/ . Embryo!, exp. Morph. Vol. 47,pp. 161-168, 1978
Printed in Great Britain (^ Company of Biologists Limited 1978
Metabolic behaviour and cleavage capacity
in the amphibian egg
By ARNALDO H. LEGNAME 1 AND MARTA I. BUHLER 1
From the Instituto de Biologia, Facultad de Bioquimica, Quimica y
Farmacia, Universidad National de Tucwndn, R. Argentina
SUMMARY
During the winter season the full grown Bufo arenarum oocyte shows the metabolic behaviour characteristic of differentiated tissues of the same species. Due to seasonal variations,
during the amplexus period, it acquires the metabolic behaviour of the segmenting egg.
Short-time-induced ovulations (5-6 h) determine germinal vesicle breakdown immediately
before the expulsion of the oocyte, without modifying the ovarian metabolism of the same.
The incidence of the operative type of metabolism upon their capacity to cleave after insemination and needle pricking, has been studied in coelomic oocytes, which have attained
nuclear maturation and have not experienced oviducal secretion effects.
The results obtained indicate that the segmenting capacity of the egg is attained only when,
through biochemical modifications, the oocyte acquires the metabolic behaviour characterizing embryonic cells.
It is postulated that the metabolic changes observed in the oocyte constitute a fundamental
aspect of cytoplasmic maturation.
INTRODUCTION
Although the concept of cytoplasmic maturation was introduced 20 years ago
(Aplington, 1957), the modifications which occur in the nucleus during amphibian oocyte maturation have always attracted the attention of many investigators
and it has been established that non-chromosomal elements such as cytoplasm
and cortex play a fundamental role in these processes (Schuetz, 1974; Brun, 1975).
The participation of cytoplasm in oocyte maturation has been mainly studied
in relation to its influence upon the rupture of the germinal vesicle (Reynhout &
Smith, 1974; Wasserman & Masui, 1975; Elinson, 1977). The fact that egg
segmentation can be normally accomplished in the absence of the nucleus
(cf. Aimar & Delarue, 1976), and that the chronological appearance of segmentation furrows depends upon the qualitative characteristics of cytoplasm
(Aimar, 1977), assign a role to this cellular component during the segmentation
processes.
The main aim of this paper is to correlate some cytoplasmic activities, mainly
metabolic, with the capacity of the egg to initiate segmentation. In this sense,
1
Authors' address: Insiituto de Biologia, Chacabuco 461 4000-S.M. de Tucuman,
R. Argentina.
162
A. H. LEGNAME AND M. I. BUHLER
it has already been demonstrated (Legname et al. 1972; Salomon de Legname,
Fernandez & Legname, 1975a); Legname et al., 1976) that the fully grown Bufo
arenarum oocyte has two different and alternative types of metabolic behaviour
closely related to the seasonal variations of pituitary activity determined by
Houssay (1947). In fact, during the fall-winter period the oocyte, like differentiated tissues, breaks down carbohydrates through the Embden-Meyerhof
route followed by a classical Kreb's cycle. On the other hand, during the reproductive period (spring-summer) the oocyte acquires the metabolic behaviour
which characterizes the early stages of development. This type of metabolism
exhibits marked activity of the pentose phosphate cycle, while the tricarboxylic
acid cycle, operating as the glutamic-aspartic cycle, provides important amounts
of precursors for nucleotide biosynthesis (Salomon de Legname, Sanchez
Riera & Sanchez, 19756).
Evidence indicating that the changes in the metabolic activity of the oocyte,
resulting from seasonal variations, represent an indispensable requisite for egg
segmentation, are reported in this paper.
MATERIAL AND METHODS
Biological. Adult specimens of Bufo arenarum were collected in the surroundings of Tucuman (R. Argentina) between May-August (winter animals) and
September-November (summer animals). Specimens were kept at 15° C until
use, which generally occurred within the 15 days of collection.
Coelomic oocytes were obtained by injecting homologous hypophysis (1 gland
per animal) into sexually mature females kept for 24 h at 25 °C. Five to six
hours after pituitary injection the animals were pithed and the oocytes removed
from the body cavity. Oocytes were maintained in Ringer's solution buffered
to pH 8 with 0-005 M Tris-HCl until the first polar body was ejected. In order to
determine the condition of the germinal vesicle, some oocytes were fixed by
boiling and observed after manual dissection under a stereo microscope, according to Cloud & Schuetz (1977).
Sperm suspension was obtained by macerating a testis in 4 ml Ringer's
solution, debris being eliminated by gauze filtration. Viability was controlled by
inseminating uterine oocytes.
Cleavage criteria. The appearing of the first furrow, disregarding whether it
was normal or atypical, was used as the cleavage criterion in coelomic oocytes
activated by glass needles. Trypsin-treated coelomic oocytes (Elinson, 1973)
were inseminated in 10% Ringer's containing the diffusible factor obtained
according to Barbieri & Oterino (1972). 100/4 of sperm suspension per ml
medium was added, and segmentation was estimated by the appearance of
normal furrows, as well as by development up to the blastula stage. In some
cases the diploidy of the eggs was determined for caryotype study.
Oocyte metabolism. The metabolic modifications occurring in Bufo arenarum
Metabolism
and cleavage in amphibian eggs
163
oocytes can be estimated on the basis of different parameters (Legname et al.
1972). Since it has been confirmed that the metabolic changes are closely related
to both functional (Salomon de Legname et al. 1915 a) and structural (Salomon
de Legname et al. 1977) mitochondrial modifications, the type of metabolism
operating in the oocyte was determined in the present paper on the basis of the
capacity of the isolated mitochondrion to oxidize citrate and fumarate. Taking
into account that with the onset of summer metabolism the mitochondrial
capacity to oxidize citrate is markedly reduced while the fumarate oxidation
rate is not significantly affected (Salomon de Legname et al. 1975a), we have
considered as metabolically immature those oocytes in which the citrate/
fumarate oxidation ratio ranged between 1-1 and 2-0. On the other hand, those
in which this same ratio ranged between 0-3 and 0-6 were regarded as metabolically mature.
Mitochondrial fraction. Body cavity oocytes were homogenized in a Potter
homogenizer with a hand-operated teflon pestle. Sucrose 0-5 M in 10~3 M EDTA
at pH 7-5 was used as the suspension medium. Ten volumes of sucrose per
volume of oocytes were used.
To homogenize ovarian oocytes, etherized animals were opened, and a
small portion of the ovary was removed. This was thoroughly washed in the
above described suspension medium and homogenized in a porcelain mortar.
The pestle was very gently pressed in order to break only fully developed
oocytes. The homogenate was then gauze-filtered. All operations were performed in an ice bath.
The homogenates, obtained as described above, were twice centrifuged at
1 085 g- for lOmin, and the decanted supernatant was again centrifuged at
10-800 g for another 10 min. The pellet obtained, resuspended in sucrose 0-25 M
without EDTA, constituted the mitochondrial fraction used in our experiments.
All operations were performed in a RC2-B Sorvall centrifuge at 0-3 °C.
Manometry. The oxidative capacity of mitochondria against citrate and
fumarate was manometrically assayed by evaluating oxygen uptake at 25 °C.
Reaction flasks of about 17 ml, shaking at 80 cycles per minute, with an amplitude of 7 cm, were used. CO2 was absorbed with 20 % KOH placed in the central
reservoir. The reaction system consisted of 2 ml mitochondrial fraction, 0-1 M
potassium phosphate buffer, 10-4/oriole cytochrome C, 1-3/tmole NAD and
3-7 /tmole ATP, in a final volume of 3-0 ml. Substrates, prepared from their
corresponding acids neutralized with KOH in the presence of phenol red, were
added at 45 /tmole, which assured the saturation of the oxidative capacity of
the system.
Protein determination. Protein concentrations were determined according to
the method of Lowry, Rosebrough & Randall (1951), using bovine serum
albumin as standard.
164
A. H. LEGNAME AND M. I. BUHLER
Table 1. Citrate Ifumarate ratio and germinal vesicle condition before
and after ovulation Induced in vivo
Ovarian oocytes
Season
Expt.
Cit./fum.
Germinal
vesicle
Coelomic oocytes
Cit./fum.
Germinal
vesicle
Autumn/winter
1
1-50
1-45
+
2
1-24
1-27
+
3
200
1-75
+
Spring/summer
1
0-35
0-37
+
2
0-42
0-46
+
3
0-33
0-32
+
Each experiment was performed on a same animal. Ovulation was induced 5-6 h after
pituitary injection. —, Intact germinal vesicle. + , Germinal vesicle breakdown.
Table 2. Citrate jfumar ate ratio and germinal vesicle condition before
and after ovulation induced in vitro
Ovarian oocytes
Season
Expt.
Cit./fum.
Germinal
vesicle
Coelomic oocytes
Cit./fum.
Germinal
vesicle
Autumn/winter
1
1 -73
—
0-54
+
2
• 1-66
0-38
+
3
1-60
0-45
+
Spring/summer
1
0-48
—
0-41
+
2
0-37
0-42
+
3
0-44
0-45
+
Each experiment was performed on a same animal. Ovulation was induced 18-24 h after
pituitary injection. —, Intact germinal vesicle. + , Germinal vesicle breakdown.
RESULTS
The results shown in Table 1 allow us to confirm that two alternative metabolic behaviours, which are season-dependent, are operative in the fully grown
Bufo arenarum oocyte. Oocyte metabolism expressing a citrate/fumarate ratio
above unity during the autumn-winter period is considered to belong to the
immature type while a ratio not reaching unity during the reproductive period
is an expression of a metabolically mature oocyte. In addition, the relatively
short periods of time required for the in vivo induction of ovulation (5-6 h) are
not long enough to modify the metabolic behaviour of the oocyte, which
exhibits, in the coelomic cavity, the same biochemical activity observed in the
ovary. From these results, it can also be assumed that, independently of the
metabolic behaviour of the oocyte, ovulation always determines the breakdown
of the germinal vesicle. On the other hand, when ovulation is induced in vitro
Metabolism and cleavage in amphibian eggs
165
Table 3. Citrate/fumarate ratio and egg cleavage after pricking
Season
Expt.
Cit./fum.
No. of
eggs
pricked
1 36
1-24
1-67
1-86
4
1
0-30
Spring summer
2
0-43
3
0-40
4
0-35
Each experiment was performed on
Autumn winter
1
2
3
No. of
eggs
cleaved
41
6
34
5
1
20
21
3
20
20
77
65
37
34
51
50
a different animal.
Percentage
cleaved
14-3
14-7
50
14-2
1000
84-4
91 8
980
Table 4. Citrate /fumarate ratio and egg cleavage after insemination
Season
Autumn/winter
Expt.
Cit./fum.
No. of
eggs inseminated
No. of
eggs
cleaved
1
1 89
126
2
1-45
100
90
3
1-60
87
4
1-45
1
120
Spring/summer
0-46
2
0-41
100
90
3
0-50
J15
4
0-44
Each experiment was performed on a different
3
0
0
0
115
83
88
108
animal.
Percentage
cleaved
2-3
0
0
0
95-8
830
97-7
93-9
(Table 2) and the ovary is under pituitary control for longer periods of time
(18-24 h), winter ovarian oocytes change their immature metabolism into a
mature one. Under such conditions, the metabolic change is associated with
ovulation and germinal vesicle breakdown. As would be expected, the metabolic
behaviour exhibited by ovarian summer oocytes is not modified by induction
of ovulation.
On the basis of the above results, we have been able to obtain coelomic
oocytes of the same nuclear maturation stage and free from oviduct influences,
although exhibiting a different metabolic behaviour. Disregarding other
variables, the influence of metabolism on the attainment of cleaving capacity
by the oocyte has been studied in this material.
In the first series of experiments needle-prick was used as the activation
stimulus and cleavage capacity assessed by the appearance of the fiist furrow.
The results, which are shown in Table 3, indicate that in oocytes regarded as
metabolically immature, pricking leads to the appearance of the first furrow in
166
A. H. LEGNAME AND M. I. BUHLER
less than 15% of cases. When similar experiments were conducted on oocytes
already metabolically mature, the onset of segmentation occurred in 80-100%
of pricked eggs.
In a second series, the oocyte capacity to initiate cleavage after insemination,
as a function of its metabolic condition, has been studied. Since it is known
(cf. Schuetz, 1974) that the vitelline membrane constitutes a barrier to the
penetration of spermatozoa in coelomic. oocytes, this membrane was partially
eliminated by trypsin treatment according to Elinson (1973). In addition, since
it has been demonstrated by Katagiri (1974) that alteration of the egg cortex
impairs normal gastrulation, we have used genuine segmentation up to the
blastula stage as the criterion of fertilization. Under these conditions, our
results (Table 4) show that, after insemination, oocytes from different ovulations
regarded as metabolically immature either do not cleave or reach very low
percentages of cleavage. When identical experiments were conducted during the
reproductive period, when the oocyte has already acquired metabolic maturity,
insemination resulted in high percentages of segmenting eggs. In our experiments, such figures ranged between 83 and 93 %. This last figure was quite close
to that found with pricking experiments.
The possibility of a parthenogenetic cleavage in embryonic oocytes due to
trypsin effects upon egg cortex, was controlled on aliquots of non-inseminated
oocytes. No case was observed of enzyme-induced cleavage.
DISCUSSION
Our results allow us to conclude that in Bufo arenarum, as in other amphibians
(cf. Schuetz, 1974), ovulation is always preceded by germinal vesicle breakdown.
Furthermore, although ovulation as well as metabolic changes are induced by
pituitary extracts, both processes can be chronologically separated by varying
the duration of the hypophyseal stimulus. This indicates that the mechanisms
which modify the metabolic behaviour of the oocyte during its maturation are
different from those involved in ovulation and germinal vesicle disintegration.
Our results also show a close correlation between the type of metabolism
operating in the oocyte and its capacity to initiate segmentation. In fact, activation either by needle pricking or by insemination indicates that the capacity to
initiate segmentation is limited to those oocytes, which, through biochemical
transformation, have acquired the metabolic behaviour operating during the
reproductive period.
Since this type of behaviour is hormone-dependent (Salomon de Legname
et ah 1975 a; Legname et al. 1976), it can be postulated that egg segmentation
requires the presence of a hormonally modified cytoplasm. This hypothesis is
in agreement with Katagiri & Moriya (1976) and Elinson (1977), who report
that normal sperm response to the egg and aster formation, depend upon a
hormonally modified cytoplasm. Although cytological studies have not been
Metabolism and cleavage in amphibian eggs
167
performed yet, it is permissible to assume that in metabolically mature coelomic
oocytes which accomplish a normal segmentation, both a normal sperm response
to the egg and aster formation from the sperm centriole occur. In fact, both
processes have been reported for the normal fertilization of this species (Manes
& Barbieri, 1976, 1977).
It is worthwhile mentioning that in our experiments on in v/iro-induced
ovulations, the winter egg metabolism changes into the summer type after
18-24 h incubation with pituitary extracts. Total maturation of Bufo bufo
oocytes after incubation during closely similar periods of time in the presence
of pituitary extracts and progesterone, have been reported by Tchou-Su & Wang
(1964) and by Katagiri & Moriya (1976), respectively.
Finally, if we accept, with lwamatzu & Chang (1972), that oocyte cytoplasmic
maturation involves the acquisition of the capacity to cleave and duplicate
D N A , the metabolic changes observed by us would constitute a fundamental
aspect of maturation processes or, at least, a measurable expression of it.
Although a correlation has not been established between the metabolism of the
summer oocyte and D N A synthesis, it has already been postulated (Salomón de
Legname, 1969; Salomón de Legname et al. 19756) that the metabolic changes
occurring in Bufo arenarum oocytes involve the triggering of fundamentally
anabolic processes, mainly leading towards nucleotide synthesis. This type of
biochemical behaviour could be regarded as a metabolic adaptation for supplying certain requirements, mainly nucleic acids, during the high mitotic activity
which characterizes segmentation (Brachet, 1969).
Appreciation is expressed to Mr Eduardo Rothe for his assistance in the preparation of
the manuscript. This work was supported in part by grants from the Consejo Nacional de
Investigaciones Científicas y Técnicas (R. Argentina) (CONICET), the Population Council,
New York, N.Y. (awarded to the CONICET) and the Fundación Lucio Cherny (R. Argentina). M.I.B. was supported by a scholarship from the CONICET.
REFERENCES
AIMAR, C. (1977). Contrôle cytoplasmique de la segmentation chez les amphibiens; modifications des rymes du clivage de l'oeuf après injection de cytoplasme hétérospecifique.
C. r. hebd. Séanc. Acad. Sci., Paris 284, 215-218.
AIMAR, M. M. C. & DELARUE, M. (1976). Modification, par injection de cytoplasme hétérospecifique, de la chronologie du premier clivage de l'oeuf d'Amphibien. C. r. hebd. Séanc.
Sci. Paris 282, 1737-1740.
APLINGTON, W. H. (1957). The insemination of body cavity and oviducal eggs of amphibia.
Ohio J. Sci. 5 7 , 91-99.
BARBIERI, F. D . & OTERINO, J. M. (1972). A study of the diffusible factor released by the
jelly of the egg of the toad, Bufo arenarum. Devi Growth Differ. 14, 107-117.
BRACHET, J. (1969). Les acides nucléiques dans la différenciation embryonnaire. Annls
Embryol. Morph. Suppl. 1, 21-37.
BRUN, R. (1975). Oocyte maturation in vitro: Contribution of the oviduct to total maturation
in Xenopus laevis. Experientia 3 1 , 1275-1276.
CLOUD, J. G. & SCHUETZ, A. W. (1977). Interaction of progesterone with all or isolated portions
of the amphibian (Rana pipiens) oocyte surface. Devi Biol. 6 0 , 359-370.
168
A. H. L E G N A M E A N D M. I . B U H L E R
ELINSON, R. P. (1973). Fertilization of frog body cavity eggs enhanced by treatments aff­
ecting the vitelline coat. / . exp. Zool. 183, 291-302.
ELINSON, R. P. (1977). Fertilization of immature frog eggs: cleavage and development
following subsequent activation. / . Embryol. exp. Morph. 3 7 , 187-201.
HOUSSAY, B. A . (1947). Ovulaciσn del sapo Bufo arenarum Hensel. I. Gonadotrofina hipofisaria. Revue Soc. Arg. Biol. 2 3 , 176-185.
IWAMATSU, T. & CHANG, M. C. (1972). Sperm penetration in vitro of mouse oocytes at various
times during maturation. / . Reprod. Fert. 3 1 , 237-247.
KATAGIRI, CH. (1974). A high frequency of fertilization in premature and mature coelomic
toad eggs after enzymic removal of vitelline membrane. / . Embryol. exp. Morph. 3 1 , 573587.
KATAGIRI, CH. & MORIYA, M. (1976). Spermatozoan response to the toad egg matured after
removal of germinal vesicle. Devi Biol. 50, 235-241.
LEGNAME, A . H . , SALOMON DE LEGNAME, H . , MICELI, D . C , SANCHEZ, S. S. & SANCHEZ
RIERA, A . N. (1976). Endocrine control of amphibian oocyte metabolism. Acta Embryol.
Morph. exp. 1, 37-49.
LEGNAME, A . H . , SALOMON DE LEGNAME, H . , SANCHEZ, S. S., SANCHEZ RIERA, A . N . &
FERNANDEZ, S. N. (1972). Metabolic changes in Bufo arenarum oocytes induced by
oviducal secretions. Devi Biol. 29, 283-292.
LOWRY, O. H., ROSEBROUGH, N. J. & RANDALL, C . J. (1951). Protein measurement with the
Folin phenol reagent. / . biol. Chem. 193, 265-275.
MANES, M. E. & BARBIERI, F . D . (1976). Symmetrization in the amphibian egg by disrupted
sperm cells. Devi Biol. 53, 138-141.
MANES, M. E. & BARBIERI, F. D . (1977). On the possibility of sperm aster involvement in
dorso-ventral polarization and pronuclear migration in the amphibian egg. / . Embryol.
exp. Morph. 40, 188-197.
REYNHOUT, J. K . & SMITH, L . D . (1974). Studies on the appearance and nature of a matura­
tion-inducing factor in the cytoplasm of amphibian oocytes exposed to progesterone.
Devi Biol. 3 8 , 394-400.
SALOMON DE LEGNAME, H . (1969). Biochemical studies on the energetics of Bufo arenarum
segmenting eggs. Archs Biol., Liege 80, 471-490.
SALOMON DE LEGNAME, H . , FERNANDEZ, S. N. & LEGNAME, A . H . (1975o). Metabolism and
mitochondrial activity in Bufo arenarum ontogenesis. Archs Sci. biol., Bologna 59, 19-29.
SALOMON DE LEGNAME, H . , FERNANDEZ, S. N., MICELI, D . C , MARIANO, M. I. & LEGNAME,
A . H . (1977). Modifications of the mitochondrial ultrastructure related to metabolic
changes during Bufo arenarum ontogenesis. Acta Embryol. Morph. exp. 2, 93-107.
SALOMON DE LEGNAME, H . , SANCHEZ RIERA, A . B. & SANCHEZ, S. S. (19576). Source of
precursors for nucleotide biosynthesis in Bufo arenarum segmenting eggs. Acta Embryol.
Morph. exp. 2, 123-136.
SCHUETZ, A . W . (1974). Role of hormones in oocytes maturation. Biol. Reprod. 10, 150-178.
SCHUETZ, A . W . (1977). Molecular mechanisms controlling cytoplasmic and nuclear matura­
tion in oocytes. Res. Reprod. 9, 3-4.
TSCHOU-SU & WANG, Y. L. (1964). The development of eggs of Bufo bufo asiatious insemin­
ated at different states of maturity. Acta Exp. Biol. Sνnica 9, 101-116.
WASSERMAN, W . J. & MASUI, Y. (1975). Effects of cycloheximide on a cytoplasmic factor
initiating meiotic maturation in Xenopus oocytes. Expl Cell Res. 9 1 , 381-388.
{Received 10 March 1978, revised 18 May 1978)