Original article

Light- and electron-microscopic observationson the relationship between prelampbrush oocytesand surrounding granulosa cells in the layingJapanese quail (Coturnix coturnix japonica)

M Callebaut

RUCA, Laboratory of Human Anatomy and Embryology, Groenenborgerlaan 171,2020 Antwerpen, Belgium

(Received 9 September 1990; accepted 3 May 1991)

Summary ― Transmission electronmicroscopic (TEM) observations demonstrated that the most su-perficial region of quail oocytes during the prelampbrush stage differs locally from the deeper oo-plasm and is an active zone which forms exooplasmic cones, ridges or knob-like protrusions in thedirection of/or in the granulosa cells. This exooplasm, in which no mitochondria were seen, is separ-ated from the endooplasm, by a narrow interrupted filamentous layer. Using a lipid-preserving meth-od of fixation, morphological evidence was found for the transport of lipid material from the granu-losa cells into the exooplasm of the oocyte. Open intercellular bridges between exooplasm and gran-ulosa cell cytoplasm were also seen. Differences between the electronmicroscopic aspect of clearand dark granulosa cells have been described.

Japanese quail / prelampbrush oocyte / avian ovarian granulosa cell / ooplasm / lipid

Résumé ― Étude avec le microscope photonique et électronique des relations existant entrel’oocyte (avant le stade en écouvillon) de caille et les cellules de la granulosa qui l’entourent.À l’aide du microscope électronique, nous avons observé que l’oopiasme le plus superficiel a unestructure qui diffère localement de l’ooplasme plus profond. L ooplasme superficiel forme par en-droits des cônes ou crêtes d’exooplasme dirigées vers ou dans les cellules de la granulosa. L’exoo-plasme ne contient pas de mitrochondries et est séparé de I endooplasme par une mince couche fi-lamenteuse interrompue, visible après l’emploi d’un fixateur qui préserve bien les lipides. Nosobservations suggèrent que du matériel lipidique provenant du cytoplasme des cellules de la granu-losa est transporté vers l’exooplasme. De plus, l’exooplasme présente localement des ponts intercel-lulaires ouverts en communication directe avec le cytoplasme des cellules de la granulosa. Les diffé-rences d’ordre ultrastructural entre les cellules claires et foncées de la granulosa sont décrites.

caille japonaise / oocyte prelampbrush / cellule de la granulosa ovarienne / ooplasme / lipide

INTRODUCTION

In vertebrates the function of the granu-losa cells surrounding the oocyte is not

completely understood and also differs

widely according to the developmentalstage of the follicle. Electronmicroscopicobservations have demonstrated that in

sauropsidian follicles the morphology ofthe granulosa cells is typical of that of bio-synthetically active secretory cells owingto the presence of abundant rough endo-plasmic reticulum, Golgi complexes andmitochondria, especially during the earlystages of intrafollicular development (Bel-lairs, 1965; Wyburn et al, 1966; Rahil andNarbaitz, 1973; Klosterman, 1987). Thetransfer of organelles (transosomes or lin-ing bodies) from the granulosa cells intothe oocyte has been observed both inbirds (Press, 1964; Bellairs, 1965; Wyburnet al, 1966) and in chelonians (Rahil andNarbaitz, 1973).

In the present work, transmission elec-tron microscopy (TEM) was used to studythe relationship between the quail pre-lampbrush oocyte and the surroundinggranulosa cells. The term prelampbrushstage was used (Callebaut, 1973) and notBalbiani or dispersed Balbiani stage asused by Bellairs (1967) since we haveshown by appropriate fixation methods

(Callebaut, 1984) that part of the elementsfrom the Balbiani complex persist in theirparanuclear position for a much longer pe-riod than is usually assumed. Indeed, its

presence near the germinal vesicle canstill be demonstrated during at least theentire phase I of the ensuing lampbrushstage (in quail oocytes with a diameter of =700 pm).

In the present study we provide evi-dence that there exist important interac-tions between the superficial ooplasm(called exooplasm) and the granulosa cellcytoplasm.

MATERIALS AND METHODS

After decapitation of laying Japanese quail (Co-turnix coturnix japonica) and opening of their ab-domen, pieces of ovary were removed and fixedin 1% glutaraldehyde in 0.05 M sodium cacody-late buffer containing 0.01% malachite green,according to the procedure of Lawton (1989). Af-ter 2 h fixation the pieces were (without rinsing)either placed directly in 70% alcohol for 3 h (fixa-tion method 1) or directly in 1 % aqueous osmi-um tetroxide for 1 h (fixation method 2).

After fixation method 1, the pieces werepassed through 50% alcohol (3 h), 30% alcohol(3 h), tap water during 1 night and then alsopostfixed in 1% aqueous osmium tetroxide for 1h. After rinsing in tap water, the tissues (bothfrom methods 1 and 2) were postfixed in 2%

aqueous uranyl acetate (1 h), followed by rins-ing in tap water. For light microscopic studies,the tissues were dehydrated in alcohol and em-bedded in paraffin or glycol methacrylate. Eight-gm thick paraffin sections and 1-gm thick glycolmethacrylate sections were made. For electronmicroscopic studies, the tissues were dehydrat-ed in alcohol and infiltration and embedding wasperformed via propylene oxide into LX-112 Res-in (Ladd).

The ultrathin sections made with an LKB ul-tratome were stained with 10% uranyl acetate inmethanol for 7 min, followed by staining withlead citrate (Reynolds, 1963). The ultrathin sec-tions were then studied with a transmission elec-tron microscope (Siemens Elmiscop 101 ).

RESULTS

Light microscopic observations

After fixation method 1, besides the intra-oocytal lipid spherules around the Balbianicomplex and germinal vesicle, numerousgroups of lipid spherules (of similar size)were seen in the granulosa layer (fig 1 ).However, on the paraffin sections it wasnot always clear whether the lipid sphe-rules were localized in the granulosa cells

or between them. After glycol methacrylateembedding, no lipid inclusions were visiblein the granulosa layer, indicating that theyhad been solubilized by the histologicalprocedure.

Transmission electronmicroscopic observations

With fixation method 1 the lipids were wellconserved, also at the electron microscop-ic level. The superficial ooplasm was seento form different kinds of exooplasmic ex-tensions in the direction of/or in the granu-losa cells. The first type was seen to pointto the junction of 2 granulosa cells and of-ten had the form of a cone or ridge with a

broad base (fig 2). The exooplasmic areaswere incompletely separated from the re-mainder of the ooplasm by a nearly planeregion of filaments (probably actin bun-

dles). By contrast to the underlying endoo-plasm, no mitochondria were seen in theexooplasmic cones (fig 2). The latter con-tain groups of lipoid elements localized be-low intercellular communications betweena granulosa cell and the exooplasm. Bel-lairs (1967) described them on a drawingas pinocytotic-like vesicles, since by herfixation and embedding method the lipoidmaterial was probably solubilized. Some-times globular material seems to be in theprocess of expulsion by the granulosa cellcytoplasm in the direction of the exoo-

plasm (fig 3).

In several places of the exooplasmiccones direct continuity between the granu-losa cell cytoplasm and the exooplasmseemed to exist thus forming true intercel-lular communications (fig 2).

The granulosa cells were also seen togive off transosomes in the exooplasmiccones (fig 2). The second type of exoo-plasmic extension was seen to form a

large knobby protrusion (sometimes start-ing from an exooplasmic cone) into a

granulosa cell (fig 4). A third smaller type,the so-called &dquo;protuberance&dquo; (probably cor-responding to the description of Bellairs,1965) also often originating from the exoo-

plasmic cones was seen to invaginate intothe neighbouring granulosa cell (fig 3). Inor around the &dquo;complex mass&dquo; (Bellairs’ de-nomination: 1965) of the granulosa cells,numerous fat spherules were occasionallyseen (fig 5). With fixation method 1 the

complex masses were sometimes seen tocontain elements resembling nucleated redblood cells and with the size range of viral

particles. No lipid material could be ob-served between the granulosa cells or be-tween the granulosa cells and the base-ment membrane.

Dark granulosa cells could be detectedlocally after fixation method 2 (fig 6).

These dark cells have a rather angular as-pect with numerous tentacle-like exten-

sions near the basement membrane, be-tween the clear rounded granulosa cellsand also extend to the exooplasmic conesof the oocyte.

Although the dark granulosa cells havea prominent nucleolus, their nucleus issmaller than that in the clear granulosacells. The nuclear wall has an irregular out-line and the nucleoplasm is as denselystained as the cytoplasm between the nu-merous vacuoles. The boundary betweennucleus and cytoplasm is therefore not

easily visible. This gives the cell a mouldywood aspect. Sometimes the ooplasm ofthe prelampbrush oocyte was not whollycovered by granulosa cells and even ex-tended to the basement membrane. Afterfixation method 2, the lipids were less wellconserved.

In the ooplasm of some of the prelamp-brush oocytes a voluminous body (partiallysurrounded by a cell membrane: trilaminarat high magnification) as large as a wholegranulosa cell (and probably representingan engulfed granulosa cell) was seen. Itseemed to be composed of remnants of

cell organelles forming vacuoles and irreg-ular clumps with variable aspect and diam-eter. A central part containing dense

granular material and resembling a nucle-ar area could sometimes be observed.

DISCUSSION AND CONCLUSION

The present TEM study offers a clear viewof granulosa cell and oocytal membranesand retains the lipids (after fixation method

1: Callebaut, 1990). This can be explainedby: i), the presence in the glutaraldehydefixative of malachite green which retains

phospholipids in the tissues (Teichmann etal, 1972; Lawton, 1989); and ii), the treat-ment with 70% alcohol immediately afterthe primary fixation which not only pre-vents solubilisation of some fixed proteins(Silverton and Anderson, 1961) but also ofsome lipids: cholesterol and triglycerides(Callebaut et al, 1991 ).

Since De Somer’s report (1905) it hasbeen known that the smallest follicles inthe ovary of the adult chicken contain nu-merous lipid spheres grouped round theBalbiani complex. The origin of these lipidspheres is not known, but the following hy-potheses may be considered: i), they maybe synthesized by the oocyte (so-calledendogenous yolk formation); ii), they maybe formed in the granulosa cells and thentransported into the ooplasm; iii), they maybe formed from precursor material in thetheca and pass between the granulosacells; iv), they may come from plasma lip-ids which penetrate through the folliclewall.

In the present study morphological evi-dence was found for the second possibility,but the other hypotheses cannot be ex-cluded. Earlier studies seemed to indicatethat some components, such as Golgi bod-ies (Brambell, 1926), were transportedfrom the granulosa cells to the ooplasmbefore the onset of the rapid oocytalgrowth period. In a recent study we alsofound electron microscopic evidence forthe delivery of lipids by the granulosa cellsto the superficial avian ooplasm in the larg-est follicles during their final yellow yolk as-semblage (Callebaut, 1991a). According toKemp (1958), intercellular bridges exist be-tween the avian granulosa and the oocyte.By contrast, Press (1959) and Bellairs

(1965) found no evidence for such intercel-lular bridges. In the present study howev-

er, using a different method of fixation in-tercellular bridges were also seen.

Among vertebrates, true intercellularcommunications between granulosa cellsand oocyte have been described by TEMonly in squamate reptiles (lizards and

snakes) (Ghiara and Filosa, 1966; Hubert,1971; Neaves, 1971; Taddei, 1972; Bou-Resli, 1974). These communications origi-nate through a secondary fusion of the oo-cyte with the granulosa cells before theybegin to differentiate into pyriform cells

(Andreuccetti et al, 1978). Let us mentionthat the class Aves also belongs to a simi-lar genome lineage as do the reptilian sub-order of the Squamata, suggesting a com-mon phylogenetic past (Ohno, 1970).

Dark granulosa cells have been de-scribed at some periods (different accord-ing to the fixative used) of avian folliculardevelopment (Holl, 1890; Brambell, 1926;Marza and Marza, 1935; Press, 1964).They have been considered as degenerat-ing cells (Brambell, 1926; Bellairs, 1965;Chalana and Guraya, 1980; Guraya,1989). However, the present study sug-gests that these angular cells with longtentacular processes between the neigh-bouring granulosa cells and extendingfrom the basement membrane to the oo-

cyte have a holding or supporting function,as has already been suggested by Holl

(1890). We have also described them atmore advanced stages of follicular devel-opment (Callebaut, 1991 b). As was thecase in the lampbrush and beginning post-lampbrush stages (Callebaut et al, 1981 ),during the prelampbrush stage of the quailoocyte we could also discern 3 major oo-plasmic zones by the trypan blue-inducedfluorescence method (Callebaut and Si-

jens, 1985). The exooplasmic zone de-scribed here where uptake of material fromthe granulosa cells seems to occur prob-ably corresponds to the narrow most su-perficial unlabelled ooplasmic zone seen

after trypan blue administration. It probablyfunctions as an oocyte transit compart-ment and also exists during the lampbrushstage (Callebaut, 1991b).

ACKNOWLEDGMENTS

I thank C De Chepper for excellent technical as-sistance and W Walscharts for typing the manu-script. I also thank L Andries for valuable sug-gestions and Prof D Scheuermann andcollaborators (RUCA, histological laboratory) forthe use of their TEM. This study was supportedby grant 3.0037.90 from the NFWO of Belgium.

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