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J. Embryol. exp. Morph. Vol. 15, 1. pp. 61-6, February 1966
Printed in Great Britain
Anaerobiosis in the segmenting eggs of
Bufo arenarum
By FRANCISCO D. BARBIERI &
HORTENSIA S. DE LEGNAME 1
From the Instituto de Biologia General, Universidad Nacional de Tucumdn
In the last few years our interest has been devoted to the energy metabolism
of the eggs of the common toad Bufo arenarum Hensel which, like some other
amphibian eggs, can cleave at a normal rate in the absence of oxygen or in the
presence of cyanide (Barbieri & Legname, 1957). Under anaerobic conditions a
rapid accumulation of lactic acid gives evidence of an intense glycolytic activity,
which is inhibited when the eggs are returned to oxygen (Pasteur effect) (Barbieri
& Salomon, 1963). Furthermore, an increase in oxygen uptake during the first
2 h of recovery has been observed (payment of the oxygen debt) (Legname,
1966). Taking into account the low value of the respiratory quotient (R.Q. =
0-5-0-7) during this period it can be assumed that most of the oxygen is not
involved in the oxidation of lactate (Legname, 1966). A similar respiratory
response has already been described in Ranafusca (Brachet, 1934) and R.pipiens
eggs (Cohen, 1955), and has served to support the idea of an 'oxidative reserve'
in the eggs of these vertebrates (Brachet, 1934).
An alternate interpretation to explain the use of the oxygen absorbed during
the payment of the respiratory debt has been proposed by Cohen (1955). Considering from a kinetic point of view the substantial amounts of lactate produced
at the end of anaerobiosis, the reaction at the onset of re-oxygenation might be so
accelerated that the synthesis of pyruvate exceeds its utilization. As this reaction
involves the reduction of nicotinamide-adenine dinucleotide (NAD+) by lactic
dehydrogenase, the heightened oxygen uptake might be due, principally, to the
re-oxidation of the coenzyme. Since in this oxidation process no CO2 is produced, a low R.Q. is to be expected. The experimental evidence presented by
Cohen in the same paper is only indirect, being restricted to showing that the
amounts of O2 absorbed and CO2 evolved are too low to be the result of the
oxidation of lactate.
According to this hypothesis, some accumulation of pyruvate must follow the
disappearance of lactate, unless it has been utilized as fast as it is synthesized
through a process not leading to its complete oxidation. Some pilot experiments
1
Authors' address: Instituto de Biologia, Facultad de Bioquimica, Quimica y Farmacia,
Universidad Nacional de Tucuman, Tucuman, R. Argentina.
62
F. D. BARBIERI & H. S. DE LEGNAME
on B. arenarum eggs seem to support the first possibility. In fact, by a semiquantitative isolation of pyruvic acid, through a chromatographic separation
of its 2,4-dinitrophenylhydrazone (Barbieri & Pascual, 1962), we have observed
an abrupt rise when the eggs are reoxygenated after a period of anaerobiosis
(Barbieri & Pascual, unpublished results). This observation compelled us to
attempt a kinetic study of pyruvate and lactate levels during the respiratory
recovery using more accurate methods. We made use of a sensitive technique
successfully proved on this biological material, which permitted us to show that
the level of pyruvate varies significantly during normal development (Barbieri &
Salomon, 1962) and that it decreases in the eggs subjected to anaerobiosis or to
fluoride action (Salomon & Barbieri, 1964). Moreover, the same method allows
us to determine simultaneously the level of a-ketoglutaric acid, one of the intermediates of the tricarboxylic acids cycle.
MATERIAL AND METHODS
B. arenarum eggs were obtained by treating adult females with homologous
hypophysis preserved in glycerine according to the method of Pisano (1956) and
fertilization was accomplished in vitro.
In order to ensure good anaerobiosis, the egg jelly was removed by ultraviolet
irradiation as described elsewhere (Legname, 1966). Preliminary tests allowed
us to exclude the possibility that this treatment could affect the level of the
metabolites studied here.
Every experiment, started after the first or second mitotic division, included
two steps: anaerobiosis and the re-oxygenation of the eggs. Anaerobiosis was
achieved by a flow of nitrogen through a series of Warburg vessels (shaking at
eighty complete strokes per minute) each of which contained 200 eggs suspended
in 2-5 ml of 10% amphibian Ringer's solution without bicarbonate. The gas
was previously washed through pyrogalate, vanadous sulfate-amalgamated zinc
(Meites & Meites, 1948) and water. After 20 min of flow, the system was closed
and kept for 3 h without further shaking. In the second step, an air flow was
established through the shaking vessels and maintained until each experiment
was finished, in order to ensure a quick and uniform re-oxygenation. The bath
was thermostatically maintained at 25 °C.
Samples were taken out every 15 min, homogenized by repeated freezing and
deproteinized with 10% metaphosphoric acid. The precipitated protein was
removed by refrigerated centrifugation and aliquots of the clear supernatant
were collected for the analysis. Lactic acid was estimated by the method of
Barker & Summerson (1941) according to the technique of Ishida & Taguchi
(1957). Pyruvic and a-ketoglutaric acids were simultaneously measured with the
spectrophotometric method of Goodwin & Williams (1952). A Hilger (Uvispek)
spectrophotometer was used.
Anaerobiosis in segmenting eggs
63
RESULTS
Several pilot experiments in which lactic acid and the two ketoacids were
separately determined confirmed our previous results (Salomon & Barbieri,
1964; Barbieri & Salomon, 1964): B. arenarum eggs under anaerobiosis accumulate lactate and lose pyruvate and a-ketoglutarate; when switching from nitrogen
to air, the reverse process is observed, i.e. a fall of lactate and a rise of both
ketoacids.
30
60
90
120
150
minutes since the onset of respiration
Fig. 1. The levels of lactate (O), pyruvate (•) and a-ketoglutarate ( 0 ) during the
respiratory recovery.
In experiments performed with eggs obtained from four different females, the
three metabolites were simultaneously determined with consistent results. The
numerical data of one of these experiments are given in the graph of Fig. 1. It
can be seen that the pool of lactate decreases in a biphasic curve. The first phase,
which lasts about 1 h, tends to have a uniform rate of fall of about 0-01 /m for
100 eggs per minute; the second phase also shows a uniform rate, but about four
times slower (0-0026/fcM/100 eggs/min). The level of pyruvate rises abruptly when
the eggs are re-oxygenated until the end of the first hour; thereafter, it decreases
at a lower rate. Hence, the pyruvate content also varies in a biphasic curve, with
an inflexion point that coincides with that found in the curve of lactate. aKetoglutaric acid increases also during the re-establishment of respiration, until
it reaches its maximum value 2 h after the beginning of re-oxygenation.
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F. D. BARBIERI & H. S. DE LEGNAME
In all the samples analysed the fall of lactate was far more pronounced than
the rise of pyruvate and a-ketoglutarate.
DISCUSSION
The kinetics of the three metabolites analysed here are in agreement with the
classical metabolic schemes.
The biphasic character of the lactate curve cannot be explained by our experimental data, since it does not depend, as far as we know, only upon the current
concentrations of lactate and pyruvate. Among the many possible factors which
may operate in vivo, we may remember the influence of the relative concentrations of oxidized and reduced NAD, as well as the influence of hydrogen-ion
concentration (Tobin, 1964).
The immediate accumulation of pyruvic acid fits well into the hypothesis
advanced by Cohen (1955) by showing that the turnover of lactate to pyruvate is
so rapid that it may exceed its rate of utilization. In any case, the increments
measured remained insufficient to compensate for the lack of balance between
the oxygen taken up and the lactate consumed (Legname, 1966). Even taking
into account the rise of a-ketoglutarate, this problem remains unsolved. Two
alternative explanations are possible: (1) the probable accumulation of other
intermediates of the tricarboxylic acids cycle before the oxidation of pyruvate
reaches its completion; (2) the participation of lactate as a precursor in some
anabolic process. We may remember in this connexion that when B. arenarum
eggs are transferred to air after anaerobiosis they seem capable of synthesizing
glycogen (Barbieri & Salomon, 1963).
It is worth while to compare our experiments with those performed by Duspiva
(1961, 1962), who studied the changes of lactic and pyruvic acids levels in eggs
treated with cyanide. When B. bufo and Triturus alpestris eggs are submitted to
the action of this respiratory inhibitor, their lactate level increases continuously,
while their pyruvate content, after an initial rise during the first hours, decreases
progressively. If these eggs are transferred to a normal medium without HCN, a
fall of lactate and a rise of pyruvate take place. These data agree, therefore, with
our observations reported here on B. arenarum eggs. We notice that Duspiva's
graph for T. alpestris is the only one which shows a good correlation between the
fall of lactate and the rise of pyruvate. This may be due either to the difficulty of
getting a rapid elimination of cyanide at the end of the treatment, or to the fact
that this inhibitor may interfere not only with the respiratory enzymes but also
with other enzymic systems.
SUMMARY
1. A description of the lactate, pyruvate and a-ketoglutarate level changes of
Bufo arenarum eggs transferred to air after anaerobiosis is presented.
2. The immediate changes after the onset of the respiratory recovery are a fall
Anaerobiosis
in segmenting
eggs
65
of lactate content and a simultaneous increase of the level of pyruvate. Subsequently, an accumulation of a-ketoglutarate occurs.
3. The increase of the pool of these ketoacids is not enough to compensate
the inbalance between the lactate consumed and the oxygen taken up.
RESUME
Anaerobiose chez les oeufs de Bufo arenarum
1. On presente une description des changements des teneurs en lactate,
pyruvate et a-cetoglutarate lorsque des oeufs de Bufo arenarum sont transferes
a l'air apres une periode d'anaerobiose.
2. Les changements immediats apres le debut de la reprise de la respiration
sont une chute de la teneur en lactate et une augmentation simultanee de celle en
pyruvate. Ulterieurement, il se produit une accumulation d'a-cetoglutarate.
3. L'augmentation du pool de ces acides cetoniques est insuffisante pour
compenser le desequilibre entre la consommation de lactate et celle d'oxygene.
ACKNOWLEDGEMENTS
One of us (H. S. de L.) was the recipient of a fellowship given by the Consejo Nacional de
Investigaciones Cientificas y Tecnicas (R. Argentina) and this work was supported by a grant
from the same institution.
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