Development 114, 185-192 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
185
Purine utilisation, de novo synthesis and degradation in mouse
preimplantation embryos
MARIA ALEXIOU and HENRY J. LEESE
Department of Biology, University of York, Heslington, York YO1 5DD, UK
Summary
The importance of de novo purine synthesis as opposed
to the reutilisation of metabolites by salvage pathways,
and the nature of the excretory product(s) of purine
degradation, have been examined in cultured preimplantation mouse embryos. In the presence of azaserine and mycophenolic acid, which inhibit de novo purine
synthesis, embryo cleavage was blocked prior to compaction, the precise stages at which this occurred
depended on whether the cultures were established on
day 1 or day 2 after fertilisation, and indicated that
salvage pathways were insufficient to fulfil the demand
for nucleotides during early preimplantation development. The end-product of purine degradation appeared
to be xanthine, which was excreted in very small
amounts on days 1, 2 and 3, with a pronounced rise from
the early to late blastocyst. Uric acid formation or
excretion could not be detected. Exogenous hypoxanthine and adenine, which partially inhibited development, were taken up by the embryos and converted to
xanthine, most probably by salvage pathways, since the
enzyme xanthine oxidase, which converts hypoxanthine
directly to xanthine and then to uric acid, could not be
detected. Exogenous guanine had little effect on development and was also converted to xanthine, but in this
case, the conversion was probably in a single step, via
the enzyme guanase.
Introduction
1964; Monesi and Sain, 1967; Epstein and Daentl, 1971;
Clegg and Piko, 1982), together with the timing of
embryonic genome expression (Young et al., 1978;
Flach et al., 1982; Bolton et al., 1984; Schultz, 1986).
Data from the incorporation of radiolabelled precursors
have shown a continually increased synthesis of new
RNA molecules of all major classes, beginning at the 2cell stage, at which time the embryonic genome is
switched on. However, little is known about purine
turnover (ie., synthesis and degradation), and excretion
during the first five days of mouse embryo development.
Thompson Ten Broeck (1968) observed increasing
inhibition of mouse preimplantation development by
adenine, adenosine and guanosine as their concentrations were raised from 10~6 M to 1CT2 M. Epstein et
al., (1971) reported studies on the uptake and utilisation of exogenous guanine and adenine by the mouse
preimplantation embryo. There was a major increase in
the uptake of radiolabelled guanine between the 2- and
8-cell stages with maximum uptake at the early
blastocyst stage; it was also shown that guanine entering
the embryo was rapidly salvaged to GMP, which was
further converted to GDP and GTP.
The aim of the present study was to assess the
importance of the de novo and salvage pathways to
preimplantation mouse embryo development, and to
Mouse preimplantation embryos may be grown in
chemically defined media consisting of ions, simple
energy sources and a macromolecule such as albumin
(Pratt, 1987). Although DNA and RNA are synthesized
during the preimplantation period, the addition of
precursors of nucleic acid synthesis to the incubation
media is not required (Whitten, 1971; Brinster, 1973).
Purine nucleotides are synthesized in adult mice as in
most other mammals either by de novo biosynthesis or
by the "salvage" pathway, whereby reutilisation of
purines derived from nucleotide degradation occurs
(Murray, 1971; Rodwell, 1988).
The purines adenine and guanine can be salvaged by
the action of the enzymes adenine phosphoribosyl
transferase (APRT) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT), respectively (Fig. 1).
Alternatively, they may be broken down, in rodents, to
allantoin (Henderson and Paterson, 1973; Hurst, 1980).
In humans, the pathway stops with uric acid production, presumably due to the loss, with evolution, of
uricase activity.
Radiolabelled purine bases, which are incorporated
into nucleotides via the salvage pathway, have been
used to study the qualitative and quantitative pattern of
nucleic acid synthesis in early mouse embryos (Minz,
Key words: purities, xanthine, hypoxanthine, azaserine,
preimplantation embryo, development, mouse, HPLC.
186
M. Alexiou and H. J. Leese
RIBOSE 5-PHOSPHATE
ATP—*
PRPP
I
-glutomlne
-glycine
DE NOVO
-HC03-formote
<
azuerfne.
-aspartete
ATP-.-ADP
GDP-^GTP
IMP-
odenoslne
• adenlne
-Inosine
hypoxanthine"
#
E5
E2
-XMP
1P - *
xanthosine
•xanthine
guanoslne
E4
-guanine'
uric acid
I
Fig. 1. Purine metabolism in
adult mouse tissues. PRPP: 5phosphoribosyl-1pyrophosphate. XMP:
Xanthosine monophosphate.
Ej: Adenine phosphoribosyl
transferase (APRT). E2:
Hypoxanthine-guanine
phosphoribosyl transferase
(HGPRT). E3: Xanthine
oxidase; E4: Guanine
deaminase (guanase); E5: IMP
dehydrogenase. * The site of
inhibition by mycophenolic
acid. A The site of inhibition
by azaserine. The bold lines
indicate the routes of the
salvage pathway.
allantofn
determine the nature of the end-products of purine
degradation. Two potent inhibitors of nucleotide synthesis, azaserine and mycophenolic acid, have been
used to inhibit the de novo pathway. It has been known
since the mid 1950's that azaserine, acting as a
competitive antagonist of glutamine, inhibits de novo
purine synthesis in mammals and avians (Skipper et al.,
1954; Levenberg et al., 1957; Moore and Le Page,
1957). There is evidence that azaserine reacts with the
enzyme converting formylglycinamide
ribotide
(FGAR) to formylglycinamidine ribotide (FGAM).
Mycophenolic acid blocks nucleotide synthesis largely
by inhibiting the enzyme IMP dehydrogenase, which is
involved in the formation of GMP (guanosine monophosphate) from IMP (inosine monophosphate)
(Franklin and Cook, 1969).
The approach to the problem of detecting excretion
products derived from purine catabolism has been to
analyse the culture medium in which embryos were
grown. Embryonic development and end-product excretion have been assessed in response to supplementation of the culture medium with hypoxanthine,
adenine, guanine, xanthine and uric acid.
High performance liquid chromatography (HPLC)
has been used to detect the purine bases, nucleosides
and nucleotides. This technique has been successfully
applied to the analysis of these compounds in various
biological fluids and tissues, such as urine, leukocytes,
erythrocytes, liver, brain (Harkness et al., 1983;
Wynants and VanBelle, 1985; Brown et al., 1982),
oocytes and cumulus cells (Eppig et al., 1985; Downs et
al., 1986), and recently, human preimplantation embryos (Leese et al., 1991).
Materials and methods
Embryo collection and culture
5-8 week old female mice of the inbred strain CBA/Ca x
C57BL/6, were superovulated with 5 i.u. PMS (Folligon,
Intervet, Cambridge, UK), between 12:00 and 13:00 hours,
followed 48 hours later by 5 i.u. HCG (Chorulon, Intervet,
UK). After the second injection they were placed with fertile
males of the same strain. Mating was checked the following
morning by the presence of a vaginal plug (Dayl). The
cumulus oophorus from embryos recovered from the oviduct
on day 1 (20-24 hours following HCG) was dispersed by
treatment for 5-10 minutes with 1 mg/ml hyaluronidase
(Sigma Chemical Co, Poole, Dorset, UK) in medium M2
(Whittingham, 1971). Subsequent embryonic stages were
flushed from the oviduct or uterus, washed in M2 and grown
in 2-5 /il droplets of M16 under 3 ml paraffin oil in Petri dishes.
The embryos were cultured in groups of 30-120 at 37°C in a
humidified atmosphere of 5% CO2 and 95% air. They were
transferred to a new droplet each day between 9:0012:00a.m. The time interval between the samples was,
therefore, approximately 24 hours. The spent-medium drops
were either analysed immediately or stored in a 5 /il microcap
(Drummond Scientific, UK) at 20°C.
The compounds to be tested (all from Sigma) were added to
the culture medium at the following concentrations: 0.05 and
0.1 mM hypoxanthine, 0.05 mM guanine, 0.05 mM adenine,
0.05 mM xanthine, 0.05 mM and 0.1 mM uric acid. The
response of the embryos to azaserine was tested at concentrations of 10, 25, 50 and 100 /ig/ml in M16. The extent of
inhibition was not significantly different at each of these
Purine metabolism in mouse embryos
187
Table 1. Effect of azaserine (50 jig/ml) and azaserine (50 fig/ml)+hypoxanthine (0.1 mM), on mouse embryo
development from the 1-cell and the 2-cell stage to the blastocyst
% Development
2-cell stage
4-cell stage
8- to 16-cell stage
blastocyst
Total no. of embryos
Azaserine
+hypoxanthine
Azaserine
Control
lc*
2ct
lc*
2ct
lc+
2ct
100
94.7113.1
87.414.6
80.916.1
100
96.112.2
94.01+2.3
44.6+14.8
0
0
0
88.315.1
0
0
13.5
0
0
0
75.613.8
0
0
100
165
143
213
59
* meanlS.E.M. of 4 independent experiments, t meaniS.E.M. of 6 independent experiments.
t meanlS.E.M. of 3 independent experiments.
concentrations, and a concentration of 50 Mg/ml was used in
the experiments shown in Table 1. The response of embryos
to mycophenolic acid at a variety of concentrations was
reported by Hoshi and Yuki (1986), and it was not considered
necessary to repeat this work. A concentration of 25 /<g/ml
mycophenolic acid was chosen as appropriate. Each of the
compounds was dissolved in 0.2 M NaOH (except for guanine
which was dissolved in 0.5 M KOH), and then added in the
smallest possible volume to M16 in order to minimise any
change in pH or osmolarity. In all cases, the media were
adjusted to maintain a pH of 1.2-1A and an osmolarity
between 285 and 300 mosmol/L. For each series of experiments the osmolarity of the controls and the test media did
not differ by more than 5 mOSM. Control cultures containing
M16 alone or M16 plus 0.2 M NaOH at a final concentration
similar to that used for test substrates were always included.
Purines and metabolic inhibitors were added either on day 1
or day 2. In each case, cultures were continued to the
blastocyst stage on day 5.
HPLC analysis
HPLC analysis was carried out on a Kontron 400 series system
(Kontron Instruments, Watford, Herts, UK) which consisted
of a solvent pump, an autosampler injector, a variable
wavelength UV detector and a computer data system MSDOS to record the analyses and determine the retention time
and peak area (in mV) of the compounds eluted.
The chromatogTaphy was performed in an isocratic mode,
with a reverse-phase Hypersil, 5 ODS (25 cmX2 mm) column
(Shandon, Runcorn, Chesire, UK) at room temperature. For
all the reference samples, the injected volume was 20 (A; for
the culture medium samples, it was 14-18 jA.
The mobile phase was 25 mM NH4H2PO4, pH 4.5,
prepared with double distilled water and filtered through a 0.4
Ijm membrane filter immediately before use. The column was
cleaned with Analar methanol after approximately 50
samples. Elution was performed at a flow rate of 0.3 ml/min,
with the detector set at 254 nm and an absorbance sensitivity
of 0.002AFS.
The peaks corresponding to each compound were identified
by comparing the retention times with those of freshly
prepared standards. Identification could be confirmed by
setting the UV detector to various wavelengths including the
one representing the highest absorption of a standard of the
pure compound. Further confirmation was obtained by the
addition of a known amount of the substance to be identified.
Analyses of results
The results were analysed using Student's Mest and Analysis
of Variance(oneway).
Results
Inhibitors of de novo synthesis
Metabolic inhibitors were added to the incubation
medium in an attempt to discover whether embryos
could develop to the blastocyst stage when de novo
purine synthesis was supressed.
Of embryos treated with azaserine from day 1, 55%
were blocked at the 1-cell stage, while the remainder
cleaved successfully to the 2-cell stage, after which
division ceased (Table 1). When the embryos were
incubated with azaserine from the 2-cell stage
(Table 1), 90% of them completed the second cleavage
division but did not develop further. Of those embryos
that reached the 4-cell stage, many attempted to
compact even though they were unable to divide
further. Although most embryos were degenerate,
some had formed a blastocoel cavity by late day 4.
Hypoxanthine, an activator of the salvage pathway,
was added to the cultures together with azaserine, to
discover whether the inhibitory effect of the latter was
reversible. The concentration of hypoxanthine used
(0.1 mM) was high enough to stimulate the salvage
pathway (Epstein, 1970; Monk, 1987). There was no
inhibition of HGPRT activity at this concentration of
hypoxanthine (data not shown). In the presence of
hypoxanthine, the proportion of embryos, either those
cultured from day 1 or day 2, that completed one
division was lower than that in the presence of azaserine
alone (see Table 1).
In order to discover whether the inhibitory effects
were reversible, the embryos were incubated in azaserine for 24 hours and then transferred to M16 or M16
supplemented with hypoxanthine; no improvement in
development was observed.
When the embryos were incubated for 4 hours only in
azaserine, there was some stage-dependent reversal of
inhibition after transfer to M16 or M16+hypoxanthine;
188
M. Alexiou and H. J. Leese
Table 2. Effect of mycophenolic acid (25 ng/ml) on mouse embryo development from the 1-, 2- and 4-cell stages
to blastocyst
% Development
Mycophenolic acid
Control
2-cell stage
4-cell stage
8-cell stage
blastocyst
Total no. of embryos
4c
lc
96.1±0.5
94.8±0.4
84.3+6.3
100
91.4
98.7±0.7
89.4±0.3
0
0
117
47
87
lc
2c
100
100
91.9±2.3
75.6+3.4
77
2c
4c
100
42±9.8
0
93.3
0
45
107
lc, 2c, 4c refer to cultures from the 1-cell (day 1), 2-cell (day 2) and 4-cell (late day 2, 54-56 hours post HCG) stages, respectively.
Values for cultures from the 1- and 2-cell stage are mean±S.E.M. of 3 determinations.
Values for cultures from the 4-cell stage are mean of 2 determinations.
when the inhibitor was added at the 2-cell stage, almost
85% of the embryos developed to blastocysts in M16
and 65% in M16+hypoxanthine; reversal of inhibition
was reduced when azaserine was added at the 1-cell
stage, where some embryos progressed beyond the 4cell stage, but less than 5% formed blastocysts after
transfer to M16 or M16+hypoxanthine. After incubation with the inhibitor for 4 hours at the 8-cell stage,
followed by transfer to M16 or M16 +hypoxanthine, all
the embryos formed blastocysts by day 5; however,
these blastocysts had fewer cells than those formed in
control cultures without azaserine treatment; 79 ±5
cells and 101 ±10 cells, respectively.
Mycophenolic acid had an inhibitory effect different
from that of azaserine. The results, reported in Table 2,
showed that the embryos developed to the 8-cell stage
after continuous exposure to the inhibitor from day 2.
However, when treated with the drug early after
fertilisation, (day 1), the number of embryos reaching
the 8-cell stage was reduced, and the main feature of
these cultures was the inhibition of compaction.
Excretion products
Incubation media were removed after each day of
development and assayed for the release of potential
excretory products of purine metabolism.
Xanthine was consistently present each day; the
amounts excreted were very low, in the range of 0.030.1 pmol embryo" 1 24 hours" 1 (Fig. 2). There was a
small but statistically insignificant fall in xanthine
excretion between day 2 and day 3, but a marked
increase (P < 0.001) from the early to the late blastocyst
stages.
Uric acid, an anticipated end-product, was not
detected at any stage. In view of this, its immediate
metabolic precursor, xanthine, was added to the culture
medium at concentrations of 0.05 and 0.1 mM.
Xanthine had no effect on embryo development and its
concentration in the medium was unchanged. No uric
acid production was detected. The addition of uric acid
similarly had no effect on embryo development, and its
concentration in the culture medium also remained
unchanged.
0.8 n
48
96
120
Time post HCG (h)
Fig. 2. Xanthine excretion by mouse embryos developing
in vitro from the 1-cell stage to the blastocyst in the
presence of 0.05 mM hypoxanthine (white hatched bars:
H), and 0.05 mM adenine (black hatched bars: A). Dark
bars (C) show the amounts of xanthine excreted in control
cultures without hypoxanthine or adenine treatment. C:
Each bar corresponds to the mean ±S.E.M. of 5-12
determinations with 30-120 embryos each, (a) significantly
different (P <0.01) from the other stages. H: Each bar
represents the mean ±S.E.M. of three to five
determinations with 22-48 embryos in each determination,
(c) statistically different (P <0.001) from the other stages
treated with hypoxanthine. A: Each bar represents the
mean ±S.E.M. of three separate experiments with 28-40
embryos each, (b) statistically different (P <0.001) from
the other stages treated with adenine.
Allantoin could not be separated in the HPLC system
used.
Addition of purines
(i) Hypoxanthine: exogenous hypoxanthine, added to
cultures on either day 1 or day 2, disappeared steadily
from the medium (Fig. 3). The hypoxanthine uptake
was accompanied by the appearance of xanthine in the
medium. The excretion pattern showed a small increase
from the 1-cell stage to the compacted morula, with a
dramatic rise following cavitation (Fig. 2). When 1-cell
embryos were exposed to 0.05 mM hypoxanthine, the
Purine metabolism in mouse embryos
189
0400-1
3-
0.300-
0.200-
0.100a
X
0
48
0.000
40
60
80
100
120
140
72
96
120
Time post HCG (h)
Time post HCQ (h)
Fig. 3. Disappearance of 0.05 mM hypoxanthine (nmol)
from cultures of preimplantation mouse embryos
established at the 1- or 2-cell stage. • cultures established
on day l(l-cell). A cultures established on day 2(2-cell).
proportion developing to blastocysts was significantly
lower (P < 0.05) than those treated from the 2-cell
stage (Table 3). For those established on day 1, 82% of
control and 65% of hypoxanthine-treated cultures
reached the blastocyst stage, whereas there was no
difference for those started on day 2.
(ii) Guanine: 0.05 mM guanine, added at the 1-cell
stage, inhibited development to hatched blastocysts by
only 10% relative to the controls, and when added at
the 2-cell stage, gave no significant inhibition (Table 3).
The guanine was rapidly utilised by the embryos, such
that after 24 hours, all the purine had been consumed.
This was true of all the developmental stages. The
amount of xanthine excreted after guanine addition
(Fig. 4), was extremely high (0.125 pmol embryo"1
hour" 1 for each stage), with the amount excreted by
control embryos less than 1% of this value. The total
amount of guanine consumed was 0.183 pmol embryo"1
hour" 1 , thus, 70% could be accounted for by xanthine
appearance in the medium. This implied that only 30%
was converted to guanine nucleotides by the salvage
pathway.
Fig. 4. Xanthine excretion by mouse embryos in culture
from the 1-cell stage, in the presence of 0.05 mM guanine.
Each bar represents the mean ±S.E.M. of three
determinations with 28-40 embryos each.
(iii) Adenine: when embryos were exposed to 0.05
mM adenine from the 1-cell stage, their development to
blastocysts was inhibited by almost 55% (Table 3). The
pattern of xanthine excretion in this case was qualitatively similar to that of hypoxanthine but the values were
lower; 0.09±0.0125 pmol embryo"1 24 hour" 1 for the
developmental stages before cavitation, and 0.47±0.05
pmol embryo"1 at the blastocyst stage (Fig. 2). The
increase in xanthine excretion was not due to degenerate or arrested embryos. This was shown by flushing
early blastocysts from the uterus, with M16, on day 4
and culturing them in the presence of adenine for 24
hours. These embryos developed to expanded blastocysts excreting xanthine in similar amounts to those
cultured continuously from day 1.
Discussion
The main conclusions of this study are that the
preimplantation mouse embryo is dependent on de
novo synthesis for its supply of purine precursors
required for nucleic acid formation, and that xanthine
appears to be the end-product of purine degradation.
Table 3. The effect of supplementation with hypoxanthine, adenine and guanine (all 0.05 mM) on the
development of 1-cell and 2-cell mouse embryos to blastocyst
% Development
Control
2-cell stage
4-cell stage
8- to 16-cell stage
blastocyst
Total no. of embryos
Hypoxanthine
Guanine
Adenine
2ct
lc*
2c*
lc*
2c*
lc*
2c
lc*
100
100
98.7±1
84.9±1 .2
100
100
84.2±3.1
100
100
94.6±0.7
65±3.3
100
98.8±1.4
94.6+4.6
100
92.4±3.3
72.4±11.4
46.6±12.8
100
97
96.2
100
97.3±1 .2
90.8±l .2
73.8±7 .7
100
96.8±0.5
89
194
93
167
101
62
104
140
lc, 2c refer to cultures from the 1-cell and 2-cell stages, respectively.
• mean±S.E.M. of 3 independent experiments.
t mean±S.E.M. of 4 independent experiments.
Values for adenine (2-cell) are the mean of 2 independent experiments.
•
100
190
M. Alexiou and H. J. Leese
The importance of de novo synthesis and the salvage
pathway were compared by the use of inhibitors.
Azaserine, which inhibits nucleotide and therefore
DNA synthesis, might have been expected to inhibit
cleavage completely. That the embryos were able to
complete one division in the presence of the inhibitor
may be due to the availability of sufficient intracellular
pools of nucleotides and/or their precursors (particularly those of guanine), which are possibly maternally
derived, or formed shortly after fertilisation. Another
possible explanation is that the embryos were exposed
to the inhibitor late in the cell cycle, when DNA
replication had started, and sufficient RNA molecules
had been synthesized, such that a reduction in the
availability of precursors did not impair cell division.
The latter hypothesis is supported by the finding that
embryos incubated in azaserine at approximately 21
hours post HCG did show a restricted ability for
division compared to those exposed three hours later.
In experiments such as these it is difficult to
distinguish true inhibition of a biosynthetic pathway
from possible non-specific toxic effects. In addition,
inhibitors may affect more than one metabolic pathway.
For example, it is possible that azaserine could exert its
inhibitory effect on glutamine uptake or metabolism as
opposed to glutamine incorporation into purines (Hundal et al., 1990). We consider this unlikely. While
glutamine is beneficial to mouse preimplantation embryo development, and can help overcome the "2-cell
block" in embryos from random-bred mice (Chatot et
al., 1990), it has to be added to the culture medium, and
in none of the present studies was this the case.
Furthermore, during the early stages of development,
when azaserine was added, utilisation of glutamine by
mouse embryos is low (Gardner et al., 1989).
One approach to the problem of non-specific inhibition is to expose the embryos for a shorter period (4
instead of 24 hours), and to attempt to reverse the
inhibitory effect. When this was done, there was partial
reversal of the effects of azaserine, following exposure
of the embryos to M16 or M16 +hypoxanthine. This
suggested that azaserine had not caused embryo
degeneration. It was also shown that supplementation
of M16 with hypoxanthine did not enhance the
reversibility of inhibition by M16 alone. Since hypoxanthine, added to the cultures treated continuously with
azaserine, was unable to reverse the effect of the
inhibitor, this further supports the proposition that
stimulation of the salvage pathway on its own is not
sufficient to fulfil the demand for nucleotides.
The suggestion was made earlier, that intracellular
pools of nucleic acid precursors may be sufficient to
sustain cell division even in the presence of inhibitors.
Support for this possibility is provided by the results of
the experiments with mycophenolic acid (Table 2),
which showed that although the conversion of IMP to
GMP was blocked by the inhibitor, the embryos could
complete at least two divisions. Hoshi et al., (1988),
similarly showed that the inhibitory effect of mycophenolic acid on embryo development could be reversed by
guanine, and by xanthine, at a concentration as low as
25 /ig/ml. This suggested that xanthine could be
salvaged by the activity of HGPRT or XPRT (xanthine
phosphoribosyl transferase), an enzyme known to be
present in E. coli able to convert xanthine to xanthosine
monophosphate (XMP), (Miller et al., 1972). The XMP
could then be converted to GMP overcoming the
inhibitory effect of mycophenolic acid.
An attempt was made to test this possibility by
incubating mouse embryos in a reaction mixture similar
to that used for HGPRT determination (Leese et al.,
1991), containing xanthine instead of hypoxanthine as
substrate. However, the results were negative in that no
XMP and/or GMP production was detected.
The appearance of xanthine in the culture medium
suggested that this oxypurine is the end-product of
purine catabolism in early mouse embryos. Xanthine is
formed from hypoxanthine by the enzyme xanthine
oxidase (EC 1.2.3.2). The same enzyme converts
xanthine to uric acid (Fig. 1). Thus the absence of uric
acid in the excretory products is intriguing. It is possible
that uric acid is produced, but not at a level high enough
to be detected. This proposition was tested by adding
xanthine to the culture medium. Neither the morphological appearance of the embryos nor their pattern of
excretory products changed as a result of xanthine
addition and no uric acid was detected. Uric acid added
to groups of embryos was neither taken up nor broken
down. Furthermore, there was no uric acid production
when extracts of embryos were incubated with xanthine
or hypoxanthine as substrate (data not shown). On the
other hand, increased amounts of xanthine were
produced when the embryos were incubated with
hypoxanthine, suggesting that xanthine oxidase was
active during the preimplantation stages.
The xanthine could however, have been derived by
an alternative route involving guanine deamination
(Fig. 1), in a reaction catalysed by guanase (EC
3.5.4.3). This enzyme is very active during the early
preimplantation stages in the mouse, before declining
in the blastocyst (Epstein et al., 1971). In support of this
proposition was the finding that guanine added to the
cultures disappeared rapidly and could be accounted for
stoichiometrically by the appearance of xanthine in the
medium. In other words, exogenous hypoxanthine
could be salvaged to form purine nucleotides which
gave rise to xanthine indirectly rather than via xanthine
oxidase.
Exposure to hypoxanthine at the 1-cell stage, blocked
subsequent cell division (Table 3), a finding reported in
other strains of mice (Nureddin et al., 1990). Hypoxanthine has also been implicated in causing a 2-cell block
in random-bred mouse embryos (Loutradis et al.,
1987), and in the maintenance of meiotic arrest in
mouse oocytes (Eppig et al., 1985; Downs et al., 1986).
Nureddin (1990) and his colleagues had suggested
that hypoxanthine is involved in the inhibition of a cell
processs occurring during the first 30 hours of development, which does not involve the conversion of IMP to
AMP or GMP via the salvage pathway. The data (Table
3) showing that the embryos develop as well as the
controls, if not better, in the presence of hypoxanthine
Purine metabolism in mouse embryos
added after the 2-cell stage, is consistant with this
suggestion. The mechanism by which hypoxanthine
blocks the first cleavage division remains obscure.
Adenine, when added at the 1-cell stage, also exerted
an inhibitory effect on embryo development, in agreement with the report by Thomson Ten Broeck (1968),
showing a concentration-dependent inhibitory effect of
a variety of nucleosides and nucleoside bases, including
adenine, on mouse preimplantation development.
When adenine was administered from day 2, almost all
the embryos reached the hatching blastocyst stage
(Table 3).
The similarity in the results of hypoxanthine and
adenine addition provides further evidence in favour of
the proposition that these two purines may be utilised in
the salvage pathway, and that the xanthine produced is
via nucleotide formation rather than direct conversion
from hypoxanthine. The lower xanthine production
with adenine could be attributed to lower levels of
APRT in comparison to that of HPRT (Epstein, 1970;
Monk, 1987; Alexiou et al., 1990).
The metabolic fate of guanine appeared to differ
from that of adenine and hypoxanthine. Mouse embryos took up and converted exogenous guanine
predominantly to xanthine. Furthermore, the embryos
were tolerant of concentrations of guanine, that, in the
case of hypoxanthine and adenine, were inhibitory to
development (Table 4). The preference for converting
guanine directly to xanthine, instead of reutilising it via
the salvage pathway was possibly due to the very high
activity of guanosine deaminase (guanase).
Why preimplantation embryos should apparently
lack xanthine oxidase activity is unclear. This enzyme,
with hypoxanthine as a substrate, is used experimentally to generate free radicals in a wide variety of
biological systems (Parks and Jacobson, 1987; Friedl et
al., 1989). It has been suggested that such oxygen
species exert a detrimental effect on early embryo
development (Loutradis et al., 1987; Nasr-Esfahani et
al., 1990; Legge and Sellens, 1991) and the capacity to
form them via xanthine oxidase is something which may
not be advantageous to the early embryo.
M. Alexiou was supported by the Hellenic State Scholarships Foundation. We thank P. G. Humpherson for expert
advice on analytical methods, S. Sgardelis for his help on data
analysis and K.L. Martin and D.R. Brison for critical
comments on the manuscript.
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{Accepted 30 September 1991)