MICROTUBULE PARTICIPATION IN THE MECHANISM OF
CORTICAL ALVEOLI EXOCYTOSIS IN FERTILIZED EGGS OF
CTENOPHARYNGODON IDELLA.
LOTUS MESTER, DRAG09 SCRIPCARIU, FLORENTINA
MARINESCU, RODICA NICHITEANU, RADU MESTER
L'btude cinbtique de l'bliberatiou des vacuoles corticales des oocyter de poisson a d6montrb que le processus d'exocytose se dbroule pendant 10 min aprbs la fbcondation. L'Ctude
ultrastructurale montre la prbsence des microtubules autour du pore d'exocytose. Nos rbsultata
sugghrent la participation des microtubules dans le processus de l'exocytose des vacuoles
corticales. .
INTRODUCTION
Cortical alveoli are specialized membrane-limited secretory granules
located under the oocyte plasma membrane of many invertebrates and vertebrates. The disappearance of the cortical alveoli has been considered a
consequence of fertilization (D e t t 1 a f f, 1962 ; A u s t i n, 1968 ; A n d e rs o n , 1974; K u d o , 1976; E p e l , 1975; S c h u e l , 1978). The activation of eggs is characterized by a wave of cortical alveoli breakdown,. starting
from the sperm penetration area onto the vegetative pole of the cell. The
most recent studies have tried to establish their origin, structure, mechanism
of breakdown and relationship with oocyte activation (D e t e r i n g et al.
1977; I w a m a t s u and K e i n o , 1978; E p e l , 1978; S c h u e l ,
1978; B r u m m e t t and D u m o n t , 1981.).
Although the process of exocytosis has been studied on a wide range
of cells, the exocytosis mechanism of the cortical alveoli has not yet been
understood. Exocvtosis i m ~ l i e sthe fusion of secretory vesicle with the cell
membrane and a temporary formation of an exocytic canal, which allows
the discharge of the secretory contents ( D e t e r i n g et al. 1977 ; E p e 1,
1978; B r u m m e t t and D u m o n t, 1981). The cell disposes several
mechanisms to specify exocytotic membrane interactions. Some of them
were reported for somatic cell exocytosis ( P a 1 a d e and B r u n s, 1968;
P a l a d e , 1975; M e l o d e s i and al., 1978; O r c i and P e r r e l e t ,
1978; P l a t t n e r, 1981). With fish oocytes, the exocytosis mechanism
238
LOTUS MESTER. A N D COLL.
of the cortical alveoli is little known. However, observations on sea urchin
eggs have shown that in the exocytosis mechanism of the corticalalveoli
several factors do participate: the release of the calcium ions from the intracellular binding site ( E p e 1, 1975 ; V a c q u i e r, 1975; G i 1 k e y et al.,
1978 ; S c h u e 1, 1978), and that of the enzymatic products released during
fertilization ( F o d o r et al., 1975 ; S c h u el, et al., 1976). Electron microsc o ~ i cstudies show that the membrane of the cortical alveoli fuses with the
plasma membrane during fertilization, and the resulting mosaic membrane
may participate in some of the characteristic permeability changes occurring
afterwards (A n d e r s o n, 1974; E p e 1, 1975).
This 1DaDer
tries to reaveal the release of the cortical alveoli in fertilized
I
fish eggs using light and transmission electron microscopy. Electron microscopic studies show that microtubules participate in exocytosis and membrane
fusion, and they polymerise during cortical reaction at fertilization.
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M A TERIALS A N D METHODS
Animals. Mature fish (Ctenophyryngodon idella) were obtained from
the Nucet Piscicultural Station.
For the fertilization experiment, the sperm suspension was added a t
room temperature to the mature eggs directly into the dry dishes. Before
and a t preset intervals of 1-20 min after fertilization, the eggs were fixed
with 2.5% glutaraldehyde prepared in 50 mM cacodylate buffer, pH 7.4, containing 6% sucrose, for 60 min a t 4OC. Then, the eggs were washed extensively with 50 mM cacodylate buffer, pH 7.4, and were h e d in a solution of
1%alcian blue prepared in acetic acid 1 N, for 2h, according t o 'R o t h m a n
(1969). The eggs were washed with cacodylate buffer and postfixed in 1%
osmium tetroxide, prepared in 50 mM cacodylate buffer, p H 7.4, containing
0.5% ruthenium red, for 6 h at cold. The samples were washed several times
with 50 m M cacodylate buffer, p H 7.4, and then dehydrated in alcohol,
propylene oxide and embedded in Epon 812. Ultrathin sections stained with
uranyl acetate and lead nitrate were examined with a Philips 201 electron
microsco~e.
FO;
histological observations, the eggs prepared for the 'electron microscone were embedded in varaffin and the thin sections were examined a t a
lig& microscope. The coAical alveoli of the unfertilized eggs and of eggs a t
predetermined intervals of 1-20 min after fertilization were also examined
histochemically. The eggs were fixed in 1.5% paraformaldehyde with 6%
sucrose and 3 mM calcium nitrate, and were processed for light microscopy.
The paraffin sections were double-stained with 1% alcian blue and Schiff
reagent according to G a n t e r and J o 1 1 e s (1969).
RESULTS
Light microscope study. The histological observations of the unfertilized eggs fixed and processed for electron microscopy reveal the presence
of several layers of cortical alveoli located in the peripheral cytoplasm of
the egg, just under the plasma membrane (Plate 1, A). The same picture
MICROTUBULE PARTICIPATION I N CORTICAL ALVEOLI
239
was obtained after the histochemical analysis of the unfertilized eggs, doublestained with alcian blue and PAS. Cortical alveoli appear as heterogeneous
intracellular structures of various size. Their basic structure consists of a
dense core and halo, containing a homogeneous material (Plate A, 1).
Kinetic analysis of the cortical alveoli breakdown was followed on
eggs fixed and double-stained at preset intervals of 1-20 min after fertilization. Histochemical observations demonstrated that the process of exocytosis of the cortical alveoli in this species of fish begins 3 min after fertilization. Figure 1, B shows the presence of some cortical alveoli discharged
into the perivitelline space, 3 min after insemination. Subsequently, 10 min
after fertilization, the cortical alveoli are completely eliminated from the
egg cytoplasm. The contents of several cortical alveoli discharged in the
perivitelline space is observed in Fig. 1, C and Fig. 2, A (see arrows). Light
microscopy observations concerning the release mechanism dynamics of the
cortical alveoli after fertilization show that the interval from the initial
breakdown to complete disappearance of these structures from the cell spanned a-oroximatelv 10 min.
Electron microscopic study. Ultrastructural observations of the unfertilized egg cortical alveoli reveal the same variety of contents a8 proved by
the histological study. Each cortical alveolus has a central spherical structure
or core, with a heterogeneous electron density seen at an ultrastructural level
(Fig. 2, B). In this fish species, the central granule exhibits a characteristic
internal fine structure, .probably in correlation with the amount and type
of the neutral - and acid muco~olvsaccharides.Between this central manule
and the cortical alveoli membrane there is a granular material, also rich in
mucopolysaccharides, as revealed by histochemical staining.
Within about 2-3 min after insemination, the cortical alveoli begin
to be discharged from the eggs. The activated eggs .show certain structural
modifications at the periphery of the cortical alveoli and plasma membrane.
In the oocyte plasma membrane appear pits, exhibiting the exocytotic canal,
by which secretory granules attach to the cell surface (Fig. 3, A, see arrow).
Ultrastructural observations indicate the presence of microtubules directly
associated with the sites where cortical alveoli are attached onto the cell
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surface of the activated fish oocyte. Microtubules are found in the cytoplasmic
matrix or in the less electron-dense regions in the cortical alveoli, surrounding
the exocytotic pore in formation. At the central point of fusion and in the
adjacent sites, a great number of microtubules can be seen (Fig. 3, A, and
Fig. 4, A). The presence and disposition of the microtubule aggregated fibres
appear very clearly at higher magnification (Fig. 3, B and Fig. 4, B). The
microtubules appear as elongated, unbranched, cylindrical elements of 20-32
nm diameter. On the cytoplasmic face of the plasma membrane, and especially in the adjacent zone of the exocytotic canal there are a mass of electrondense material or connecting material (Fig. 3, B and Fig. 4,. B,
arrow head).
10 min after fertilization. the membrane of the r u ~ t u r e dcortical
alveoli becomes integrated in thk original egg membrane. ~ C t h i stage,
s
the
egg surface is covered with numerous cytoplasmic projections and the cell
is devoid of cortical alveoli.
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240
LOTUS ME$TER A N D
COLL.
DISCUSSION
The kinetics of release of the cortical alveoli contents was monitored
by light microscopy with double-stained sections and by transmission electron microscopy. Our histochemical and electron-microscopic observations
concerning the rate of cortical alveoli breakdown following sperm contact
with egg, showed that the interval from the initial breakdown to the complete
disappearance of these structures from the egg spanned approximately 10
min. The discharged contents of the cortical alveoli diffused into the perivitelline mace from the ene surface. The rate of cortical alveoli breakdown
in the eggs of fish species exhibits some variation. I w a m a t s u and K e i n o
(1978), studying the rate of cortical alveoli release after fertilization of Oryzias latipes eggs, established that the process began after 45 sec and was
completed in 5 min. At Fundulus heteroclitus activated eggs, the interval
from the initial breakdown to the complete disappearance of the cortical
alveoli spanned approximately 40-90 sec (B r u m m e t t and D u m o n t,
1981). However, the exocytosis of the cortical alveoli in Clupea palasii
eggs was slower - between 2 min and 30 min (Y a n a g i m a c h i, 1957).
*The exocvtosis of cortical alveoli beeins
a t the site of sDerm ~enetration
"
and spreads over the entire surface of the egg. It is a functional process,
controlled bv
, manv intracellular factors. For somatic .cells. exocvtosis covers
a wide range of different biochemical, biophysical and ultrastructural features
( P a l a d e , 1975; P l a t t n e r , 1981).
Our ultrastructural observations on the cortical alveoli exocytosis of
fish eggs demonstrated the interaction of these structures with plasma membrane and the formation of an aperture a t this point. The egg cell has several
'mechanisms to specify the exocytotic membrane interaction. I n our case,
one of the mechanisms is the formation of the microtubule structures. consideredss a coupling signal between the cortical alveoli and the cell membrane.
There is probably some relationship between the change of the intracellular
pH, the plasma membrane permeability and the calcium im concentration
occurring upon activation, and the changes in the organization of the cortical
cytoplasm, which allow the aggregation of tubulin into microtubules. The
microtubules are never seen in the cortical cytoplasm or in the peripheral
zone of the unfertilized eggs. Our data indicate that the functional .coupling
of the cortical alveoli to the plasma membrane is established after egg insemination.
The mechanism of cortical alveoli exocytosis in fish eggs is not well
understood. However, several information on invertebrate eggs do exist.
In unfertilized eggs of the sea urchin, actin and tubulin are present in abundance
in an unpolymerized form ( H a r i s , 1979; C o f f e et al., 1982). A spiral
cortical fibre system consisting of microtubules is formed in sea urchin eggs
about 10 .min after fertilization or after the parthenogenetic activation.
Parthenogenetic activation induces fluctuations in the cohesiveness of the
sea urchin egg ,cytoplasm, as they display a different organization of the
microtubule system. Thus, unfertilized oocyte tubulin may be incompetent
to polymerize until fertilization or activation, a t which time the tubulin
becomes modified to a polymerization competent form.
(7"
Fig. 1. A) Photo~r~icrograph
of cortical alveoli in the mature egg of fish. Note the presence of
several layers of cortical alveoli a t the periphery of the cell. x 10/10.B) View of an egg 3 mi11
after fertilization. S o t e the presence of some cortical alveoli in the perivitelline space (arrow)
x 10/10. C) View of an egg 10 min after fertilization. This figure shows the disappearance of
cortical alveoli from the cytoplasm of the oocyte. Some cortical alveoli appear in the perivitelline space (arrows) :., 10/36.
- 0 9 9 ~x sauoz 1at1!1s!p OM^ aqi 30 ~ ~ I ~ ~ I ~~ernaanrasna~[n
JIIRJJE
aqa % u ! ~ o q s'B%a paz!l!aaajun u n j o snloaala 1ca!iro3 n +noaqa tlc,!iaas u!q~, (a .gb9/sx (SMOJJV)
aaads eu!l[aa!a!rad
aqa u! naas ara !Ioaaln Ina!laoa Iaraaas .[la3 aql jo msu[doa6a aqa w o r j
sarnaanrls asaqa jo aauasqn aqi aaoN yoaalu [aa!iroa aqi 30 UO!~C~IJ!III~P! aql aoj s v d - a n l q ue!ale
qa!n\ uo!aaro[oa u!aas-alqnoa .uopsz!l!aiaj aaijs u!~u 02 B%a un jo sna!a uopaas s!q& ( v .Z 931s
Fig. 3. A) Electron micrograph of a cortica! alveolus 3 mi11 after fcrtilizntion. 11 dense array of
microtubules (arrows) is concentrated in the vicinity of the esocytotic pore (large arro\v). 8 o t e
the plasma membrane invagination on the surface of the cell. x 18.400. B) Higher magnifirnti~n
of the figure 3 A, revealing the disposition of microtubules, the esocytotic pore (large itcrow)
ant1 the presence of a n electron-dense material (arrows). x 110.000.
Fig. 4. .I) Electron-micrograph of a cortical nlveolus 5 tninafter fertilization. Two pits in the
plrcsrna membrane are seen in opposition with the cortical alveoli. Nurnerous micrntl~bules
are seen in the vicinity of the pores (small arrows). x 14.400. B) Higher magnification of the
figure 4 A, showing the dispo itiorl of the microtubllles in the vicinity of the pore and the pre110.000.
sence of an electron-dense material (arrows).
MICROTUBULE PARTICIPATION I N CORTICAL ALVEOLI
2 41
S u p r e n a n t and R e b h u n (1984) studying oocyte cytoplasmic
tubulin of surf clam presented evidence that oocyte ,tubulin is maintained
in the unassembled state bv a combination of intracellular com~artimentalization and specific inhibiiors of microtubule assembly. Fertilization may
trigger t h e release or synthesis of microtubule-associated protein or microtubule organizing centers necessary for microtubule assembly (B r y a n et
al., 1975; N a r u s e and S a k a i , 1977; W e i s e n b e r g and R o s e n f e 1 d, 1975). Thus microtubule polymerization is controlled b y many intracellular factors.
The formation of microtubule in the activated fish egg seems t o depend
on several factors, which are triggered a t fertilization. We conclude that the
presence of microtubules near the contact zones of t h e membrane fusion and
exocytotic pores is a main event occurring after egg activation and a n intracellular factor t h a t controls the process of the cortical alveoli exocytosis.
The relationship between cortical alveoli breakdown and tubulin polymerization deserves further study.
PARTICIPAREA MICROTUBULILOR I N MECANISMUL D E EXOCITOZA AL VACUOLELOR CORTICALE A OVOCITELOR FECUNDATE
D E CTENOPHARYNGODON IDELLA.
REZUMAT
f n ovocitele mature de Ctenopharyngodon idella s-au identificat mai
multe stratmi de vacuole corticale. Fiecare vacuolg cortical5 prezintg un
granul central cu o structurg heterogeng, mgrginit de un material granular.
Analiza cinetic5 a elibergrii vacuolelor corticale a reliefat faptul cg procesul
de exocitozg incepe la aproximativ 3 min dupg fecundare gi se terming in
urmgtoarele 10 min.
Studiul electrono-miscroscopic a precizat prezenta a numerogi microtubuli in jurul porului de exocitozg. S-a apreciat cg polimerizarea tubulinei
in microtubuli are loc rapid dupg fecundarc gi este asociatg cu participarea
acestor straturi in procesul de exocitozg a1 vacuolelor corticale din ovocitele
de pe!te.
REFERENCES
ANDERSON (E.), 1974 -Comparative aspects of the ultrastructure of the female gamete.
Int. Rev. Cytol., 4: 1-70.
AUSTIN (C.), 1968 - Ultrastructure of fertilization New York.
BRUMMETT (A. P.), DUMONT (J. N.), 1981 -Cortical vesicle breakdown in fertilized eggs
of Fundulus heteroclitus. J. Exp. Zool., 2161 63-79.
BRYAN (J.), NAGLE (B. W.), DOENGES (K. H.), 1975 - Inhibiton of tubulin assembly
by RNA and other polyanions: evidence for a required protein. Proc. Nat. Acad.
Sci., 72: 3570-3574.
COFFE (G.),ROLA (F. H.), SOYER (M. O.), PUDLES (J.), 1982 -Parthenogenetic activation of the sea urchin eggs induced a cyclical variation of the cytoplasmic resistence to hexylene glycol-Triton X-100 treatment. Exp. Cell Res., 137: 63-72.
242
LOTUS YESTER, AND COLL.
DETERING (N. K), DECKER (G. L.), SCHMELL (E. D.), LENNARZ (J.), 1977- Isolation and characterization of plasma membrane associated cortical granules from
sea urchin eggs. J . Cell Biol., 75: 899-914.
DETTLAFF (T.A.), 1962 Cortical changes in acipenserid eggs during fertilization and artificial activation. J. Embryol. E m . Morphol., 10: 1-26.
EPEL (D.), 1975 The program and- mechakisms of fertilization in the echinoderm egg.
Amm. Zool., 15: 507-522.
EPEL (D.).,. 1978 -Mechanisms of activation of sDerm and eee durine fertilization of sea
urchin gametes. Cur. Top. Devel. ~ i d l . 1
, 21 18&>46.
FODOR (E. J.), AKO (H.), WALSH (K. A.), 1975 Isolation of a proteaae from sea urchin
eggs before and after fertilization. Biochem., 14: 49234927.
GANTER (P). JOLLES (G.), 1969 - Histochimie normale et pathologique. 1, 2, Paris.
GILKEY (J. C.), JAFFE (L. F.), RIDGWAY (E. B.), REYNOLDS (G. T.). 1978-A free
calcium wave traverses the activating egg of the.Medaka, Oryzias latipes. J. Cell
Bwl., 76: 448-466.
HARRIS (P.) 1979 -A spiral cortical fiber system in fertilized sea urchin eggs. Deuelop. Biol.,
68: 5254532.
KUDO (S.), 1976 -Ultrastructural observations on the dischargd of two kinds of grandea
in the fertilized eggs of Cyprinus carpio and Carassius auratus. Dev. Growth Diff.,
18: 167-176.
IWAMATSU (T.), KEINO (H.), 1978 - Scanning electron microscopic study on the surface
change of eggs of the teleost, Oryzias latipes a t the time of fertilization. Dev.
Growth Diff.., 20: 237-250.
MELODESI (J.), BORGESE (N.), DE CAMILLI (P.) CECCARELLI (B.), 1978 - Cytoplasmic membranes and the secretary process. I n : Membrane Fusion, Poste
G., Nicolson G. L. (eds.), Amsterdam; 509-627.
NARUSE (H.), SAKAI (H.), 1977 -Microtubule assembly inhibitory factor from sea urchin
egg cortex. J Cell Biol., 64: 242-245.
ORCI (L.), PERRELET (A.), 1978-Utrastructural aspects of exocytotic membrane fusion.
I n : Membrane Fusion, Poste G., Nicolson G. L. (eds), Amsterdam: 629-656.
PALADE (G. E.), 1975 - Intracellular aspects of the process of protein synthesis. Science
(Wash.), 189I 347-358.
PALADE (G.E.), BRUNS (R. R.), 1968 - Structural modulation of plasmalemmal vesicles.
J. Cell Biol. 37: 633-649.
PLATTNER (H.),
1981 -Membrane behaviour during exocytosis. In :Cell Biology Int. Rep.,
5: 435-459.
ROTHMAN (A. H.), 1969 - Alcian blue as an electron stain. Exp. Cell Res., 581177-179.
SCHUEL (H.), TROLL (W.), LORAND (L.), 1976 -Physiological responses of sea urchin
eggs to stimulation by calcium ionophore A-23187 analyzed with protease inhibitors. Exp. Cell Res., 103: 4 4 2 4 4 7 .
SCHUEL (H.), 1978 - Secretory functions of egg cortical granules in fertilization and development. A critical review. Gamete Res., 1: 299-382.
SUPRENANT (K. A.). REBHUN (L. I.), 1984 - Purification and characterization of oocvte
cytoplasmic tubulin and m&otic spindle tubulin of the surf clam Spisula solidissima. J. Cell Biol., 98: 253--266.
VACQUIER (V. D.), 1975 -The isolation o f intact cortical granules from sea urchin eggs:
calcium ions trigger granule discharge. Devel. Biol., 43: 62-74.
WEISENBERG (R. C.), ROSENFELD (C. A.). 1975 In vitro ~olvmerizationof microtnbdes in& asters and spindies in'h~mo~enates
of suri clam eggs. J. Cell Biol.,
64: 1 4 6 1 5 8 .
YANAGIMACHI (R.), 1957 - Studies of fertilization in Clupea palasii. 111. Manner of sperm
entrance into the egg. Zool. Mag. (Tokyo), 66: 226-233.
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Facultarea de Biologie
Splaiul Independenjei 91-95
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