/ . Embryol. exp. Morph. Vol. 59, pp. 175-186,1980
Printed in Great Britain © Company of Biologists Limited 1980
175
Changes in the oxidative metabolism during
maturation of amphibian oocytes
ByARNALDO H. LEGNAME1 AND
HORTENSIA SALOMON DE LEGNAME1
From the Instituto de Biologia del Desarrollo (INBIODE) Universidad Nacional
de Tucumdn and Consejo Nacional de Investigations Cientificas y Tecnicas,
R. Argentina
SUMMARY
Some aspects of oxidative metabolism during cytoplasmic maturation of the Bufo arenarum
oocyte have been studied.
During the autumn-winter period (immature oocyte), carbohydrates are degraded through
the glycolytic pathway, followed by the classical tricarboxylic acid cycle.
During the spring-summer period (mature oocyte), carbohydrates are mainly used through
the pentose phosphate cycle, while the tricarboxylic acid cycle operates as the glutamicaspartic cycle.
The oxidative phosphorylation of ADP does not seem to change during oocyte maturation. Although maturation does not alter the absolute values of ATP and citrate in the oocyte,
it determines their different compartmentalization, which, through phosphofructokinase, in
turn regulates the glycolytic activity of the oocyte.
Oxygen uptake decreases by about 40 % during maturation, while simultaneously, a
marked increment in respiratory stimulation by 2,4-dinitrophenol is observed.
INTRODUCTION
In amphibians, the end of oogenesis is marked by cessation of growth and
blocked meiosis associated with a state of metabolic inhibition. During the
amplexus period, this quiescent period is ended by an increase in gonadotropic
activity, which triggers completion of the first meiotic division, maturation of
cytoplasm and ovulation (Merriam, 1972). During this period, the oocyte
reaches a state of physiological maturation, not necessarily coincident with
nuclear (meiotic) maturation (Smith, 1975).
In Bufo arenarum, Legname & Buhler (1978) demonstrated that cytoplasmic
maturation, which can be chronologically separated from germinal vesicle
breakdown, constitutes an indispensable requisite for segmentation. Even
though a close relationship between modifications of biochemical activity and
the maturational state of the oocyte can be established, neither the nature of
1
Authors' address: Instituto de Biologia del Desarrollo, Chacabuco 461, 4000 - S. M.
Tucuman R. Argentina.
12-2
176
A. H. LEGNAME AND H. SALOMON DE LEGNAME
these modifications nor the way they act upon so called 'cytoplasmic maturation' has been accurately determined.
To the best of our knowledge, biochemical aspects of the amphibian oocyte
have only been studied in relation to progesterone-induced nuclear maturation
(Brachet et ah 1974; Brachet, Pays-De Schutter & Hubert, 1975) or to the
metabolic modifications of winter coelomic oocytes induced by oviduct secretions (Legname et al. 1972). The main concerns of the present paper are aspects
of intermediate metabolism in the full-grown Bufo arenarum oocyte during two
different seasonal periods: autumn-winter and spring-summer. These periods
represent the extreme conditions for both maturational state and gonadotropic
pituitary activity (Houssay, 1947).
Our results show that during the reproductive period important modifications
in the relative participation of the main routes of the oxidative metabolism
occur at the time of cytoplasmic maturation of the oocyte. In addition, our
results shed some light on the mechanisms through which these biochemical
differences are controlled.
MATERIALS AND METHODS
Biological. Adult specimens of Bufo arenarum were collected near San Miguel
de Tucuman (R. Argentina) between May-August (winter animals) and
September-December (summer animals). Specimens were kept at 15 °C until
used, generally within 15 days of collection. Animals were spinal pithed and
oocytes removed from follicles with watchmakers' forceps. Only fully grown
oocytes (exhibiting a pigment-free vegetal pole) were used. Oocytes were kept
in buffered Ringer solution at pH 8-0 with 0-005 M Tris HC1.
Respiratory activity. Oxygen uptake was measured at 25 °C following Warburg's direct method in reaction flasks of about 17 ml. Shaking rate was 80
cycles per minute at an amplitude of 7 cm. CO2 was absorbed by 20 % KOH
placed in the centre well. Determinations were performed on 200-oocyte batches
placed in 3-3 ml of Ringer solution. When necessary, 2,4-dinitrophenol was
added, to a final concentration of 1.5.10~4 M. This concentration produced maximal respiratory stimulus in eggs and embryos of this species (Legname, 1968).
Isotope experiment. The shunt index was determined by incubating 300
oocytes in a Warburg vessel containing either 3 [id of 14C-1-glucose or 14C-6glucose in 3 ml of Ringer solution. After 90 minutes, 0-05 ml of 1 N - H 2 S O 4 was
tipped in from the side arm and the vessels were kept for 15 min for equilibration. 14CO2 produced was absorbed by 0-15 ml of 1 N-NaOH contained in the
center well. NaOH was quantitatively transferred into a vial containing 15 ml of
Bray's fluid, and the radioactivity was measured in a Packard liquid-scintillation spectrometer. Yields of 14CO2 from 14C-1- and 14C-6-glucose were finally
calculated from radioactivities.
14
C-l-glucose (7-16 mCi/mol) and 14C-6-glucose 52-8 mCi/mol) were purchased from New England Nuclear.
Biochemistry of amphibian oocytes
111
Enzymatic assays. Fifty oocytes were homogenized in 2 ml sucrose 0-25 M in
EDTA 10-3 M at pH 7-5 in a Potter homogenizer, and the homogenate was
centrifuged at 13300 g for 20 minutes. Aliquots of the supernatant were used
to assay the specific activity of phosphofructokinase (PFK) (Underwood &
Newsholme, 1965) and glucose-6-phosphate-dehydrogenase (G6PDH) (Glock
& McLean 1953).
Mitochondrial fraction. Oocytes were homogenized in sucrose 0-25 M in
EDTA 10~3 M at pH 7-5 in a Potter homogenizer. The suspension was centrifuged at 1085 g for 10 min, and the decanted supernatant again centrifuged at
13300 g for 10 min. The pellet obtained was resuspended in sucrose 0-25 M
without EDTA and constituted the mitochondrial fraction. All operations were
performed in a RC2-B Sorvall centrifuge at 0-3 °C.
Tricarboxylic acid cycle. The capacity of isolated mitochondria to oxidize
pyruvate, citrate, succinate and fumarate was assayed manometrically. The
reaction system consisted of 2 ml of mitochondrial suspension, 0-1 M phosphate
buffer, 10-4 /tmoles MgC12, 0-12 /tmoles cytochrome C, 1-3 /tmoles NAD and
3-7 /unoles ATP, in a 3-3 ml final volume. Forty-five ^moles of metabolites were
added as the potassium salts, except pyruvate, which was used as the sodium
salt.
Citrate. In order to determine total citrate levels, oocytes were homogenized in 6 % perchloric acid and the protein-free supernatant, separated by
centrifugation, was neutralized at pH 7-4 with 5 M-K 2 CO 3 . The precipitate of
KC1O3 was eliminated by centrifugation and the citrate content evaluated from
the limpid supernatant according to Williamson & Corkey (1969). The intramitochondrial content of this substrate was determined from the mitochondrial
fraction isolated as described above. Supernatant values were estimated from the
difference between total values and the mitochondrial fraction.
ATP. Aliquots of the supernatant used to determine the total and intramitochondrial citrate content were also used to assay ATP. The determinations
were performed according to Lamprecht & Trauschold (1965).
NADH 2 and NADP extinction changes were followed in a Beckman DV
spectrophotometer, at 340 nm.
Oxidative phosphorylation. The oxidative capacity of the mitochondrial
fractions was determined according to Slater (1953). The reaction mixture
contained 0-03 M phosphate buffer at pH 7-4, 0-01 M sodium malonate, 0-04 M
sodium fluoride, 002Mglucose, 6,7-10~ 4 M-ADP(potassium salt), 6,10 4 M-AMP
(potassium salt), 0005 M-MgCl2, 0-01 M potassium a-ketoglutarate, 5.5.10~ 5
M cytochrome C and 112 units hexokinase per mg of mitochondrial protein.
The final volume of the system was adjusted to 1 ml for a 7 ml Warburg flask.
In order to prevent the precipitation of magnesium fluorophosphate, MgCl2 was
added after the addition of hexokinase and immediately before the mitochondrial preparation, which had been resuspended in sucrose 0-25 M - E D T A 10~3 M.
The CO2 formed during the reaction was absorbed with 20 % KOH placed in
178
A. H. LEGNAME AND H. SALOMON DE LEGNAME
the central reservoir, in which a folded filter paper had been placed in order to
increase the absorption surface. Reaction was allowed to continue at 25 °C for 30
minutes, after which 0-1 ml of 35 % perchloric acid was tipped in from the side
arm. Ten minutes after stopping the reaction, the flask content was poured into
a centrifuge tube and the protein precipitate was separated by centrifugation.
The supernatant was neutralized with KOH and kept for 30 minutes in an ice
bath, then separating potassium perchlorate by centrifugation.
Hexose monophosphate was enzymatically determined in the supernatant
fluid by following NADP reduction at 340 nm, according to Slater (1967). This
reaction mixture contained 50 mM Tris-CIH buffer at pH 7-4, 10 mM MgCl2,
2 mM EDTA and 1 mM NADP in a 2-8 final volume. The reaction was triggered
by adding 0-002 IUB of glucose-6-phosphate dehydrogenase in serum albumin
solution (1 mg/ml).
Proteins. They were assayed according to Lowry, Rosebrough, Farr &
Randall (1951), using bovine albumin as standard.
RESULTS
Respiratory activity. In order to express the respiratory activity of the oocyte
the following terminology, proposed by Gregg (1960) for Ranapipiens eggs and
embryos, will be used.
Respiratory norm. The respiratory rate exhibited by intact embryos at a given
stage and under standard conditions.
Respiratory potential. The respiratory rate exhibited by intact embryos at a
given stage and under maximal stimulation by 2,4-dinitrophenol (DNP).
Respiratory control quotient. The quotient obtained by dividing the respiratory potential by the corresponding respiratory norm.
Our results, expressed in Table 1, show that oxygen uptake, under standard
conditions (respiratory norm) of ovarian oocytes changes with season. During
the autumn-winter period the respiratory rate is about 40 % above that observed during the spring-summer period. The maximal respiratory stimulation
by DNP also shows significant differences between both types of oocytes. The
uncoupling agent causes a less than two-fold increase in the respiratory activity
of the winter oocyte, while during the amplexus period the oxygen uptake of the
oocyte is raised five to six times by DNP.
These respiratory differences are clearly indicated by the respiratory control
quotient, which reaches 1-8 for winter oocytes and 5-7 for spring-summer
oocytes. These values, obtained during the embryogenesis of this species
(Legname, 1968) allow us to relate the respiratory behaviour of the summer
oocyte to that of the segmenting egg, and that of the winter oocyte to the respiratory behaviour of more advanced stages of development.
Since our determinations were performed on oocytes mechanically removed
from the follicles, their follicular cell layer was retained. In order to establish
Biochemistry of amphibian oocytes
179
Table 1. Respiratory activity in ovarian oocytes at different seasons
Season
Expt.
Respiratory
norm
Respiratory
potential
Respiratory
control
quotient
1
2
3
4
1-8
0046
0085
1-7
0042
0073
1-8
0047
Autumn-winter
0084
1-7
0043
0076
1
0026
0148
6-2
2
5-1
0034
0175
3
51
0036
0186
Spring-summer
6-8
4
0-204
0030
Values of respiratory norm and respiratory potential are expressed as/*l oxygen/oocyte/h.
Each experiment was performed on a different animal.
Table 2. Respiratory activity in ovarian and coelomic oocytes obtained during
autumn-winter
Expt.
Respiratory
control
quotient
Oocytes
Respiratory
norm
Respiratory
potential
Ovarian
Coelomic
0040
0038
0064
0068
Ovarian
Coelomic
0042
0041
0075
0065
1-6
1-8
1-8
1-6
Ovarian
Coelomic
0044
0050
0074
0080
1-7
1-6
Values of respiratory norm and respiratory potential are expressed as /*1 oxygen/oocyte/h.
Each experiment was performed on the same animal.
whether these cells affect the results, experiments were conducted using both
ovarian and oviposited oocytes from the same animal. Coelomic oocytes are
free from follicular cells, and since it is known (Legname & Buhler, 1978) that
in vivo induction of ovulation does not modify the metabolic behaviour of the
oocyte, it could be established (Table 2) that the presence or absence of the
follicular cells did not alter the respiratory levels of the oocyte (under our
working conditions).
The seasonal difference in respiratory activity exhibited by the oocyte suggests
differences in relative participation of metabolic routes involved in the oxidative
processes.
Glycolysis and pentose phosphate cycle
The relative participation of these metabolic pathways in the utilization of
glucose by Bufo arenarum oocyte, has been estimated by the shunt index, i.e.
180
A. H. LEGNAME AND H. SALOM6N DE LEGNAME
the relative yields of 14CO2 produced from 14C-6-glucose or 14C-1-glucose
(Isono & Yasumasu, 1968). Our results (Table 3) indicate that in the winter
oocyte almost 80 % of this sugar would be degraded via the glycolytic EmbdenMeyerhof pathway and only 20 % via the pentose phosphate cycle. During the
amplexus period (spring-summer), the main metabolic route would be the
pentose cycle (70-80 %), and only the remaining 20-30 % would be channelled
through the glycolytic pathway. The different relative participation of these
metabolic routes in carbohydrate degradation, during the different seasonal
periods can be correlated with the specific activities of the key enzymes of these
metabolic routes (Table 4). The high specific activity of phosphofructokinase
(PFK), key enzyme of the glycolytic pathway (Shigeo Kondo & Gerna-Torsellini, 1974) and the relatively lower activity of glucose-6-phosphate dehydrogenase (G6PDH), key enzyme of the pentose phosphate cycle (Egglesten & Krebs,
1974) observed during the autumn-winter period, would account for the
greater participation of the glycolytic pathway during this season. During the
amplexus period. PFK activity decreases significantly, while that corresponding
to G6PDH increases, indicating a greater utilization of sugars through the
pentose phosphate cycle in the spring-summer oocyte. These results can be
observed by the index resulting from dividing PFK/G6PDH activities, which
shows figures of almost 2-0 during the autumn-winter period and of about 0-3
during the reproduction season.
Tricarboxylic acid cycle
The presence of an operative tricarboxylic acid cycle in the Bufo arenarum
oocyte is shown by the capacity of the isolated mitochondria to oxidase intermediates such as pyruvate, citrate, succinate and fumarate. The relative utilization rates of these substrates during both periods show that seasonal changes do
not significantly modify the oxidation rate of fumarate, succinate and pyruvate.
However, citrate, which is oxidized at rates similar to that of fumarate, during
the winter is oxidized at a considerably slower rate during the amplexus period.
Differences in the utilization rate of citrate can be readily indicated by an
index of citrate oxidation rate/fumarate oxidation rate. As shown in Table 5, in
winter oocytes the values of this index are above unity, while during the reproductive period it oscillates around 0-5.
Variations in the fumarate/citrate index in relation to changes in metabolic
activity have previously been found in Bufo arenarum eggs and embryos (Salomon de Legname, 1969, Legname et al. 1972, Salomon de Legname, Valdez.
Toledo & Legname, 1979). It has also been postulated (Salomon de Legname,
1969; Salomon de Legname, Sanchez Riera & Sanchez, 1975) that a citratefumarate ratio at or above unity would show that a classical Kreb's cycle was
operative, while values ranging between 0-3-0-6 would indicate predominance
of its variant known as the glutamic aspartic cycle.
Since it is known (Salomon de Legname, Fernandez & Legname, 1975;
Biochemistry of amphibian oocytes
181
Table 3. Relative participation of glycolytic pathway and pentose phosphate cycle
in glucose utilization by ovarian oocytes at different seasons
Season
Expt.
Glycolytic
pathway
(Re/Ri)
Pentose
phosphate
cycle
(1-Re/RO
1
0-77
0-23
2
0-81
019
Spring-Summer
1
0-24
0-76
2
016
0-84
14
14
Re/Ri express the ratio of CO2 formation from C-6-glucose to 14CO2 formation from
14
C-1 -glucose. Each experiment was performed on a different animal.
Autumn-winter
Table 4. Phosphofructokinase (PFK) and glucose-6-phosphate dehydrogenase
(G6PDH) activity in ovarian oocytes at different seasons
Season
Expt.
PKF
G6PDH
Autumn-winter
1
2
3
1
2
3
0110
0098
0095
0045
0038
0055
0055
0051
0048
0131
0121
0160
Spring-summer
PFK/G6PDH
200
1-92
1-98
0-35
0-31
0-34
PFK and G6PDH values are expressed as /*-moles of product formed per mg/proteins/min.
Each experiment was performed on a different animal.
Salomon de Legname et al, 1977), that in Bufo arenarum embryogenesis the
utilization rate of citrate depends upon mitochondrial permeability, associated
with variations of energy metabolism, the phosphorylative capacity of the
mitochondria as well as ATP and citrate levels have been studied.
Our results (Table 6) show that the oxidative phosphorylation of ADP,
expressed by the P:O ratio, shows similar values (close to 2-0) during both
quiescent and reproductive periods. Absolute values of ATP and citrate do not
seem to change during oocyte maturation (Table 7). However, as shown in the
same table, the intracellular distribution of these metabolites is markedly
different in the two types of oocytes. During the reproductive period a marked
increment in the extramitochondrial content of ATP and citrate occurs at the
expense of a concomitant diminution of intramitochondrial content. This
different compartmentalization, more evident for citrate than for ATP, is
accompanied by a marked reduction in the specific activity of phosphofructokinase.
Since allosteric inhibition of this enzyme by ATP and citrate constitutes an
182
A. H. LEGNAME AND H. SALOMON DE LEGNAME
Table 5. Citrate/fumarate ratio in ovarian oocytes at different seasons
Season
Expt.
Citrate
Fumarate
Autumn-winter
1
2
3
4
Spring-summer
1
2
3
4
0073
0090
0064
0078
0045
0036
0030
0031
0056
0089
0056
0067
0075
0066
0062
0070
Citrate/
fumarate
1-30
101
114
116
0-61
0-54
0-48
0-44
Citrate and fumarate values are expressed as /^-moles of oxygen uptaken per mg proteins/30
min. Each experiment was performed on a different animal.
Table 6. Phosphorylative activity (P:O ratio) in ovarian oocytes at different
seasons
Season
Expt.
Oxygen
HMP
P:O
Autumn-winter
1
2
0-76
0-60
1-60
1-20
2-1
20
3
0-70
1-70
2-4
1
2
3
068
0-78
0-52
138
1-40
114
20
1-8
21
Spring-summer
Oxygen values are expressed as //-atoms. Hexose monophosphate (HMP) as /rnioles. Each
experiment was performed on a different animal.
important regulating mechanism of glycolytic activity (Passoneau & Lowry,
1964; Rapoport, 1970; Katyare & Howland, 1974) it could be postulated that
the lower relative participation of this metabolic route in the mature oocyte
would be determined by the increased extramitochondrial concentrations of
ATP and citrate. This hypothesis would agree with that of Wales (1975) for rat
embryos.
DISCUSSION
From the results presented above, we can conclude that the physiological
maturation of Bufo arenarum oocyte is related to important modifications in
intermediate metabolism. Such changes involve different relative participation
of the main oxidative routes. During the quiescence period (autumn-winter),
the degradation of carbohydrates seems to be accomplished mainly through the
Embden-Meyerhof glycolytic pathway followed by a classical tricar boxy lie
1
2
3
1
2
3
2-79(100)
308(100)
3-55(100)
0-66 (100)
0-66 (100)
0-67 (100)
Total
2-58(92-4)
2-86(92-8)
3-37(94-9)
018 (29-3)
0-23 (34-8)
0 0 2 (29-8)
Autumn-winter
Mitochondria
0-21(7-6)
0-22(7-2)
0-18(5-1)
0-48 (70-7)
0-43 (65-2)
0-47 (70-2)
Supernatant
2-76(100)
314(100)
2-44(100)
0-67 (100)
0-65 (100)
0-66 (100)
Total
110(400)
109(35 0)
0-89(36-5)
004 (5-9)
0 0 7 (10-7)
0 0 5 (7-5)
Spring-summer
Mitochondria
1-66(60-0)
205(650)
1-55(63-5)
0-65 (941)
0-58 (89-3)
0-61 (92-5)
Supernatant
Citrate values are expressed as /*g/100 oocytes. ATP as /*g/oocyte. ( ) = indicates percentages. Each experiment was performed on a different animal.
ATP
Citrate
Expt.
Table 7. Citrate and ATP compartmentalization in ovarian oocytes at different seasons
OO
o
S
S
%'
,-s
^
^
|
'§M
§•
g-
D
184
A. H. LEGNAME AND H. SALOMON DE LEGNAME
acid cycle. During the breeding season (spring-summer) the pentose phosphate
cycle as well as the variant of the tricarboxylic acid cycle known as the glutamic
aspartic cycle, acquire major importance.
The parameters studied indicate a marked similarity in the metabolic behaviour of the immature oocyte and the more differentiated tissues of embryogenesis on the one hand, and between the physiologically mature oocyte and
segmenting stages on the other (Salomon de Legname et al. 1975, Salomon de
Legname et al., 1977).
On the basis of these considerations it can be postulated that the fully grown
oocyte, which has not yet accomplished maturation, maintains the metabolic
pattern of the tissues from which it originates. Through endocrine induction of
maturation, as a response to seasonal variations, the oocyte acquires a new
metabolic behaviour which will remain unchanged until cleavage is accomplished. From the gastrula stage onwards and as ontogenesis progresses, this
type of metabolic behaviour is gradually replaced by the metabolism characteristic of adult tissues,
The metabolic change occurring during the cytoplasmic maturation of the
oocyte is required not only for onset of egg segmentation (Legname & Buhler,
1978) but also for the whole pregastrular period which is characterized by high
mitotic activity (Brachet, 1969).
It has already been postulated (Salomon de Legname, et al. 1975) that the
metabolic behaviour which characterizes the mature oocyte and segmenting
egg, represents a metabolic adaptation needed to satisfy the requirements for
precursors, mainly nucleotides, during cleavage. It has been reported that the
deoxynucleotides present in the amphibian embryo may be involved in controlling the initial stages of cell division and cell differentiation. (Lovtrup,
Landstrom & L0vtrup-Rein (1978).
H. S. de Legname is a member of the Carrera del Investigador Cientifico, Consejo Nacional
de Investigaciones Cientificas y Tecnicas (R. Argentina). Appreciation is expressed to Mr
Eduardo Rothe and Mr Hugo Gomez for their assistance in the preparation of the manuscript.
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{Received 23 October 1979, revised 20 March 1980)