ULTRASTRUCTURAL PECULIARITIES OF THE CORTICAL
ALVEOLI FROM THE OOCYTES OF SOME FISH SPECIES
LOTUS MESTER, DRAG05 SCRIPCARIU, AURELIA SCARLAT, RADU MEVTER
Let auteurs ont btudib In structure Blectrono-microeropique des vacuoles corticales des oocyLcs chrz trois espbces de poiseons (Esox lucius, Carossius auratus gibelio ct
Perca jluviatilis). E n utilisant trois techniques diffcrentes d'histochimie on a constat6
qur les vacuoles corticales des poissons ont une structure interne hbtbrogibue, due ii la
prbsencc d'un melange des mucopolysaccharides. D'autre part, on remarque la prbsencr
dans la cytoplasmr corticale des oocyteq des g r a ~ ~ u l rcorticales.
s
Ce cont des formations
sphbriques, d'une taille comprise entre 0,s c t 3 microns et q l ~ iprBsentent u n materiel
interne dense e t opaque aux Blectrons.
The cortical alveoli ftom the peripheric cytoplasm of the oocytes are
specialized membrane-limited secretory organelles, which play an important
role in the morpho-functional processes produced after the fertilization.
The whole modifications are generally named "cortical reaction" and represent a preliminary condition for the normal development of the activated
4
oocyte.
The study of the cortical alveoli was the aim of many investigations,
made on invertebrates and vertebrates (E n d o, 1961 ; P a s t e e 1 s, 1965 ;
K e m p & I s t o c k , 1967; A u s t i n , 1968; A n d e r s o n , 1974;
D e t e r i n g et al., 1977 ; S c h u e 1 1978). The structure and topographical
distribution of cortical alveoli were studied in some fish species too, both a t
fotonic and electronic microscope (K u s a, 1956 ; Y a m a m o t o, 1961,
1962; K u d o, 1968, 1976 a, h).
Taking into consideration the importance of cortical alveoli in t h e
fertilization process of fishes and the dispute concerning the presence of
several types of cortical alveoli in teleostean oocytes, we followed a t the
ultrastructural level, using more cytological convergent techniques, the
cytological peculiarities and the distribution of cortical alveoli in some fish
species.
234
LOTUS MESTER. DllAGOS SCRIPAHIU, A U R E L l A SCAItLAT, RADU MESTER
M A T E R I A L A N D METHODS
The experiments were carried out on three fish species, collected from
the Danube Delta, namcly: perch (Percafluoiatilis), prussian carp (Carassius
auratus gibelio) and pike (Esox lucius).
The cortical alveoli were examincd using the following cytochcmical
techniaues :
1. Alcian hluc staining of the mucopolysaccharides, after Rothman
(1969). The oocytes were fixed in glutaradehyde 3 % which contained 1,5%
paraformaldehyde, 6,5% sucrose and 3 mM calcium nitratc, for one hour
ancl a half. After being washed for 2 hours with cacodylate h u f f ~ r50 mM,
p l l 7,4, containing sucrose, the fixed material was stained with a 1 % solution
of alcian b111c in acetic acid 1 N for another 2 holirs. Afterwards, t h e pieces
were washed with acetic acid solution 1% with 6,5% glucose, rewashed with
cacodylate-sucrose buffer and postfixed for 12 hours in 1 % OsO, buffered
washed with a solution of sucrosewith cac~clvlate.The material was again
"
cacodylatc buffer, dehyclrated in ct'hanol an propylene oxide ancl cmbeddecl
in E ~ o n .
2. Staining technique of mucopolysaccharides with methenamine silver,
after the method described b y D e M a r t i n o and Z a m 1, o n i [1967].
The pieccs were fixed and rinsed as in the previously mentioned technique.
Thrn. the matrrial was transferred in a solution of ~ e r i o d i cacid 1 % for 30
minutes and washed with threc or four changes with cocodylate buffer 50 mM,
p H 7,4. The staining was done with methenaminc silver for 2 hours, in darkness. -After heing washecl with cacodylate buffer, the material was postfixed for
2 hours in OsO, solution 1% )>uffered with cacodylate, p H 7,4. Then the pieces
werc processed for the electron microscopy and embedded in Epon.
3. Technique for the demonstration of glycoproteins with pyroantimonium, after the method described b y K a 1 t and T a u d 1 e r (1971). The
oocytes were fixed in a mixture containing glutaraldehyde 3%, paraformaldehyde 1,5% sucrose 6,5%, and pyroantimonium 1%. After fixation (6 hours)
the material was washed with cacodylatc buffer 50 mM, pH 7,4 containing
sucrose 6,5% and postfixed for 2 hours in OsO, solution 1% buffered with
cacodylate. Afterwards, the pieces were rinsed several times in the same
bilffrr solution, dehydrated and embedded in Epon.
R E S U L T S AIVD D I S C U S S I O N
1. Ultrastructural peculiarities of the cortical alveoli from the pike's
oocytes. The cortical alveoli from the pike's oocytes appear as very large
cytoplasmic structures, with a diameter t h a t varies between 8 a n 35 y, located
a t the periphery of the egg, immediately under the radiate membrane (Plate 1,
A and R). Indifferent of the staining technique used, their inner structure
appears very heterogeneus. We can consider t h a t there is an ultrastructural
polymorphism of the cortical alveoli content, consisting of electron-densc
zones, with a heterogeneous distribution and of zones with variahle densities.
The comparative studies with many staining methods, specific t o different
categories of mucopolysaccharides did not permit t o establish some ultra-
ULTRASTRUCTURE O F CORTICAL ALVEOLI FROM OOCYTES OF FISHES
235
structural qualitative differences. The use of these techniques permits however
the identification of a c o m ~ l e xorganization
of cortical alveoli in fishes.
"
Previous investigations on cortical alveoli ultrastructure in fishes (I w am a t s u & 0 h t a. 1976. K u d o. 1976 a).
,, carried out i n standard technical conditions could not' indentifi the complex nature of glycoproteic
material from the cortical alveoli.
In the cortical cytoplasm of oocytes, in the vicinity or close t o cortical
alveoli, were identified smaller vacuolar structures, granules (Plate 1, A, B,
C, arrows). These structures have a much smaller diameter, between 0,5-3 p
and differ deeply as ultrastructure in comparison with t h e typical cortical
alveoli. Cartical granules show a homogeneous or finely granular inner structure. On the basis of their staining affinity, we have identified in pike's
oocytes the presence of two types of cortical granules: some of them electron-dense (Plate 1, A and C. sce arrows) and others with a medium density
structure (Plate 1, C long arrow and D short a ~ r o w ) .They appear to be
morphologica1l;v idez:tical, with a round or oval shape and limited by a
membrane.
K u d o (1971, 1976 a) suggested the presence, in the cortical cytoplasm of fish oocytcs, of some structures differing from cortical alveoli. The
author mentioned in gold fish and carp's eggs the presence of three types of
cortical structures, named cortical alveoli, cortical granules A and cortical
granules B. The distinctive criterion hetween cortical structures was their
size and appears t o he relative enough. I n Oryzias latipes were identified
LLgranules a" too, disposed around the cortical alveoli (Y a m a m o t o,
1961). The nature of glycoprotcic material and the functional significance
of cortical granules in the fertilization mechanism and cytodifferentiation
process are little known. After the observations of K u d o (1976 b), the
cortical granules open in the privitelline space before the cortical alveoli.
At t h e same time, it was suggested that they are elaborated b y Golgi apparatus and contain acid mucopolysaccharides. Except this observation, there
isn't any indication on the chemical composition of the cortical granules.
Some of them could be pigmentary cortical granules.
2. The ultrastructure o f cortical nl~eoliin ~ r u s s i a ncarw oocrtes. I n t h e
oocytes cytoplasm of prussian carp, in the vicinity of plasmalemma, were
identified cortical alveoli with a verv heterogeneous inner structure. The
cortical alveoli appear as big organelies, with a diameter between 6 and
25 p. A part of cortical alveoli were identified in a deep layer too, among
yolk platelets (Plate 11, A). The inner structure of the cortical alveoli in
prussian carp is characterized 1)y n great polymorphism as in pike's oocytes.
One can observe electron-dense zones with an irregular disposal and zones
with variable electronic densities, that occupy the largest part of t h e alveoli's
surface. Inde~endentlvof the nature of dve utilized for the identification
of mucopolysaccharides, the inner content of cortical alveoli appears t o be
heterogeneous, suggesting the existence of different glycoproteins which
are in a variable proportion in every structure. Previous ultrastructural
data made evident in t h e oocytes of old fish-cortical alveoli with a homogeneous and dense inner structure, limited by an outer translucent material
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0
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236
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LOTUS MESTER, DRAGOS .SCRIPCARIU. AURELIA SCARLAT. RADU MESTER
(K u d o , 1976 a). It must be specified that the author employed standard
fixations, without supplementary
staining
- - for glycoproteins.
- .
Beside typical cortical alveoli, in the cortical cytoplasm of the oocytes
of prussian carp, were identified some ovoid structures also, named cortical
granules (Plate 11, A and Plate 111 A, C, D, E, arrows). Like those described
in pike's oocytes, these cortical granules are disposed topographically in
the vicinity of cortical alveoli. Their inner structure appears to be relatively
homogeneous, heing boundered by a membrane. The cortical granules differ
on the basis of their inner structure with variable density: some of them show
a n electron-dense (Plate IT.A and Plate I11 A1 and others have an inner
content which appear to be weak electron-dense or even translucent (Plate
111, C and D). After Yamamoto (1962) the small granules (a granules) identified near the alveoli from the oocytes of Oryzias latipes would contain an
enzyme capable to solve the cortical alveoli membrane. This hypothesis is
controversed, because even the presence of the granules is disputed (I w am a t s u & 0 h t a, op. cit.).
3. The rcltrastructure of cortical alveoli i n perch oocytes. As it can be
observed from the Plate 11, B and Plate 111, B, in the perch oocytes there is
a population of cortical alveoli relatively heterogeneous in shape and structure. The size of cortical alveoli varies between 8 and 3 0 ; ~and their inner
content is very heterogeneous. Like the other two fish species, the cortical
alveoli have a n inner structure constituted from electron-dense zones and
zones with weak electron densities, with a variable disposal. It is possible
that their content be a mixture of acid and neutral glycoproteins in variable
proportions, though the idea is not excluded that every species presents a
certain type of glycoproteins in a predominant quantity.
Like the images obtained in the oocytes of pike and prussian carp,
in perch eggs too were identified small cortical structures, rounded or oval,
located in the vicinity of cortical alveoli (Plate 11,B and Plate 111, B). There
are also two typcs of cortical granules: some electron-dense and others more
translucent.
CONCLUSIONS
The electron microscope study of the cortical alveoli from the fish
oocytes points out the following peculiarities:
1. I n all the three investigated fish species, the cortical alveoli appear
as organelles variable in size and with a n inner heterogeneous structure,
composed of electron-dense and electron-translucent zones. Their disposal
in the cortical alveoli is not peculiar and reflects a variable content in acid
and neutral muco-polysaccharides.
2. I n all fish species were identified -in the cortical cytoplasm of the
eggs -smaller structures, named cortical granules, located in the vicinity
of cortical alveoli. The cortical granules present an inner structure homogeneous, but variahle, that permits their grouping in two different populations.
ULTRASTRUCTURE OF CORTICAL ALVEOLI FROM OOCYTES O F FISHES
237
PARTICULARIT~J'ILE ULTRASTRUCTURALE ALE VACUOLELOR
CORTICALE DIN OVOCITEJt'; UNOR SPEC11 D E PEST1
REZUMAT
Autorii au studiat ultrastructura vacuolelor corticale din ovocitele
a trei specii de pegti (Esox lucius, Carassius auratus gibelio gi Percafluviatilis).
Pentru a preciza structura acestor organite celulare, au fost utilizate trei
tehnici diferite de evidentiere a glicoproteinelor: albastru alcian, argint
metaminat gi piroantimoniat. Indiferent dc tehnica utilizatii, la toate cele
trei specii de peyti cercetate, vacuolele corticale apar sub forma unor structuri variabile ca m5rime pi cu o compozifie intern5 foarte heterogeng. Dispozitia intravacuolarg a zonelor electrono-dense gi electrono-translucide nu
apare particular5 gi reflect5 prohabil un continut variabil Pn mucopolizaharide
acide qi neutre.
La toate speciile de pepti analizate s-au identificat in citoplasma cortical& a ovocitelor, structuri ovoide mai mici cu un diametru cuprins intre 0,s
gi 3 microni, denumite granule corticale. Prezenta lor a fost mult timp controversatg. Granulele corticale sint frecvent asociate cu vacuolele corticale
qi prezint5 o structur5 intern5 omogeng. Pe baza densitgtii structurii interne
s-au descris doug populatii de granule corticale. Rolul acestor structuri ovocitare este foarte putin cunoscut.
REFERENCES
ANDERSON (E.), 1974 - Comparative aspects of the ultrastructure of the female gamete.
Int. Rev. Cytol., 4 : 1-70.
AUSTIN (C. E.), 1968 Ultrastructure of fertilization, New York, Rinehart and Winston, 1969
DE MARTIN0 (C.), ZAMBONI (L.), 1967 Silver methenamine stain for electron microscopy. J. Ultrastruc. Res., 19: 273-282.
DETERING (N. K.), DECKER (G. L.), SCHMELL (E. D.), LENNARTZ (W. L.), 1977Isolation and characterization of plasma membrane-associated cortical granules
from sea urchin eggs. J. Cell Biol., 75: 899-914.
E N D 0 (Y.),1961 Changes in the cortical layer of the sea urchin eggs a t fertilization as studied with the electron microscope. I . Clypeaster japonicus. E x p . Cell Res., 25:
383-397.
IWAMATSU (T.), OHTA (T.), 1976 -Breakdown of the cortical alveoli of medaka eggs a t
the time of fertilization, with a particular reference to the possible role of spherical
bodies in the alveoli. Wilhelm Roux's Archives, 180: 297-309.
KALT (M. R.), TAUDLER (B.), 1971 -Method of fixation and staining of the glycoproteins
for electron microscopy. J. Uhrastruc. Res., 3 6 : 633-440.
KEMP (N. E.), ISTOCK (N. L.), - Corticale changes in growing oocytes and in fertilized or
picked eggs of Rana pipiens. J. Cell Biol., 34: 111-122.
KUDO (S.), 1968 -Electron microscope observations on the cortical changes in the egg of
Carassius auratus I. The release of granules. Sci. Rep. Tohoku Univ. Ser. IV,
33: 185-195.
KUDO (S.), 1976 a
Ultrastructural observations on the discharge of two kinds of granules
in the fertilized eggs of Cyprinus carpio and Carassius auratus. Dev. Growth and
Differentiation, 18: 167-176.
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238
LOTUS MESTER. DRAGOS SCRIPCARIU, A U R E L ~ ASCARLAT, RADU MESTER
KUDO (S.), 1976 b - Studies on the discharge of two kinds of granules in fertilized eggs of
Carassiw carassius. Annot. Zool. Jap., 49: 105-119.
PASTEELS (J. J.), 1965 - Gtude au microscope Clectronique de la reaction corticale. J . Embryol.
E z p . Morphol., 13: 327-339.
KUSA (M.), 1956- Studies on cortical alveoli in some teleostean eggs. Emrbyofogia, 3 r 105-129.
ROSENBAUM (R. M.), 1958 - Histochemical observations on the cortical region of the oocytes
of Rana pipiens. Quart. J . Micr. Sci., 99: 159-169.
ROTHMAN (A. M.), 1969 Alcian blue as an electron stain. Exp Cell Res. 58: 177-179.
SCHUEL (H.), 1978 - Secretory functions of egg cortical granules in fertilization and development: a critical review. Gamete Res., 1: 299-382.
YAMAMOTO- (T.), 1961 - Physiology of fertilization in fish eggs. Int. Rev. Cytof., 12:
361-405.
YAMAMOTO (T.), 1962 - Mechanism of breakdown of cortical alveoli during fertilization
in the medaka Oryzius latipes. Embryologia, 7 : 228-251.
-
.
A. Section through I>rripl~eralo o l ~ l a s ~ofn Esox llrcil~sooryte showing cortical nlvcoli
hcterogeneons strnctnre. Notr tllc ~ ~ r r s c nof
r r cortical granules (CG, arrow) loc:t~t.~l
n r ~ t l r rthe vitellinc ~ n c t n h r : ~ n(VM);
r
Z R - sona radiata : YI-'- yolk platelets. Pyroantimonium
stain. M;~gnificntionx 1300. I3. Srction throug11 cortex of Esow llicius oocyte showing t h r
(CA) \%it11;I
strncturc of cortical :~lveoli(Ch). /\lcian hlne stain. Magnification x 1300. C. Section through ;I
tnature cortical alveolns of .F,'sox I I I C ~ Ioocyte
IS
etainrcl with silver methmamine. R'ote t h e haterogencons internal content of t h r cortiral~:~lveolns
:lntl the preeenre of cortical granules (C(;.
arro\\.e) i n the rortic.nl r?t~)l)l;rms.Magniiirntion :-:1300. D. \i higher magnification 0: 21600)
of t h e rortir:tl rytnl>l:tsn~nl' fi,'so.u /rrrirrs nocytc* stainetl wit11 silver methenamine sho\r.ing t h e
e ~ ~ t l o ~ ~ l a s n irrtirulnrn
atic
(IIER) ant1 two types of secretory
llrewnce of ~ n i t ~ r h o n t l r i i(AZ),
t
. .
:rrnnules with unifornl electron tlensity a t the ultrastructural level (arrows).
A. Sertioti through peril)lieral ooplas~nof C~rmssiuanrtrtrtrts gibelio oocyte stained ~ i t h
alcian hlnc. Bote the presence it1 tlic cortex of 1n:iny rortirel alveoli (CA) with n hcteropeneous
structure and of cortical granules (CG, arrows) ; VM - vitellirie metrihrane ;%R - zoria radiatu;
YP - yolk platelets. Mngnification x 3870. B. The cortical cytoplasm of PrrcaJuriatilis oocyte
stained with pyroantimonium: CA - cortical alveoli: CG - cortical granules; ZR - zonn
radiata. Magnification x 8100.
r\. Sectio~ithrough the cortical cytopliisrn of Grrnssircs nurntcis gibrlio ooryte stai~recl
\\it11 ;rlrin~iLjlne, showing sorne cortical granules (CC. i~rro\\s)interspersed a111011gthe cortical
i~lveoli(CA). Mag~~ificatiori
>< 6420. B. Sectio~ithrough peripheral ooplas~nof Prrcnfluointilis
oocytc stainctl with alcian blue. Kotc the heterogeneous strrrctlrrc of the cortical alveoli (CA)
illid the presence in the cortical cytoplasn~of cortical granules (CG, arrows); Y P - yolk platelets. Magnificatior~x 2700. C. The cortical cytoplasn~of Cnrassius aurntos gibelio oocyte stained
\vith i11cia11hlue showing the cortical granules (CG) with compact internal structure. Mngnificnti011 >: 5600. D. The same, allother image. Magnification X 5600. E. \1 higher inagriificatio~~
( X 8100) of the cortical cytoplasn~of Curasiccs trrtrnttts gibrlio oocyte showing t h e existence of
a ~jnlmlatia~l
of rortiral g r a r ~ ~ i l (eCs G , arrows) with a hnmogrneons innrr structure of variablr
i ~ ~ t e ~ ~ Alri;u~
s i t y . 11l11cS I ; I ~ I I . Aotc the 1,rcxellcc of rn:cny ~r~itocho~rdriu
(ill) ant1 the e ~ i c l o ~ ~ l i ~ s l ~ ~ n t i ~
I eticulnni (RER).