MITOCHONDRIAL DIFFERENTIATION DURING THE EARLY
DEVELOPMENT OF THE AMPHIBIAN EMBRYO
by
L e n n a rt Nelson
AKADEMISK AVHANDLING som med tillstå n d av r e k to r s ­
äm betet v id Umeå u n iv e rs ite t fö r erh ållan d e av filosofie
dokto rsex am en ,
fram lägges
till
o ffen tlig
g ra n sk n in g
to rsd a g e n den 17 decem ber 1981 kl 10.00 vid FysiologiB otanik H ufo, sem inarierum B.
Exam inator: P ro fe sso r S0ren L 0 v tru p , Umeå
O p ponent: D ocent B a rb a ra C annon, Stockholm
T itle :
M itochondrial d ifferen tiatio n d u rin g th e early developm ent
of th e am phibian em bryo.
A u th o r:
L e n n art N elson.
A b s tra c t:
M itochondria from X enopus laevis and Ambystoma mexicanum em bryos betw een fertilizatio n and th e b eg in n in g of
feed in g w ere stu d ie d : th e form er with re s p e c t to m etabo­
lic b e h av io u r, enzyme p a tte r n an d c a r rie r a c tiv ity , and
th e la tte r w ith re s p e c t to m orphological p ara m e te rs.
The m etabolic b eh av io u r of m itochondria was stu d ie d
by a s se ssin g th e ra te s of oxygen u p ta k e in p re se n c e of
v ario u s s u b s tr a te s . The ra te s of oxidation of most s u b ­
s tr a te s change d u rin g developm ent. The only s u b s tr a te
to be read ily m etabolized is glutam ate (in p re se n c e of
m alate), whose ra te of oxidation p re s e n ts a peak d u rin g
g a stru la tio n and declines d u rin g la rv a l developm ent. T he
h igh ra te of oxidation of glutam ate and a high a s p a rta te
am in o tran sferase ac tiv ity indicate th a t th e glutam atea s p a rta te cycle may be predom inant in early em bryonic
m itochondria.
The activ ity of enzym es from the m atrix , the in n e r
m em brane and th e o u te r m em brane w ere s tu d ie d . D uring
early developm ent activ ities of enzym es in th e v ario u s
com partm ents ch an g e in d ep en d en tly of each o th e r. F u r ­
th erm o re, enzym es w ithin one com partm ent may v a ry
in d e p e n d e n tly . M easurem ents of c a r rie r activ ity rev eal
th a t th e c a r rie r fo r dicarboxylic acids d isp lay s a high
ac tiv ity b efo re g a stru la tio n and d ecrease s th e r e a fte r ,
while th e tric a rb o x y lic acid , p y ru v a te and glutam ate/O H
c a rrie rs show th e opposite p a tte rn of ch an g e, th e ir
activ ities b ein g low o r u n d etectab le d u rin g early develop­
m ent.
T his implies th a t a m itochondrial d ifferen tiatio n tak es
place ' d u rin g developm ent, b eg in n in g at g a stru la tio n
when th e f ir s t d iffe re n tia te d cells a p p e a r. In o rd e r to
co rre la te m itochondrial an d cellular d iffe re n tia tio n , m or­
phological p aram eters of m itochondria from u n d iffe re n tia ­
ted and d iffe re n tia te d cells - R uffini cells and epiderm al
cells - w ere an aly zed . M itochondria from the d iffe re n tia ­
ted cells are sig n ifican tly d iffe re n t from those in u n ­
d iffe re n tia te d cells. T h u s the p ro c e sse s of cell d iffe re n ­
tiation are accom panied by m orphological and biochemical
d ifferen tiatio n of th e m itochondria.
Key w ords:
A m phibia, X enopus laevis, Ambystoma mexicanum , mito­
c h o n d ria , d iffe re n tia tio n , enzym es, c a r r ie r s , oxidation,
d ev elo p m en t.
ISBN 91-7174-093-7
Umeå 1981. D istrib u te d by th e D epartm ent of Zoophysiolo g y , U n iv ersity of Umeå, S-901 87 UMEÅ, Sw eden.
MITOCHONDRIAL DIFFERENTIATION DURING THE EARLY
DEVELOPMENT OF THE AMPHIBIAN EMBRYO
by
L e n n a rt Nelson
AKADEMISK AVHANDLING som med tillstå n d av r e k to r s ­
äm betet vid Umeå u n iv e rs ite t fö r erh ållan d e av filosofie
do k to rsex am en ,
fram lägges
till
o ffen tlig
g ra n sk n in g
to rsd a g e n den 17 decem ber 1981 kl 10.00 vid FysiologiB otanik H ufo, sem inarierum B.
Exam inator: P ro fe sso r S ören L ö v tru p , Umeå
O p ponent: D ocent B a rb a ra C annon, Stockholm
T itle :
M itochondrial d iffe re n tia tio n d u rin g th e early developm ent
of th e am phibian em bryo.
A u th o r :
L e n n a rt N elson.
A b stra c t :
M itochondria from X enopus laevis and Ambystoma mexicanum em bryos betw een fertilizatio n and the b eg in n in g of
feed in g w ere stu d ie d : th e form er w ith re s p e c t to m etabo­
lic b e h a v io u r, enzyme p a tte r n an d c a r r ie r a c tiv ity , and
th e la tte r w ith re s p e c t to m orphological p a ra m e te rs .
The m etabolic b e h av io u r of m itochondria was stu d ie d
b y a s se s sin g th e ra te s of oxygen u p ta k e in p re se n c e of
v a rio u s s u b s tr a te s . T he ra te s of oxidation of most s u b ­
s tr a te s ch an g e d u rin g developm ent. The only s u b s tr a te
to be read ily m etabolized is glutam ate (in p re se n c e of
m alate), whose r a te of oxidation p re s e n ts a p eak d u rin g
g a stru la tio n and declines d u rin g la rv a l developm ent T he
h igh ra te of oxidation of glutam ate an d a high a s p a rta te
am in o tran sferase ac tiv ity indicate th a t th e glutam atea s p a rta te cycle may be p redom inant in early em bryonic
m ito ch o n d ria.
The ac tiv ity of enzym es from th e m atrix , th e in n e r
m em brane and th e o u te r m embrane w ere stu d ie d . D u rin g
ea rly developm ent a ctiv ities of enzym es in th e v ario u s
com partm ents ch an g e in d e p en d en tly of each o th e r. F u r ­
th erm o re, enzym es w ithin one com partm ent may v a ry
in d e p e n d e n tly . M easurem ents of c a r rie r activ ity rev eal
th a t th e c a r rie r fo r dicarboxylic acids d isp lay s a high
ac tiv ity b efo re g a stru la tio n and d ecrease s th e r e a fte r ,
while th e trica rb o x y lic acid , p y ru v a te and glutam ate/O H
c a rrie rs show th e o pposite p a tte r n of c h an g e, th e ir
ac tiv itie s b ein g low o r u n d e te c ta b le d u rin g e arly develop­
m ent.
T his implies th a t a m itochondrial d ifferen tiatio n tak es
place d u rin g developm ent, b eg in n in g a t g a stru la tio n
when th e f ir s t d iffe re n tia te d cells a p p e a r. In o rd e r to
co rre la te m itochondrial and cellular d iffe re n tia tio n , m or­
phological p aram eters of m itochondria from u n d iffe re n tia ­
te d an d d iffe re n tia te d cells - Ruff ini cells and epiderm al
cells - w ere an aly zed . M itochondria from th e d iffe re n tia ­
te d cells are sig n ifican tly d iffe re n t from those in u n ­
d iffe re n tia te d cells. T h u s th e p ro c e sse s of cell d iffe re n ­
tiation a re accom panied by m orphological and biochemical
d ifferen tiatio n of th e m itochondria.
Key w ords:
A m phibia, X enopus laev is, Ambystoma m exicanum, mito­
c h o n d ria , d iffe re n tia tio n , enzym es, c a r r ie r s , oxidation,
d ev elop m en t.
ISBN 91-7174-093-7
Umeå 1981. D istrib u te d by th e D epartm ent of Zoophysiolo g y , U n iv ersity of Umeå, S-901 87 UMEÅ, Sw eden.
1
CONTENTS
LIST OF PUBLICATIONS
2
INTRODUCTION
3
CARBOHYDRATE METABOLISM
6
E n e rg y so u rces
6
G lycolytic pathw ay
7
P entose p h o sp h ate pathw ay
10
AMINO ACID METABOLISM
11
PROPERTIES OF EMBRYONIC MITOCHONDRIA
13
O xygen consum ption
13
M itochondrial enzym es
16
M itochondrial c a r rie r s
17
M itochondrial m orphology
18
MITOCHONDRIAL DIFFERENTIATION
20
REFERENCES
25
ACKNOWLEDGEMENTS
34
2
LIST OF PUBLICATIONS
T he p re s e n t th e sis
is b a sed on the following p u b lica­
tions :
I.
L 0 v tru p -R e in ,
H.
an d
L. N elson.
C hanges
in
e n e rg y metabolism d u rin g th e early developm ent of
X enopus laevis. Subm itted fo r p ublication.
II.
L 0 v tru p -R e in ,
H.
and
L. N elson.
C hanges
in
m itochondrial re sp ira tio n d u rin g th e developm ent of
X enopus laevis. S ubm itted fo r p ublication.
III.
N elson,
L.
and
H.
L 0 v tru p -R ein .
C hanges
in
ac tiv ity of m itochondrial enzym es d u rin g th e d ev e­
lopm ent
of X enopus
laev is.
Subm itted
fo r p u b li­
cation .
IV.
N elson,
L.
C hanges in th e activ ities of m itochon­
d ria l c a r rie r s
d u rin g
th e
developm ent of X enopus
laevis. S ubm itted fo r p u b lication.
V.
N elson,
L 0 v tru p
L .,
R.
Loren tz o n , L.
B oquist
and
(1982).
M orphological
d ifferen tiatio n
S.
of
m itochondria in th e early am phibian em bryo. E x p tl,
Cell R e s ., 137, 1.
T he p a p e rs will be re fe rre d to by th e ir Roman num erals
given above.
3
INTRODUCTION
In th e c o u rse of em bryonic developm ent th e f e rtili­
zed egg, a m orphologically simple e n tity , is tran sfo rm ed
into a la rv a which u su ally p o ssesses th e main f e a tu re s ,
m orphological as well as physiological, of th e body d is tin ­
g u ish in g th e m em bers of some major animal tax o n . T his
m arvellous tra n sfo rm atio n has been th e su b jec t of in q u i­
sition fo r se v e ra l c e n tu rie s .
From
o b se rv a tio n s
v e r te b ra te
em bryos,
made
on
p a rtic u la rly
th e
developm ent
those of vario u s
of
am­
p h ib ia , it has been p o ssib le to sub d iv id e th e em bryonic
developm ent into th re e p h a se s:
n e sis
and
la rv a l
developm ent.
b la stu la tio n , m orphoge­
T his
su b d iv isio n
was
originally b a se d on v isib le c rite ria . T h u s , b lastu latio n is
c h a ra c te riz e d b y ra p id m itoses leading to a fragm entation
of th e eg g in a num ber of cells, w ithout o th e r obvious
m orphological c h a n g e s . B lastulation ends when m orpho­
g en esis b e g in s, as ev id en ced by the ap p earan ce of th e
b lasto p o re. As implied by th e name, th e em bryonic body
is
c o n s tru c te d
d u rin g
th e
p h ase
of
m orphogenesis.
L arval developm ent is th e p h ase d u rin g which v ario u s
physiological fu n ctio n s
s ta r t.
It is d ifficu lt to give an
ex act m orphological definition of th e tra n sitio n betw een
th e se two p h a s e s, b u t th e b e a tin g of th e h e a rt (s ta g e
33/34) may be tak en as evidence th a t larv a l developm ent
h as b e g u n .
It is p o ssib le by m eans of chemical indices to ch a­
ra c te riz e
th re e p h ases
almost coincident with b la stu la -
4
tio n , m orphogenesis and e m b ry o g en esis. T h u s th e re are
th re e
p h ases
in
th e
p ro te in
s y n th e s is ,
d istin c t
both
q u alitativ ely and q u a n tita tiv e ly (L d v tru p , 1974). B ut still
more
c h a ra c te ris tic
a re
th e
d ifferen ces o b tain in g with
re s p e c t to th e sy n th e sis of inform ational RNA: mRNA is
sy n th e siz e d a t low and c o n sta n t ra te d u rin g cleavage and
b lastu latio n (B ach v aro v a, D avidson, A llfrey and M irsky,
1966; Brown and L ittn a , 1966). B ut th is sy n th e sis seems
to be in d ep en d e n t of th e n u c le u s, and u n n e c e ssa ry fo r
m itoses since actinom ycin D is w ithout e ffect on develop­
m ent up to th e late b la stu la sta g e (B ra c h e t and D enis,
1963; Wallace an d E lsdale, 1963). The p ro te in s y n th e sis
tak in g place m ust th e re fo re dep en d upon m aternal mRNA.
T his m eans th a t no cell d ifferen tiatio n can take place in
th e e arly em bryo; all th e cells a risin g th ro u g h division
m ust belong to th e same ty p e , and th ey may in p rin cip le
be c h a ra c te riz e d as "u n d iffe re n tia te d " cells.
The b lasto p o re is form ed by a new k ind of cells,
th e R uffini c e lls , which pull th e em bryonic cells into the
in te rio r,
th e re b y
in titia tin g
g a s tru la tio n .
T his p h en o ­
menon m ust th e re fo re be p reced ed by and be d ep en d en t
upon a p ro ce ss of cell d iffere n tiatio n . In agreem ent with
th is
in fe re n c e it is found th a t the sy n th e sis of mRNA
in crea ses in th e late b lastu la some time befo re g a s tr u ­
lation
M irsky,
d u rin g
se ts
in
1966;
th is
(B ach v a ro v a ,
Brown
p eriod
D avidson,
and L ittn a ,
1966).
A llfrey
and
F u rth e rm o re ,
a new sta g e -sp e c ific population of
u n sta b le mRNA molecules ap p e a rs which can no longer be
d ete c te d at la te r sta g e s of developm ent (D en is, 1968).
5
T he s y n th e s is of mRNA which ta k e s place a t th e
o n se t of la rv a l developm ent is both q u a n tita tiv e ly and
q u alitativ e ly d iffe re n t from th a t going on in th e p re c e d ­
in g p h a se ; f ir s t now is th e inform ation re sid in g in th e
genome e x ten siv e ly ex p lo ited , re s u ltin g in th e form ation
of sta b le a d u lt mRNA molecules (B row n an d L ittn a , 1966.
D en is,
1968).
In
th e following te x t th e a tte n tio n
has
b een fo cu sed mainly on th e two f ir s t p h a s e s. F or th e
sak e
of
sim plicity
o p e rate w ith
g a s tr u la tio n " .
th e following discu ssio n
will mostly
th e co n cep ts "p re g a stru la tio n " an d "p o st-
6
CARBOHYDRATE METABOLISM
E n erg y so u rces
The n a tu re of th e e n e rg y so u rces consum ed d u rin g
am phibian developm ent h as been stu d ie d since th e b eg in ­
n in g
of
th e
c e n tu ry .
G regg
(1948),
and
shown
th a t
Rana
d isa p p e a r
in
b efo re
B ra c h e t
and
Needham
B a rb ie ri and Salomon
an d
(1963) have
Bufo no glycogen
g a s tru la tio n ,
th ough
at
(1935),
seems to
th e
time of
h a tc h in g ab o u t 50 p e r cen t has been consum ed. G reg g 's
re s u lts show th a t th e loss in to tal red u c in g c a rb o h y d ra te
is n ea rly
id en tical w ith
th e d ecrease in gly co g en , th e
la tte r th u s b ein g th e main e n e rg y so u rce.
T he two o th e r common e n e rg y
s o u rc e s , lipid and
p ro te in , have also been in v e stig a te d (Wills, 1936; G regg
and B allen tin e, 1946; M es-H artree and A rm stro n g , 1980).
No
m easurable
ch an g es
could
be
o b se rv e d
u n til
late
d u rin g developm ent. It a p p e ars th a t th e e n e rg y so u rces
a re u se d in th e su ccessio n c a rb o h y d ra te s , lip id s, p ro ­
te in s; p ro te in s may be com busted only when no food is
su p p lied (L o v tru p , 1974).
The chemical an aly ses have been com plemented by
m easurem ents
of
th e
re s p ira to ry
q u o tie n t
(R Q ).
T he
value of RQ was found to be low d u rin g early develop­
m ent, re ac h in g th e value of u n ity only a fte r g a stru la tio n
(B a rth and B a rth , 1954; B ra c h e t, 1934; P e tru c c i, 1961;
Legname an d B a rb ie ri, 1962). T his s u g g e s ts th a t glyco­
gen
is
not
com busted
to
any
g r e a te r
e x te n t
before
7
g a s tru la tio n ,
th u s
in agreem ent with th e c a rb o h y d ra te
d ete rm in a tio n s.
T he low RQ values may be in te rp re te d as an in d i­
cation of (a ) lipid o r p ro te in com bustion, (b ) an incom­
p lete c a rb o h y d ra te oxidation o r (c ) com bustion of o th e r
m etabolites. T he f ir s t a lte rn a tiv e does not conform with
th e chemical a n a ly ses.
The second p o ssib ility , if tr u e ,
m akes th e custom ary in te rp re ta tio n of the RQ d eterm in a­
tions void of s e n se , since th ey a re b ased on the assum p­
tion th a t th e oxidation is com plete. The th ird a lte rn a tiv e
will be d isc u sse d la te r on (p ag e 11).
G lycolytic pathw ay
T he fa ct th a t to tal c a rb o h y d ra te
rem ains ro u g h ly
c o n sta n t b efo re g a stru la tio n does not n ecessarily imply
th a t th e re is no tu rn o v e r of c a rb o h y d ra te s d u rin g the
e a rlie st
involved
sta g e s
in
of
developm ent,
v ario u s
s y n th e tic
c a rb o h y d ra te s
re a c tio n s.
One
may be
way
to
s tu d y th is q u estio n involves the application of in h ib ito rs.
It has been found th a t sodium fluoride and iodoacetate,
in h ib ito rs
of
g ly co ly sis,
do
not
affect
e ith e r
oxygen
consum ption o r developm ent up to the late b lastu la stag e
(B a rth and B a rth , 1954; Salomon and B a rb ie ri, 1964).
Among th e glycolytic enzym es, hexokinase (P e tru c c i
and M iranda, 1972; Wesolowski and L y erla, 1979), p h o sphoglucom utase,
p h o sphoglycerom utase
and
enolase
(Salomon de Legnam e, Sanchez Riera and S ånchez, 1971)
and la ctate d eh y d ro g en a se (Adams and F in n eg an , 1965)
8
have been s tu d ie d . H exokinase is the only enzyme clai­
med to be u n d e te cta b le
u n til m etam orphosis. H ow ever,
th is is not in agreem ent w ith th e re s u lt of Salomon de
Legname e t al (1971), Thoman and G e rh a rt (1979) and
our
own
index"
(I),
since
th e
d eterm inations of th e
" sh u n t
show th a t even at th e e a rlie st sta g e s about 15
p e r cen t of th e glucose is m etabolized in th e glycolytic
p ath w ay ,
an d th e
sh a re in c rea ses co n tin u ally . We may
th e re fo re conclude th a t alth o u g h th e em bryo p o sse sse s
th e
enzym es
h a rd ly
re q u ire d
exploited
fo r g ly c o ly sis, th is
a t all b efo re
g a s tru la tio n ,
pathw ay is
and
early
developm ent is in d ep e n d e n t of g ly colysis.
The
reaso n
fo r th e low ac tiv ity
of th is
pathw ay
d u rin g e arly developm ent is not know n. H ow ever, it is
known th a t th e p h o sp h o ry latio n of fru c to s e -6 -p h o sp h a te
is th e most im p o rtan t co n tro l p o in t in g ly co ly sis. Phosp h o fru c to k in a se , th e enzyme cata ly sin g th is reaction step
is in h ib ited by high co n cen tratio n s of ATP (L e h n in g e r,
1975). The early am phibian em bryo contains high levels
of ATP (L p v tru p -R e in , Nelson and L o v tru p , 1974; Salo­
mon de Legnam e, F e rn a n d e z , Miceli, Mariano and L eg­
nam e, 1977) with an e n erg y c h arg e of about 0.95 (T h o ­
man and G e rh a rt,
14
CC>2 p ro d u ctio n
time
w hereas
possible
th e
mechanism
glycolysis ( I ) .
1979). As a p p ears from fig u re 1 the
.
14
from
[6-
C ]-g lu co se in c re a se s
ATP
level
to
explain
d e c re a se s,
th e
with
s u g g e stin g
a
low in itial ra te of
9
• ------ • C6-14C}- GLUCOSE
O A T P - LEVEL
- 150
C P M / EMBRYO
ng / EMBRYO
- 250
DEVELOPMENTAL STAGES
F ig. 1.
C hanges in th e glycolytic activ ity ( I) and the
level of ATP (L o v tru p -R ein
th e X e n o p u s em bryo.
e t a l .,
1974) in
10
P entose p h o sp h ate pathw ay
T he im portance of th e p en to se p h o sp h ate pathw ay
fo r
em bryonic
developm ent
was
f ir s t
d em o n strated by
L in d b erg and E rn s te r (1948) in the sea u rch in em bryo.
L a te r it has been found in many o th e r em bryos, among
which those of am phibian species (H erm ann and Tootle,
1964; B royles and S trittm a tte r, 1973).
C onfirm ing
p rev io u s
o b se rv atio n s
by
Salomon
de
Legname et al. (1971) we h av e e sta b lish e d th a t th e re is
a tu rn o v e r of glucose d u rin g developm ent b efo re g a s tr u ­
latio n , b u t th a t most of th e glucose p a sse s th ro u g h th e
p en to se p h o sp h a te pathw ay
(I).
Since th e re is no n e t
loss of re d u c in g c a rb o h y d ra te s it may be p resum ed th a t
th is pathw ay is en g ag ed in th e p ro d u ctio n of rib o se -5 p h o sp h ate fo r th e sy n th e sis of nucleic acid s.
11
AMINO ACID METABOLISM
The main su b sta n c e oxidized in th e early em bryo
m ust
be
looked
fo r
o u tsid e
th e
ran g e
of
tra d itio n al
e n e rg y so u rc e s. V arious k in d s of evidence s u g g e st th a t
th e amino acids glutam ic and a sp a rtic acid are involved
in th e oxidative metabolism . T h u s , it h as been o b se rv e d
re p e a te d ly th a t th e egg contains larg e am ounts of th ese
amino
acids
which
d ecrease
d u rin g
th e
early
p a r t of
developm ent (D e u ch a r, 1956; M etafora, 1967).
Isotope
exp erim en ts
show
th a t
glutam ate
is
the
p r e fe r r e d s u b s tr a te d u rin g early developm ent, th e oxi­
dation
of
glutam ate
b ein g
h ig h e st d u rin g g a stru la tio n
( I ) . F u rth e rm o re , u n til th e late g a s tru la ( I I ) , glutam ate,
in th e p re se n c e of m alate, is oxidized f a s te r th an any
o th e r
s u b s tr a te
by
iso lated
m itochondria.
Salomon
de
Legnam e, Sanchez R iera and S anchez (1975) have shown
th a t when hom ogenates of Bufo b lastu lae are in cu b ated
with rad io activ e glu tam ate, th e isotope is found in a s p a r­
ta te and in u rid in e m o n o -, di- and tri- p h o s p h a te s .
T hese o b se rv atio n s s u g g e st th a t glutam ate is tr a n s ­
form ed
(F ig .
de
to
a s p a rta te
in
th e
g lu ta m a te -a sp a rta te
cycle
2 ). T his cycle has an RQ value of 0.67 (Salomon
Legnam e,
1969),
actu ally o b se rv e d .
which co rre sp o n d s
well with th a t
12
PYRUVATE
/
/
!
I
7
OXALOACETATE
ASPARTATE
GLUTAMATE
MALATE
2-OXOGLUTARATE
FUMARATE
/
SUCCINATE
F ig. 2.
The g lu ta m a te -a sp a rta te cycle an d th e p ro d u c ­
tion of p y ru v a te .
13
PROPERTIES OF EMBRYONIC MITOCHONDRIA
O xygen consum ption
Since G odlew ski's o b serv atio n s
(1900) it is known
th a t th e ra te of re sp ira tio n in creases d u rin g th e co u rse
of am phibian developm ent. T his has been confirm ed la te r
innum erable
times
( e .g .
Legname
and B a rb ie ri,
1962;
Landström and L d v tru p , 1974).
The ratio betw een ATP and ADP + P. is known to
i
be e s se n tia l fo r th e reg u latio n of the r e s p ira to ry chain
(H olian, Owen and Wilson, 1977). As m entioned e a rlie r,
th e ATP level in X e n o p u s is high d u rin g cleavage stag e s
(L 0 v tru p -R e in e t a l ., 1974). By com paring th e shape of
th e ATP c u rv e w ith th a t e sta b lish ed by Landström and
L d v tru p
(1974)
fo r
th e
oxygen
u p ta k e
d u rin g
early
developm ent of X e n o p u s it is obvious th a t a re v e rs e d
relatio n
e x ists
betw een
ATP p e r em bryo (F ig .
e sta b lish ed
fo r
Bu fo
re sp ira tio n
3 ).
by
and th e
amount of
A sim ilar relation has been
Salomon
de
Legname
et
al.
(1977). T he p re g a s tru la sta g e s have th e low est r e s p ir a ­
tio n , in agreem ent with th e h igh level of ATP. When the
am ount of ATP d ec rease s th e re sp ira tio n in c re a se s .
S tu d ies of th e effects on developm ent of the r e s p ir a ­
to ry in h ib ito rs , cyanide and azide, have been conflicting
(B ra c h e t,
1945;
1934;
C raw ford
B a rn e s,
and
1944;
Wilde,
Spiegelman
1966;
Lamy
and Moog,
and
Melton,
1972). H ow ever, cyanide and azide do not seem to in h ib it
oxygen u p ta k e
in th e same way.
C yanide stro n g ly in-
14
ni
0 2 / EMBRYO
AND
HOUR
OXYGEN CONSUMPTION
O ATP-LEVEL
DEVELOPMENTAL
F ig. 3.
C hanges
in
and L p v tru p ,
oxygen
STAGES
consum ption
(L andström
1974) and ATP level (L p v tru p -
Rein e t a l ., 1974) in th e X enopus em bryo.
15
h ib its oxygen consum ption in in ta c t em bryos at all stag e s
of developm ent, w hereas th e in h ib ito ry effect of azide is
low
d u rin g
(B ra c h e t,
early
developm ent and in creases
g rad u ally
1934; Spiegelman and S tein b ach , 1945; C raw ­
fo rd and Wilde, 1966). T hese re s u lts may be explained
by th e se n sitiv ity of th e m itochondrial re s p ira to ry chain
tow ards
th e
in h ib ito rs .
C yanide
in h ib its
m itochondrial
oxygen u p tak e d u rin g th e e n tire em bryonic developm ent,
while th e se n sitiv ity tow ards azide in creases ( I I ) .
F u rth e rm o re ,
oxid ativ e
oxygen
th a n
d in itro p h enol (D N P), an u n co u p ler of
p h o sp h o ry la tio n ,
u p tak e
to
stim ulates
th e
in crease
a la rg e r e x te n t before g a stru la tio n
in la te r developm ental sta g e s (G re g g ,
nam e,
1968a;
in
Legnam e,
F ern an d ez
and
1960; Leg­
Miceli,
1971),
in d icatin g a " re s p ira to ry p otential" exceeding th e oxygen
u p ta k e
which
co n d itio n s.
is
ex h ib ited
Since
it
by
is known
em bryos
under
th a t DNP has
norma]
A T P :ase
a c tiv ity , and f u r th e r th a t th e ratio of ATP and ADP+Pj
e x e rts
a re g u la to ry function on re s p ira tio n ,
the effect
m entioned h e re may be ex p lain ed .
A num ber of o b serv atio n s indicate th a t m itochondria
in the early em bryo are m etabolically d iffe re n t from those
in la te r developm ent. O ur re s u lts co n cern in g the oxida­
tion of glutam ate (+ m alate) b ear out th is d ifferen ce. As
re g a rd s
shown
th e
th a t
c itric
a rse n ite
acid
cycle,
stim ulates
Legname
(1968b)
m itochondrial
has
oxygen
u p tak e up to th e b lastu la s ta g e , b u t in h ib its it d u rin g
la te r s ta g e s.
This double b eh av io u r could be a s c rib e d ,
on th e one h an d to its role as u n co u p ler and A T P :ase
16
Stim ulator e ffe c t, on th e o th e r h a n d , to its capacity to
in h ib it oxid ativ e d ecarboxylation (Salomon de Legname et
a l ., 1977).
D urin g early developm ent th e level of ATP is high
and th e ac tiv ity
of th e trica rb o x y lic acid cycle is low
(Thoman and G e rh a rt,
1979). T h u s , the main effect of
a rse n ite is p ro b ab ly re late d to th e A T P :ase effect which
lowers
th e ATP/ADP+P^ ratio which in
re sp ira tio n .
H ow ever,
tu rn stim ulates
th e la te r phase of developm ent is
c h a ra c te riz e d by relativ ely low ATP levels and an active
K rebs
cycle
(Salomon de Legname et a l .,
1977).
The
effect of a rse n ite may th en be re lated to its action on
oxid ativ e
d ecarb o x y latio n ,
which
is
e x p re ss e d
as
an
may
be
inhibition of re sp ira tio n .
M itochondrial enzym es
T he
change
in
m itochondrial
metabolism
looked fo r b o th a t th e re g u la to ry level and at th e level
of m itochondrial c h an g e s. V arious fin d in g s in d icate th a t
m itochondria in th e early em bryo are d iffe re n t from those
in th e la te r em bryo, q u a n tita tiv e ly as well as q u a lita tiv e ­
ly.
F or exam ple, q u a n tita tiv e an alyses of m itochondrial
fa tty acids rev eal th a t th e ir composition changes d u rin g
early
developm ent
(Bonini
de
Romanelli,
Alonso
and
B azån, 1981).
Wallace (1961) su g g e ste d th a t iso citrate is oxidized
by th e NADP+-d e p e n d e n t enzyme d u rin g early am phibian
developm ent and P e tru c c i, Amicarelli, Di Cola and Papo-
17
n e tti
(1975)
iso c itra te
r e s u lts
w ere
unable
d e h y d ro g en a se.
could
iso c itra te
not
be
to
In
d etect
NAD+-d e p e n d e n t
the p re s e n t stu d ie s th ese
c o rro b o ra te d ,
d e h y d ro g en ase is p r e s e n t,
NAD+-d e p e n d e n t
although th e a c ti­
v ity is q u ite low, th e low est among th e enzym es in v e s ti­
g ated ( I I I ) .
In
many
o th e r
re sp e c ts
early
m itochondria
are
indeed d iffe re n t (II; III and IV ). T h u s , malate d e h y d ro ­
g en ase an d a s p a rta te am in o tran sferase, enzym es involved
in
th e g lu ta m a te -a sp a rta te
cycle,
are both v e ry active
b efo re g a s tru la tio n , b u t d ecrease la te r on. The "ro ten o n e -in se n sitiv e "
a fte r
NADH
g a s tru la tio n ,
re d u c ta se
d e crease s
d rastically
and b egins to in crease only d u rin g
la rv a l developm ent. Monoamine oxidase is a b s e n t in early
m itochondria, and becomes d etectab le only at th e tailbud
sta g e .
Cytochrom e oxidase and succinate d eh y d ro g en ase
d ecrease s lig h tly , b u t s ig n ific a n tly , d u rin g developm ent.
M itochondrial c a rrie rs
A sim ilar p ic tu re o b tains with re sp e c t to the c a r ­
rie rs
(IV ).
Thus
th e
ac tiv ity of the malate c a r rie r is
h igh
in th e early em bryo, and d ecreases g rad u ally .
A
fum arate c a r r ie r , a b se n t in m itochondria from the em bryo
of la te r developm ental s ta g e s , and from th e ad u lt liv e r,
is p re s e n t in m itochondria isolated from th e early em­
b ry o . On th e c o n tra ry , the g lu tam ate/h y d ro x y l c a r rie r is
a b se n t
in
early
m ito ch o n dria,
and
becomes
d etectab le
only at th e tailb u d sta g e . The activ ity of the tric a rb o ­
xylic acid c a rrie r is relativ ely low in early m itochondria,
and in crea ses s u b s e q u e n tly .
18
M itochondrial m orphology
It
was
m entioned
above
th a t
cell
d ifferen tiatio n
begin s in th e g a s tru la and it is th u s a reasonable a s ­
sum ption th a t th e o b se rv e d m itochondrial d ifferen tiatio n
is c o rre la te d w ith cell d iffere n tiatio n .
It has been p o s­
sible to confirm th is co n jectu re as fa r as the m itochon­
d rial m orphology is co n cern ed . W orking with th re e d iffe­
ren t
cell
ty p e s ,
re p re s e n tin g
th e
cell
d ifferen tiatio n
p a tte r n s th a t a rise sp o n tan eo u sly in th e am phibian em­
b ry o ,
it was shown th a t in u n d iffe re n tia te d
m itochondria
condensed
a re
small,
conform ation.
almost
In
sp h erical
d iffe re n tia te d
and
cells
the
with
a
cells, r e p r e ­
s e n tin g R uffini cells and epiderm al cells, th e m itochond­
ria a re much la rg e r and elo n g ated , and with an orthodox
conform ation. T he m itochondria a re sig n ifican tly d iffe re n t
in th e two ty p e s of d iffe re n tia te d cells (V ).
Since it is know th a t m itochondria a re capable of
ch an g in g th e ir shape and size by m ovem ent, fusion and
fragm entation
th e se
o b se rv a tio n s
raise
th e
issu e
of
w h eth er tru e
d ifferen tiatio n o r tem porary m orphological
change is b ein g o b s e rv e d . In o u r case th e re is no r e a ­
son to dou b t th a t a real d ifferen tiatio n o c c u rs, since the
change in m orphology is in good co rrelatio n
with
the
of
ATP
functional ch a n g es.
C ondensed
m itochondria
and
high
levels
p rev ail in the p re g a s tru la em bryo (L d v tru p -R ein et a l .,
1974; Salomon de Legname e t a l ., 1977). It is tem pting
to s u g g e st th a t the high level of ATP is resp o n sib le for
19
th is conform ation. T his assum ption would receive some
s u p p o rt from Poliak (1975) an d S utton and Poliak (1980),
who
re p o rte d
a co n d en sed
conform ation
in
foetal
rat
liv e r m itochondria in co rrelatio n w ith a tra n s ie n t high
co n cen tratio n of ATP.
20
MITOCHONDRIAL DIFFERENTIATION
T he
re s u lts
m itochondria
d isc u sse d
u n d erg o
a
so
d istin c t
fa r
dem onstrate
d ifferen tiatio n
th a t
in
the
c o u rse of developm ent, and much of th e evidence s u g ­
g e s ts th a t it is p o ssible to d istin g u ish two p h ases with
re s p e c t to m itochondrial d iffe re n tia tio n .
T he f ir s t p h a se , d ep en d in g on an em bryonic ty p e of
m itochondria
o c c u rrin g
in
u n d iffe re n tia te d
cells,
is
c h a ra c te riz e d by a p a rtic u la r metabolism d ire c te d mainly
tow ards s y n th e tic a c tiv itie s. T he trica rb o x y lic acid cycle
is coupled to tra n s am inative reactio n s catalyzed by a s p a r ­
ta te tran sam in ase. T his aty p ical cy cle, w hich re p re s e n ts
th e
main
oxidative
pathw ay
d u rin g
early
developm ent
(I),
p ro d u ce s a s p a rta te while some of th e in term ed iates
of th e g lu ta m a te -a sp a rta te cycle such as m alate seem to
be
ch an n eled
tow ard
th e
s y n th e s is
(P e tru c c i, Amicarelli an d P a p o n etti,
of
p y ru v ic
acid
1977). Both a s p a r ­
ta te and p y ru v a te a re u se d as p re c u rs o rs in th e p a th ­
ways leading to th e form ation of p u rin e and pyrim idine
bases (F ig . 2; page 12).
The second p h a s e ,
o c c u rrin g
d u rin g
acquisition
of
g a s tru la tio n ,
an
involved
in
e n erg y
1969;
and
I I) .
I
"em bryonic"
in itia te d by m etabolic changes
"ad u lt"
ty p e
p ro d u ctio n
is c h a ra c te riz e d by the
of m itochondria,
(Salomon
As cell d ifferen tiatio n
de
more
Legnam e,
p ro ceed s
the
ty p e of m itochondria is g rad u ally replaced
by an "ad u lt" ty p e .
21
The ch an g es in m orphology and biochemical p ro p e r ­
ties of m itochondria d u rin g
early
developm ent may be
cau sed e ith e r b y ch an g es in p re e x istin g m itochondria in
th e u n d iffe re n tia te d cells o r by th e replacem ent of th is
population of m itochondria by a n o th e r one with d iffe re n t
p r o p e r tie s .
T hese
h y p o th e se s
a lte rn a tiv e s
put
r e p re s e n t
fo rw ard
to
actually
explain
th e
two
of the
mechanism
of
m itochondrial b iogenesis in all living c e lls . As y et we
have no indication of which ty p e of mechanism is in ­
volv ed .
Some inform ation, h ow ever, is w orth m entioning
w hich may fa v o u r th e second a lte rn a tiv e .
As we have see n , th e m itochondria of d iffe re n tia te d
cells are s u b sta n tia lly la rg e r th an those of u n d iffe re n ­
tia te d cells (V ),
so th e f ir s t a lte rn a tiv e would re q u ire
th a t m itochondrial p ro te in p e r em bryo in creases sig n ifi­
c a n tly . In fa c t, it d ecre ase s by th irty p e r c e n t from the
g a s tru la to th e tailb u d stag e ( I I ) . T h e re fo re , th e num ­
b e r of m itochondria o u g h t to d ec re a se ,
and if some of
th e early m itochondria a re bound to d e g e n e ra te , it seems
likely
th a t th ey
all do so,
being
rep laced by
a new
p o p u la tio n .
T he
c u rv e
re p re s e n tin g
the
changes
in
p ro tein
co n ten t p e r em bryo ( II) may be reso lv ed into two c u rv es
(F ig . 4 ), which make it p o ssible to d istin g u ish two mito­
ch o n d rial p o p u latio n s: one which begins to decline and
th e o th e r
to rise
aro u n d
population r e p re s e n ts
th e
g a s tru la tio n .
"em bryonic"
The d e c reasin g
ty p e ,
while the
(pg/EM BRYO)
22
O PROTEIN
O
>
mDNA SYNTHESIS
-30
&
5
UJ
QC
D
PROTEIN
01
-
20
aUi
co
c/>
MITOCHONDRIAL
UJ
X
>■
0)
0
10
20
30
40
DEVELOPMENTAL STAGES
F ig. 4.
C hanges in m itochondrial p ro te in co n ten t (II)
and mtDNA sy n th e sis (C hase and D avid, 1972)
d u rin g
th e
developm ent
of
X enopus.
The
d ash ed lines r e p re s e n t th e s u g g e ste d resolution
of th e p ro te in c u rv e .
23
in c re a sin g
population
r e p re s e n ts
th e
"adult"
ty p e
of
m ito ch o n d ria.
S tu d ies of cytochrom e oxidase activ ity in am phibian
nucleocytoplasm ic h y b rid s reveal th a t the activ ity of th is
enzyme fails to in crease d u rin g p o s t-n e u ru la r develop­
m ent as it does in co n tro l em bryos (L iepins and H ennen,
1977). T his re s u lt s u g g e sts th a t th e m aternally in h e rite d
m itochondria a re able to m aintain a functional population
of m itochondria up to n e u ru la tio n .
T his population may
co n stitu te th e "em bryonic" m itochondria.
M itochondrial DNA is sy n th e siz e d in co rrelatio n with
th e
in c re a sin g
"ad u lt"
population
(C hase
and
Dawid,
1972). T he s y n th e tic ac tiv ity is too low to explain th e
in c re a se in th e "ad u lt" p o p u lation, b u t th e m ethod yields
a minimum estim ate of th e s y n th e tic r a te . R eplication of
m itochondrial DNA is known to take place d u rin g early
developm ent of sea u rc h in (B re sc h , 1973), as well as of
fish em bryos (M ikhailov and G ause, 1974), with a doub­
ling time of seven h o u rs , although the m itochondrial DNA
co n ten t rem ains c o n sta n t.
The
two m itochondrial
populations
have
d iffe re n t
p ro p e rtie s . T he sw elling in am monium-fum arate (IV ) is a
good m ark er fo r th e "em bryonic" pop u latio n , while mono­
amine
oxidase
ac tiv ity
(III)
and glutam ate/O H
c a r rie r
activ ity (IV ) may be u sed fo r th e "adult" ty p e of mito­
ch o n d ria.
24
Replacem ent
of
m itochondria
o ccu rs
concom itantly
with th e p ro c ess of cellu lar d iffe ren tiatio n and may th u s
be causally re la te d to it. T his assum ption receiv es some
s u p p o rt
from
L andström ,
L d v tru p -R ein
and
L dv tru p
(1976), who re p o rte d th a t an in h ib ito r of m itochondrial
p ro te in s y n th e s is , chloram phenicol, may affect th e d iffe­
re n tia tio n
of R uffini cells,
an d from P ritc h a rd (1981),
who re p o rte d th a t d istu rb a n c e s of m itochondrial m etabo­
lism
may
affect
th e
d iffere n tiatio n of chicken
n e u ra l re tin a into pigm ent epithelium .
em bryo
25
REFERENCES
Adams, E. and C .V . F innegan (1965). An in v estig atio n
of la ctate d eh y d ro g en a se activ ity in e arly am phibian
developm ent. J . E x p tl. Z ool., 158, 241-251.
B ac h v aro v a, R ., E .H . D avidson, V .G . A llfrey and A .E .
M irsky
(1966).
A ctivation of RNA sy n th e sis asso ­
ciated w ith g a s tru la tio n .
P roc.
N atl.
Acad.
S c i.,
55, 358-365.
B a rb ie ri,
F .D .
an d
m ental stu d ie s
H.
Salomon (1963).
Some e x p e ri­
on glycogen and lactic acid in th e
developing eg g s of Bufo a r e n a r u m .
Acta Em bryol.
M orphol. E x p e r ., 6, 304-310.
B a rn e s , M .R.
Ran a
(1944).
pipiens
The metabolism of th e developing
as rev ealed by specific in h ib ito rs . J .
E x p tl. Z ool., 95, 399-417.
B a rth , L .G .
an d L .J .
B arth
(1954). T he e n e rg e tic s of
developm ent. U niv. P re ss .
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(1981).
P h o sp h atid ic
acid,
p h o sp h atid y lin o sito l,
p h o sp h a tid y lse rin e and cardiolipin in the co u rse of
early em bryonic developm ent. F atty acid composition
and
co n ten t in whole toad em bryos and in mito­
ch o n d rial fra c tio n s .
561-571.
Biochim.
B iophys.
A cta, 664,
26
B rä c h e t, J .
(1934). É tude du métabolisme de l'oeuf de
gren o u ille (R ana fu sc a ) au co u rs du développem ent.
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34
ACKNOWLEDGEMENTS
I wish to e x p re ss my sin ce re g ra titu d e to:
Dr
H u g u ette
L 0 v tru p -R ein
fo r
her
s u p p o rt
and
stim u latin g criticism th ro u g h o u t th e w ork.
P ro fe sso r S0ren L 0 v tru p fo r valuable s u p p o rt and
en co u rag em en t.
Mr L a rs -E rik Sandström fo r tech n ical a ssis ta n c e .
Mrs Mabel Jo n sso n fo r skilled ty p in g of th e m anu­
s c rip t .
My o th e r frie n d s and colleagues at- th e D epartm ent
of Zoophysiology fo r th e ir cooperation
sh ip .
and com panion­