J. Embryol. exp. Morph. Vol. 71, pp. 1-9, 1982
Printed in Great Britain © Company of Biologists Limited 1982
The effect of cathepsin inhibitor on
rat embryos grown in vitro
By FELIX BECK 1 AND ADAM LOWY
From the Department of Anatomy, The Medical School, University of Leicester
SUMMARY
The addition of leupeptin to New cultures of rat embryos produces growth retardation and
abnormalities of embryonic development. The effect is probably due to inhibition of the
maternal protein breakdown necessary for embryonic growth. This function is carried out by
the visceral layer of the yolk sac which shows distinct morphological changes akin to storage
disease when leupeptin is added to the culture medium. We have not found it possible to
reverse the effects of leupeptin by addition of amino acids to the culture medium.
INTRODUCTION
It is known that prior to the development of the chorioallantoic placenta
the postimplantation rat embryo obtains its nutrition by breakdown of macromolecules present in its immediate uterine environment. The principal site of
macromolecular breakdown is the visceral layer of the yolk-sac epithelium
(Beck, Lloyd & Griffiths, 1967; Freeman, Beck & Lloyd, 1980) and its source
is the maternal plasma proteins (Everett, 1935; Freeman, Beck & Lloyd, 1980;
Cockcroft, 1980). Merker & Villegas (1970) have shown that a sinus containing
sluggishly flowing maternal blood lies within the trophoblast immediately
surrounding the parietal wall of the yolk sac so that serum transudate merely
has to traverse Reichert's membrane on which the scattered cells of the parietal
yolk sac lie in order to enter the yolk-sac cavity and lie against the highly
endocytic cells of the visceral yolk-sac epithelium. In cultures of the visceral
yolk-sac epithelium [125I]albumin has been shown to be degraded to the level
of amino acids, there being little - if any - release of oligopeptides or even
dipeptides from protein taken into the endodermal epithelial cells (Williams,
Kidston, Beck & Lloyd, 1975). Recently, morphometric studies of the endodermal cells of the visceral yolk sac obtained in vitro after 48 h of culture by
New's method (New, Coppola & Terry, (1973)) have shown that both the
volume and surface area of the vacuolar compartment of the cells give measurements which are identical to those from specimens obtained from the in vivo
situation (Gupta, Gulamhusein & Beck, 1982) and this provides strong evidence
1
Authors' address: Department of Anatomy, The Medical School, University of Leicester,
Leicester LEI 7RH, U.K.
2
F. BECK AND A. LOWY
in support of the contention that similar nutritional processes are operative
both in vivo and in culture. In the present experiment the effect of leupeptin
(Aoyagi & Umezawa, 1975, Ikezagoa, Aowagi, Takeuchi & Umezawa, 1971)
which specifically inhibits the lysosomal proteolytic enzymes Cathepsin B H
and L and therefore prevents normal yolk-sac function has been studied.
MATERIALS AND METHODS
Rat embryos were grown between 9\ and 11^ days of development by the
roller culture technique devised by New and his co-workers (New, Coppola &
Terry, 1973). Pregnancy was timed from midnight preceding the morning on
which a vaginal plug was observed. In each roller tube six embryos were
cultured in 6 ml of culture serum. In various experiments 1, 2 and 4mg of
leupeptin per ml of culture fluid were added to the culture medium at the time
of explantation (9£ days) and in a further series, cultured embryos were observed
until they had completely invaginated into their own yolk sac at which point
the vitellointestinal duct is said to be sealed (Denker, 1977). This occurred
after 36 h of culture and 10 mg leupeptin/ml culture medium was then added.
In all experiments controls were grown at the same time. Embryos were removed
from the culture medium exactly 48 h after explantation and the diameter
of the yolk sac was measured by means of a measuring eye piece in a dissecting
microscope. After the presence of a brisk vitelline circulation had been observed,
the yolk sac was opened and the embryo examined with respect to the features
enumerated in Table 1. A number of embryos were then fixed in Bouin's
solution in order to confirm the morphology observed in living specimens.
Other embryos were solubilized in 1 ml N NaOH and their protein content
was determined by the method of Lowry et al. (1951). In all cases small portions
of the yolk sac were fixed in 3 % gluteraldehyde in phosphate buffer as soon as
they had been removed from the culture medium. Following osmication these
specimens were dehydrated and embedded in Araldyte. Sections were stained
with lead citrate (Reynolds, 1963) and uranyl acetate and observed in a Jeol
100 CX electron microscope at an accelerating voltage of 80 kV.
RESULTS
The effects on the embryos of experiments in which various doses of leupeptin
were present in the culture medium for 48 h are given in Table 1. They show a
distinctly dose-related embryopathic effect. Control embryos grew well in
every series. Typical abnormalities of external form and neural tube closure
are compared with a control embryo in Figs 1 and 2. At 9\ days the putative
embryonic endoderm is exposed to the culture medium (Fig. 3) but after 36 h
of culture the embryo is invaginated into its own yolk sac so that it is not
exposed to direct contact with leupeptin (Fig. 4). Addition of leupeptin at a
Effect of leupeptin on cultured rat embryos
Table 1. The effect of leupeptin on embryo culture
Dosage
Dosage
Dosage
A
Experimental
Control
Experimental
Control
Experimental
(„ = 40)
in = 38)
in = 21)
in = 20)
Heartbeat (%)
100
100
90
100
Vitelline
circulation (%)
38
100
88
92
Yolk-sac
315±OO8* 404±011* 3O7±O11* 429±010*
diameter (mm)
P<001f
P<0-01f
66
100
Fusion of allantois
88
100
(with ectoplacental
cone) %
62
100
Closure of neural
80
89
tube (%)
90
Normal external form
38
95
9
81
100
Presence of forelimb
93
100
buds (%)
95
80
97
38
Optic vesicles (%)
21-43 + 0-73* 24-58±0-35* 18-27 ±0-97* 24-7 ±0-26*
Somite number
P<0-01f
p<o-oit
Crown-rump
2-67±0078* 3-19±0-67* 2-32 ±008* 3-25 ±0-43*
P<0-01f
P<001f
length (mm)
in = 11)
in = 10)
in = 5)
in
Protein content
142-8±101* 191-0±3-39* 93-7 ±3-39* 194•5 ±7-3*
P<0-01f
P<0-01f
(MS)
in = 16)
81
Control
in = 15)
100
38
100
2-28±009* 405±007*
P<0-01f
69
100
0
100
0
6
100
100
0
100
7-2 ±1-46* 24-93 ±0-21*
P<0-01f
1-8±OO7* 3-32±OO5*
P<0-01f
in = 11)
in = 10)
50-9± 817* 169-9± 1002*
P<0-01f
* Standard error of the mean.
t Student's ,t test.
dose of 10 mg/ml of culture medium at 36 h of culture, followed by incubation
between 36 and 48 h in the presence of leupeptin resulted in the embryopathic
effects seen in Table 2.
The series of electron micrographs shown in Figs. 5-8 clearly illustrate the
dose-dependent effects of leupeptin on the morphology of the visceral yolk
endodermal cells. A slight embryopathic effect is seen at doses of 1 mg/ml
when few obvious morphological changes in the yolk-sac micrographs are
apparent (Fig. 6) but at 4 mg/ml a very obvious increase in the size of the
secondary lysosomes is seen which distorts the whole internal cellular
architecture.
The addition of the 13 essential amino acids at concentrations of up to twice
those used in standard Eagles medium to the serum of leupeptin-treated embryo
cultures at 9\ days did not reverse or ameliorate the embryopathic effect of
F. BECK AND A. LOWY
Fig. 1. Normal rat embryo explanted at 9|- days and grown for 48 h by the method
of New, Coppola & Terry, 1973. x 15.
Fig. 2. Grossly abnormal rat embryo explanted at 9\ days and grown for 48 h by
New's method in the presence of 4 /ig leupeptin/ml serum culture medium. The
neural tube (N) is open and the embryo is concave dorsally. x 35.
Fig. 3. Rat embryo 9\ days old. The embryonic endoderm at this stage (E) is on the
surface. x25.
Fig. 4. Rat embryo explanted at 9\ days and cultured for 36 h. The embryo is
entirely invaginated into the visceral layer of the yolk sac and embryonic endoderm
is no longer exposed to the surface, x 10.
Effect of leupeptin on cultured rat embryos
Table 2. The effect of culture with leupeptin between 36 and 48 h
Treated
(« = 5)
Untreated
in = 5)
Student's
Mest
3-54
402
0-05 > P > 0-02
Yolk-sac diameter (mm)
±
014
22-4
S.E.
0-5
3 08
S.E.~6-24
S.E.
Somite number
S.E.
Crown-rump length (mm)
014
24-6
P<001
P<001
3-58
±
Protein content (/*g)
S.E. 0 1
S.E. 0 1
143-5
221-9
P<001
±
S.E.
8-7
S.E.
6-6
Table 3. Effect of amino acid supplementation on the action of leupeptin
Treatment
Control
(n = 8)
Crown-rump length (mm)
3-5±006*
Yolk-sac diameter (mm)
4-4±015*
Somite number
24-38±0-26*
1 fig/m]
leupeptin
(n = 8)
Amino
acidsf
(n = 8)
Amino acids
+ leupeptin
(/i = 8)
2-5±0-3*
3-23±O17*
18-43±2-3*
3-3±007*
3-91 ±016*
24-38±0-65*
2-23±006*
2-66±008*
17-33±O-89*
* Standard error of the mean.
t The quantities of amino acid in /*g/ml of serum were as follows: L-arginal, 42; L-cysteine
(disodium salt), 28; L-glutamine, 584; L-histidine, 21; L-isoleucine, 52; L-leucine, 52; L-lysine,
73; L-methionine, 14; L-phenylalanine, 33; L-threonine, 48; L-tryptophan, 8; L-tyrosine, 36;
L-valine, 47.
leupeptin. This is shown in Table 3. Higher concentrations of amino acids
were found to be toxic.
DISCUSSION
Leupeptin is the name given to a group of tetrapeptides produced by several
strains of actinomyces; it includes propionyl- (or acetyl) L-leucil-L-leucil
arginal and analogues where leucine is replaced by isoleucine or valine. The
compounds are specific inhibitors of Cathepsin B H and L but have no effect
on Cathepsin D. The drug is a proven inhibitor of the lysosomal pathway of
protein degradation in the rat hepatocyte (Hopgood, Clarke & Ballard, 1977)
as well as in other tissues. It is not surprising, therefore, that it has embryopathic effects on embryos cultured by the New technique (New, Coppola
& Terry, 1973). The embryo throughout this culture period is known to use
the protein in its histiotroph to build up its own proteins after having first
broken down the maternal biopolymers in the lysosomal system of the visceral
F. BECK AND A. LOWY
Effect of leupeptin on cultured rat embryos
Fig. 8. Visceral yolk sac of an embryo explanted at 9i days and grown for 48 h
with 4 /*g leupeptin/ml culture fluid. A giant vacuole (V) is seen greatly distorting a
visceral yolk-sac endodermal cell, x 3000.
yolk-sac endoderm. It is interesting to observe, however, that the roles of the
Cathepsin B H and L complex are essential. Cathepsin D is unaffected by
leupeptin (Ikezawa, Agoyaki, Takeuchi & Umezawa, 1971) and non-lysosomal
mechanisms for protein breakdown are known to exist (see Dean, 1978 for
a simple review). These mechanisms are obviously not sufficient to sustain the
embryo even if there is only partial inhibition of the Cathepsin B H and L
(i.e. at doses of 1 mg/ml serum). Even more intriguing is the observation that
the embryopathic effect cannot apparently be abolished by supplementing the
culture serum with the 13 essential amino acids in the way we have tried to
do it.
It is known that in the mammalian small intestine there are a number of
carrier-mediated absorption mechanisms for different groups of L-amino acids
apparently with various groups of amino acids capable of competing for their
particular carrier. In general, basic amino acids are actively absorbed when
the luminal concentration is low (2 mM) whereas neutral and acidic acids
require a higher luminal concentration (20 mM). Acidic amino acids are transaminated in the process of transport (see Christensen, 1975, for review).
Fig. 5. Electromicrograph of visceral yolk sac explanted at 9\ days of gestation
and cultured for 48 h. The vacuole (V) is representative of the vacuolar system probably a secondary lysosome. x 2500.
Fig. 6. Visceral yolk sac of an embryo explanted at 9J days and grown for 48 h in
the presence of 1 /*g leupeptin/ml culture fluid. The vacuoles of the lysosomal
system (V) are very slightly enlarged, x 1800.
Fig. 7. Visceral yolk sac of an embryo explanted at 9J days and grown for 48 h
with 2 fig leupeptin/ml culture fluid. The vacuolar system (V) is distinctly hypertrophied. x 1600.
8
F. BECK AND A. LOWY
Free & Leonards (1944) have pointed out that the limiting factor in ingested
protein utilization in mammals is the rate of uptake of amino acids by the gut,
but excessively high plasma amino acid levels (induced by intravenous amino
acid injection) may be lethal if the amino acid is not rapidly eliminated in the
urine. It may, therefore, be that the visceral yolk sac does not have the appropriate mechanisms for allowing an adequate balance of essential amino acids
to penetrate it in order to sustain the embryo. Randomly increasing the amino
acid concentration in the incubating medium produces toxic effects and for
successful culture a plentiful supply of protein in conjunction with the active
lysosomal system of the visceral yolk-sac endoderm is therefore necessary.
The electron microscopic appearances resulting from leupeptin treatment
are dramatic. Clearly there is little, if any, inhibition of endocytosis and
apparently little possibility of cellular excretion of the undigested protein. The
effect depends upon the dose administered, from which it may be surmised
that at the lower embryopathic doses the inhibition of Cathepsin B-, H- and
L-mediated protein degradation is still far from complete. It may also be worth
bearing in mind that the gross distortion of the cell seen at high doses might of
itself produce secondary effects supplementing those of leupeptin alone. The
wider problem of the effects of some storage diseases in human pregnancy are
of interest in this connexion.
Consideration must be given to the possibility that leupeptin may have a
direct effect upon the embryonic tissues. This has not formally been disproven
by the experiments reported here and, indeed, the low but distinct endocytic
capacity exhibited by the embryonic endoderm at 9\ days, when leupeptinladen serum is in contact with these cells, makes it likely that a certain amount
of the inhibitor does indeed reach the embryonic tissue at this early stage. The
effect on the extraembryonic endoderm of the visceral yolk-sac epithelium is,
however, probably much more important. In the first place the nutritional role
of this tissue is well established and clear morphological proof is presented
showing a gross effect on this tissue. Secondly, leupeptin produces an effect
even if administered after invagination of the embryo into the yolk sac. At this
stage the drug could only reach the embryo by passing through the yolk-sac
endoderm and this is unlikely because of its size, charge and above all its
extreme affinity for certain Cathepsins. The K values governing this reaction
are such that the leupeptin would be effectively immobilized by some of the
enzymes which it would be likely to encounter when endocytosed. The higher
dose levels necessary to establish an embryopathic effect after embryonic
inversion are probably due to the much shorter period of exposure compared
to that when the drug is given at 9^ days rather than to the absence of direct
exposure of the embryonic endoderm to leupeptin.
We are grateful to Miss M. Reeve for rapidly and efficiently typing the manuscript and
to Mrs S. Bulman for expert technical assistance.
We should also like to thank the MRC for support.
Effect of leupeptin on cultured rat embryos
9
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(Received 18 February 1982, revised 29 May 1982)