, is most easily interpreted on the hypothesis
that initial embryo-coded LDH expression occurs earlier in ICM-derived, than
in the trophoblast-derived tissues, extra-embryonic ectoderm and ectoplacental
cone; see Gardner & Papaioannou, 1975). Alternatively, or in addition, the
relative rates of production of A and B subunits could change as development
proceeds, and be regulated differently in different embryonic tissues. Regional
differences in esterase patterns (Sherman, 1912 a) and in acid and alkaline
phosphatase patterns (Solter, Damajanov & Skreb, 1973; Sherman, 1912b)
also occur in postimplantation embryonic development.
We have provided evidence that ICM cells are required for the expression
of new LDH activity in trophoblast cells following attachment and outgrowth.
Gardner & Johnson (1972) have shown previously that the presence of an
ICM is required for proliferation of neighbouring trophoblast cells to form
the ectoplacental cone. However, alternative explanations of our results cannot be excluded. For instance, it is possible that giant trophoblast cells never
exhibit LDH-5 either in vivo or in vitro. Our in vitro control blastocyst outgrowths include an inner cell mass; our in vivo analysis of trophoblast-derived
tissues show that ectoplacental cone cells express LDH-5 but we have not
tested trophoblast giant cells alone.
Further investigation of the in vitro system as a model for implantation
in vivo, could provide more rigorous evidence for the supposition that implantation, or outgrowth, represents a 'trigger' event in development for the
expression of embryonic LDH activity. LDH-5 would then be a convenient
marker function in the analysis of the nature of this event.
We thank Hugh Hensleigh, Hugh Paterson and Anne McLaren for their encouragement
and many helpful discussions, and Peter McPartland for technical assistance.
Lactic dehydrogenase isozymes in mouse embryos
661
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(Received 30 June 1976, revised 9 August 1976)