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J. Anat. (1978), 128, 3, pp. 619-631 With 1O figures Printed in Great Britain

Observations on the primordial oocyte of the bandicoot Isoodon macrourus (Peramelidae, Marsupialia) SUZANNE L. ULLMANN

Department of Zoology, University of Glasgow, Glasgow G12 8QQ, Scotland

(Accepted 3 May 1978) INTRODUCTION

Although the follicular oocytes of eutherian mammals have been intensively studied (Hadek, 1965; Srivastava, 1965; N0rrevang, 1968; Biggers & Schuetz, 1972; Anderson, 1974), those of marsupials have been almost totally neglected. Morgan (1943) describes the development of the ovary in the opossum Didelphis virginiana, but does not deal with the oocyte specifically. Alcorn (1974) reports on the gross structure of the ovary in a brief note, and gives a comprehensive account of ovarian development in the Tammar wallaby, Macropus eugenii (Alcorn, 1976); while Lintem-Moore, Moore, Tyndale-Biscoe & Poole (1976) have studied the growth characteristics of the oocyte and follicle in a number of marsupials, but excluding the bandicoot. The only published account dealing specifically with the egg appears to be a paper by Guraya (1968 a) on the histochemistry of the oocyte of the opossum Didelphis virginiana. In view of the paucity of information on oogenesis in marsupials a systematic investigation of germ cell origins and oocyte development has been undertaken. In this paper observations on the bandicoot primordial follicle and oocyte (growth stage I of Baker, 1972) will be reported. A preliminary note of this work, in abstract form, has appeared elsewhere (Ullmann, 1977). MATERIALS AND METHODS

Animals were obtained by trapping on Mount Tamborine and St Lucia, Brisbane, Queensland. The ovaries and portions of the reproductive tract were removed under chloroform anaesthesia and immediately placed in fixative. The animals were then killed. Where possible, material from the same animal was prepared for both light and electron microscopy. This investigation is based on the examination of six bandicoots, which included mature and immature, parous and non-parous females. Maturity was assessed by size and weight (mature 800 g; immature around 500 g), and parity by the condition of the teats in the pouch (non-parous, teats small; parous, teats swollen). For light microscopy, tissues were fixed in aqueous Bouin's solution and, after dehydration, double embedded by soaking overnight in 2 % low viscosity nitrocellulose in methyl benzoate before blocking in 56 °C paraffin wax (Culling, 1974). Sections were cut at 5-7,um and stained either with Harris's haematoxylin and eosin, or Mallory's or Masson's trichrome stain. For electron microscopy, tissues were fixed for 2-3 hours at 4 °C in 3 % glutaraldehyde buffered at pH 7T2 with phosphate and adjusted to 300 mOsm with sucrose.

SUZANNE L. ULLMANN 620 The tissues were then washed in buffer overnight, post-fixed in 1 %0 OSO4 in phosphate buffer for 1-2 hours at room temperature, stained in the block with 5 %0 uranyl acetate for 1 hour, dehydrated in ethanol and embedded in Epon. Thick sections, for light microscopy, were stained with 1 %0 toluidine blue. Thin sections were cut on an LKB ultramicrotome, stained with lead citrate and viewed with either an AEI Corinth 500, an EM801 or a Zeiss EM95 microscope. OBSERVATIONS

The follicle The bandicoot ovary has a well-developed cortex of dense stromal tissue, bound by a collagenous tunica albuginea of irregular thickness which is most extensively developed in the hilar region. Peripherally it is invested by a cuboidal 'germinal' epithelium. The primordial follicles are located in a zone just internal to the tunica albuginea. They may be distinguished by the irregular shape and distribution of the epithelial cells, which tend to be polarized, cap-like, about each quiescent oocyte. At the light microscope level the epithelium appears incomplete, allowing adjacent oocytes to come into direct contact (Fig. 1 c). Electron microscopy, however, reveals elongate, pseudopod-like projections from the follicle cells, which complete the investment around the oocyte and may interdigitate with one another. The follicle cells rest on a basement membrane (Fig. 4). The follicle nuclei are ovoid or elongate, and the chromatin forms a coarse meshwork in the nucleoplasm, adhering to the nuclear envelope in irregular masses (Fig. 2). The mitochondria are elongate, occasionally dumb-bell-shaped, with transverse or oblique cristae, and lack the dense granules found in oocyte mitochondria. Ribosome-studded profiles of the endoplasmic reticulum are relatively abundant and, occasionally, elongate stacked cisternae lacking ribosomes and reminiscent of annulate lamellae are encountered. The Golgi apparatus is well developed and large, while moderately electron-dense, lipid droplets also occur (Fig. 4). Small organelles, circular or rod-shaped in section, and measuring 0 1-0 2 ,um in diameter, are also to be found in the cytoplasm. These organelles, which will be termed 'dense bodies', are bounded by a membrane which is separated from the electron-dense core by a narrow, semitranslucent zone (Fig. 6).

The oocyte The quiescent oocyte is subspherical or oval in shape and is invested by the oolemma which in places forms small caveoli; in these regions the membrane is more electron-dense and bears short projections on its ooplasmic surface (Fig. 5). The oolemma is closely applied to the follicle cell membrane in some regions where desmosomes may link them. In other places the membranes become somewhat separated, and into the intervening space project small microvilli from the oolemma (Figs. 4, 5, 7). Occasionally a few microvilli may also extend from the follicle cells. A large portion of the oocyte volume is occupied by the germinal vesicle, which is eccentrically located, subspherical and includes a prominent nucleolus beside several subsidiary ones. The nuclear envelope contains numerous pores, and the two membranes comprising it are separated by a conspicuous, somewhat irregular space. The structure of the nucleolus appears fairly complex. Thick plastic sections, stained with toluidine blue, show a dense more or less spherical region which stains

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a deep blue; while an irregularly shaped area, seemingly emanating from the latter region, stains purple. These regions may, perhaps, correspond to the pars amorpha and the pars nucleolonema which, at EM level, are well defined. The relationship of the two regions to each other, however, appears to be inconstant. The chromatin forms a network of coarse strands with a beaded appearance, and radiates from the nucleolus to be dispersed beneath the nuclear envelope (Fig. 1). The ooplasm contains the following organelles: a conspicuous particulate paranuclear complex; a vesicle-microtubule complex; an aggregate of tubular cisternae; 'coated vesicles'; mitochondria, lipid droplets, the Golgi apparatus, 'dense bodies'; and sCattered profiles of the endoplasmic reticulum. In paraffin sections the paranuclear complex (PNC) appears as an eosinophil body which is usually ovoid in outline and situated close to the nucleus, but variations in its shape, size, number and position are not uncommon (Fig. 1). At the ultrastructural level the PNC is seen to consist of one or more groups of Golgi-like smooth surfaced membranous profiles surrounded by a large number of lipoidal particles. These central membranes are generally in the form of flattened, loosely stacked cisternae (Fig. 4), but occasionally extended vesicles are encountered. The following particles or bodies may be recognized in the PNC: (a) Irregularly shaped electron-dense particles with smooth or crenulated contours which generally contain areas of diminished electron opacity. At high magnification these bodies appear laminated, the less dense areas resulting from a looser arrangement of the fibrils composing them (Fig. 7). () Moderately electron-dense subspherical particles composed of whirled fibrillar lamellae. Locally the lamellae may adhere together, forming electron-opaque areas

(Figs. 4, 7).

(y) Oval bodies with an electron-translucent interior, and consisting of very regular, tightly packed, concentric arrays which are composed of small spherical subunits. In transverse sections they show a crystalline lattice structure (Figs. 7, 8). These bodies are generally found complexed with the lipid (L) bodies. The differences in the appearance of this organelle in the Figures are presumed to be due to the plane of sectioning. (L) Amorphous, rounded, apparently membrane-bound bodies of moderate electron density which are usually complexed to other types of particles in the PNC. These bodies, whose general characteristics suggest that they are lipids, are not confined to the PNC but may also be found scattered in the ooplasm (Figs. 4, 7). (A) A moderately electron-dense, granular substance is also found, but only complexed with other types of particles. This substance may be found within or around the other particles in the PNC (Fig. 7). The crystalline (y), lipid (L) and granular (A) bodies are complexed together to form a compound granule which appears to be held together in regions by strands of tightly packed fibrous lamellae (Fig. 7F). Vesicles and cisternae of the smooth endoplasmic reticulum, though not abundant, may be seen scattered among the particles of the PNC, which is quite devoid of mitochondria and rough endoplasmic reticulum (Fig. 7). The second most conspicuous region in the ooplasm is that occupied by a group of vesicle-like organelles and associated microtubules (VMC) (Figs. 9, 10). The structure of these organelles and their relationship to each other is complex. The vesicles, which may have pseudopod-like extensions, are bounded by a well defined membrane which bears, on its cytoplasmic surface, an amorphous investment of a 40-2

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less electron-dense material. This arrangement gives the impression that the organelle is bounded by two membranes, like mitochondria. The membrane may appear smooth (Fig. 10 C), or decorated with granules or rodlets (Fig. 10a, b). Bundles of microtubules arise from the walls of the vesicles and often appear to have a bipolar origin from the latter (Fig. 10). They extend between the vesicles, and bundles of up to a dozen microtubules, running a fairly straight course of considerable length, and not obviously associated with vesicles, are also encountered (Fig. 9, MT). Much smaller decorated vesicles, often occurring in groups or in close proximity to the vesicles under discussion, are also frequently encountered in the VMC (Fig. 10b). At first these were taken to be 'coated vesicles' derived from the oolemma (Fig. 5), but they are now interpreted as transverse sections through the pseudopod-like extensions of the vesicles (compare Fig. 10a and b). The vesicles, measuring about half the size of the mitochondria, contain an irregularly distributed, amorphous, electron-dense material generally resembling the ooplasmic matrix in which these organelles are embedded (Figs. 9, 10). A third region of ooplasmic localization is formed by a well defined aggregate of tubular cisternae (ATC). The cisternae are orientated without apparent order, and contain a homogeneous substance more electron-dense than the surrounding ooplasm (Figs. 2, 3, T). The specializations of the oolemma have already been referred to. The electrondense caveoli become invaginated as micropinocytotic 'coated vesicles' bearing characteristic projections on their outer surfaces (Fig. 5). They migrate into the ooplasm, and have been observed in the vicinity of the Golgi apparatus. The mitochondria are mostly spherical, with a few concentric or arched cristae (Figs. 3-5). The matrix frequently contains electron-dense granules, and, sometimes, a granular electron-dense substance is seen between the mitochondria (Fig. 5, IM). Scattered globules of lipid, of moderate density, may be found sparsely distributed in the ooplasm. Occasionally these occur near the oolemma in the vicinity of similar globules in the adjacent follicle cells (Figs. 2-4, L). The Golgi apparatus is juxtanuclear and consists of a number of flattened cisternae and vesicles which may have intraluminal contents of low electron density (Fig. 9). 'Dense bodies', identical in structure to those described in the follicle cells, have also been observed in the ooplasm. They give the impression of fusing to form rodlike structures (Fig. 6, DB). Occasionally the dense core shows a less dense central

region. Elements of the endoplasmic reticulum are not abundant in the ooplasm, and the few tubules and vesicles present are often associated with mitochondria (Figs. 5, 7, ER). The profiles are mostly of the smooth variety, but are occasionally irregularly decorated with ribosomes (Fig. 5, ER). Fig. 4. Electron micrograph through follicle cell-oocyte junction and adjacent PNC. Locally the oolemma forms microvilli. Lipid droplets occur in both the follicle cell and the oocyte. Among the various particles of the PNC, flattened Golgi-type profiles are seen (arrow). Parous adult. Inset: f8-type bodies of the PNC, composed of concentric, fibrillar lamellae. In the dense region (arrow) the lamellae adhere together. Fig. 5. Electron micrograph through the oocyte-follicle cell interface showing: (i) interdigitations of follicle cell extensions; (ii) caveoli, bearing short projections on their ooplasmic surfaces; (iii) mitochondria, with dense 'intermitochondrial substance' between them. Fig. 6. Electron micrograph of oocyte-follicle cell interface, showing dense bodies in both the follicle cell and the ooplasm.

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Bandicoot primordial oocytes

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DISCUSSION

Though the structure of the primordial oocyte of the bandicoot resembles that of eutherian mammals in many respects (Zamboni, 1972), it differs from the latter in possessing a number of ooplasmic localizations, namely, the paranuclear complex (PNC), the vesicle-microtubule complex (VMC) and the aggregate of tubular cisternae (ATC). The PNC and the VMC possesses ultrastructural features which have not, apparently, been described before. Moreover, specialization of the oolemma in the form of localized caveoli and microvilli appears to be somewhat precocious in the bandicoot. A paranuclear complex (PNC) has been described not only in mammals, but in the oocytes of a number of lower vertebrates and invertebrates. It has been variously termed the 'yolk nucleus', 'archoplasm', 'idiosome', 'Golgi field', 'cytocentrum' and 'Balbiani's vitelline body' (see Raven, 1961; Hertig, 1968; Anderson, 1974). Several components of diverse morphology and histochemical constitution have been identified in the PNC, and it is now clear that the organelles comprising this structure in different animals are not homologous (N0rrevang, 1968). Although not universally present in eutherian oocytes, a PNC has been described in the rat (Sotelo, 1959; Guraya, 1964), guinea-pig (Anderson & Beams, 1960; Adams & Hertig, 1964; Guraya, 1964), rabbit (Blanchette, 1961; Guraya, 1964; Zamboni & Mastroianni, 1966), hamster (Odor, 1965; Weakley, 1966) and Indian buffalo (Guraya, 1964), and is particularly conspicuous in primate primordial oocytes (Hertig & Adams, 1967; Hertig, 1968; Guraya, 1974). As far as the author is aware, this is the first account of the ultrastructure of the PNC in any marsupial. Most studies of the PNC have been at the light niicroscope levei, where the structure is identified as a differentially staining region of the ooplasm in the vicinity of the germinal vesicle. As pointed out by Hertig & Adams (1967), electron microscopists have frequently confused the PNC in mammalian oocytes with the Golgi apparatus. In the case of the bandicoot, no ambiguities arise in the identification of the PNC. It forms a conspicuous juxtanuclear structure at both the light and electron microscopical levels. 'The bodies composing it are of diverse and unsual type, and are not encountered in the PNC of any mammal hitherto investigated. The bulk of the bodies appears to be lipoidal in nature, and composed of loosely or tightly coiled fibrillar lamellae (Figs. 4, 7). The y bodies, which appear to have a crystalline structure (Fig. 8), are particularly interesting in that they resemble the phosphorylated phosphoprotein yolk platelets of amphibian oocytes (Karasaki, 1963). Similar crystalline bodies, associated with lipid, are figured by Lyne (1976) as constituents of the eliminated yolk in bandicoot blastocysts. Whether they are directly derived from the PNC, or are later reconstituted, is unknown. As the eliminated yolk is said to be later reabsorbed by the blastocyst, the crystalline bodies may play a role in embryonic nutrition. A cyto-centrum within the PNC, such as may be associated with this region in other vertebrates (Hertig & Adams, 1967; Raven, 1961), has not been located in the bandicoot. Guraya (1964, 1968a, b) carried out histochemical investigations on the PNC of a number of fish, reptiles, birds and mammals, including the opossum, and found that it was invariably composed of RNA, protein and lipoprotein. Preliminary histochemical observations on the bandicoot show that the PNC is PAS-negative and slightly sudanophil. The a, ,6 and L bodies of the PNC (p. 623) give the ultrastructural appearance of lipids; the crystalline body resembles protein yolk, while the granular

628

SUZANNE L. ULLMANN

Bandicoot prinmordial oocytes

629

A body may represent an RNA component. There is thus circumstantial evidence that, in spite of ultrastructural dissimilarity between the PNC of the bandicoot and that of other vertebrates, its chemical composition is nevertheless similar. Although the PNC was first described over a century ago (Balbiani, 1864), we still have no idea as to its significance. Modern studies have failed to confirm it as the site of yolk formation as was surmised by the early embryologists. Whether a more indirect relationship exists between the degree of differentiation of the PNC and the amount of yolk formed, as suggested by Guraya (1964), may also be questioned, as the human oocyte has a well developed PNC but little yolk. The latter feature can, of course, be regarded as secondary. Neither a VMC nor an ATC appears to have been described in oocytes previously. The present study has not altogether clarified the three dimensional configuration of the VMC: careful serial sectioning will be necessary to achieve this. It is not clear whether all the microtubules in the VMC take their origin from 'vesicles', or whether they form bridges between them, thereby establishing an intercommunicating system within the ooplasm analogous to the endoplasmic reticulum. The presence of elements of the endoplasmic reticulum scattered among the ATC suggests that the latter may be derived from the former. The functions of the VMC and ATC are unknown. The 'dense bodies' which occur in both the follicle cell cytoplasm and the oocyte periphery form yet another puzzle. They are reminiscent of the lining bodies and oocyte bodies of avian oocytes (Paulson & Rosenberg, 1974). The association of electron-dense material and endoplasmic reticulum with mitochondria, such as occurs in the bandicoot oocyte, has also been described in rat spermatogonia (Andre, 1962) and human primordial oocytes (Hertig & Adams, 1967), and is particularly conspicuous in the oocytes of the hamster (Odor, 1965). Intermitochondrial material has also been reported for amphibian oocytes, where there are indications of a nuclear derivation (Balinsky & Devis, 1963; Al-Mukhtar & Webb, 1971). The role of this intermitochondrial material remains unresolved. The presence of these various unusual organelles in the bandicoot oocyte poses intriguing developmental problems with regard to their genesis, fate and morphogenetic significance. SUMMARY

An ultrastructural study of bandicoot primordial follicles and oocytes was undertaken, as information on this subject is lacking in marsupials. Conspicuous features of the ooplasm are a paranuclear complex (PNC), a vesiclemicrotubule complex (VMC) and an aggregate of tubular cisternae (ATC). The PNC appears as one or, more rarely, several homogeneous eosinophil bodies at the light microscope level. Ultrastructurally it is particulate, consisting of five distinct types of bodies, most of which are composed of concentric fibrillar whorls, Fig. 9. Electron micrograph of the VMC. Part of the nuclear envelope and Golgi apparatus are visible on the right. Bundles of microtubules are seen intermingled with the vesicles. Fig. 10. Electron micrographs showing a vesicle-microtubule complex. (a) Vesicles showing

(i) pseudopod-like extension decorated with granules; (ii) origin of microtubules, from vesicles; (iii) decorations on membrane. (b) Large and small decorated vesicles: the latter are probably transverse sections through the pseudopod-like extensions of the larger organelles (arrows). (c) Vesicle with pseudopod-like extension lacking decorations. The membrane is coated with a layer of an amorphous substance, giving it the appearance of being double.

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SUZANNE L. ULLMANN

but others appear homogeneous, granular or crystalline. Embedded among the particles is a group of Golgi-like vesicles. The bandicoot PNC - unlike similar structures found in the ooplasm of a variety of vertebrates, and known variously as 'Balbiani body', 'yolk nucleus', etc. totally lacks mitochondria. The VMC consists of vesicle-like organelles which may be drawn out into tubular extensions, while the bounding membrane may be decorated with granules. Bundles of microtubules ramify between the vesicles, from which they appear to originate. The vesicles contain a matrix similar to the ooplasm. The ATC contains a homogeneous substance more electron-dense than the surrounding ooplasm. 'Dense bodies' occur in the cytoplasm of both the follicle cells and the oocytes. These are elongate membrane-bound organelles, circular in cross section. An electron-dense core is separated from the membrane by a narrow, less dense zone. The genesis and morphogenetic significance of these various organelles is unknown. This work was carried out during a year spent at the School of Anatomy, University of Queensland. I wish to express my sincere gratitude to the following: Professor G. Molyneux and his staff, especially Dr R. L. Hughes, for the warm hospitality received; the technical staff for processing material; Mr J. Hardy, for his help in the Electron Microscope Unit; Mr P. Rickus, Mss C. Morrison and M. Gardener of Glasgow University, for photographic assistance; and Mr R. Gemmell (Queensland) and Professor D. R. Newth (Glasgow) for commenting on the manuscript. REFERENCES ADAMS E. C. & HERTIG, A. T. (1964). Studies on guinea pig oocytes: I. Electron microscopic observations on the development of cytoplasmic organelles in oocytes of primordial and primary follicles.

Journal of Cell Biology 21, 397-427. ALCORN, G. T. (1974). Growth and development of the ovary of the Tammar Wallaby Macropus eugenii. Australian Mammalogy 1, 292. ALCORN, G. T. (1976). Development of the ovary and urogenital ducts in the tammer wallaby Macropus eugenii. Ph.D. Thesis, Macquarie University, Sydney. AL-MUKHTAR, K. A. K. & WEBB, A. C. (1971). An ultrastructural study of primordial germ cells, oogonia and early oocytes in Xenopus laevis. Journal of Embryology & Experimental Morphology 26, 195-217. ANDERSON, E. (1974). Comparative aspects of the ultrastructure of the female gamete. International Review of Cytology Suppl. 4, 1-70. ANDERSON, E. & BEAMS, H. W. (1960). Cytological observations on the fine structure of the guinea pig ovary with special reference to the oogonium, primary occyte and associated follicle cells. Journal of Ultrastructure Research 3, 432-446. ANDRE', J. (1962). Contribution a la connaissance du chondriome. etude de ses modifications ultrastructurales pendant la spermatogenese. Journal of Ultrastructure Research Suppl. 3, 1-185. BAKER,T. G. (1972). Oogenesis and ovulation. In Reproduction in Mammals. L Germ Cells and Fertilization. pp. 1"45. Cambridge University Press. BALBIANI, E. G. (1864). Sur la constitution du germe dans l'aeuf animal avant la fecondation. Compte rendu des seances de la Societe de biologie 58, 584-588. BALINSKY, B. I. & DEVIS, R. J. (1963). Origin and differentiation of cytoplasmic structures in the oocytes of Xenopus laevis. Acta embryologiae et morphologiae experimentalis 6, 55-108. BIGGERS, J. D. & SCHUETZ, A. W. (eds) (1972). Oogenesis. Baltimore: University Park Press. BLANCHETrE, E. J. (1961). A study of the fine structure of the rabbit primary oocyte. Journal of Ultrastructure Research 5, 349-363. CULLING, C. F. A. (1974). Handbook of Histopathological and Histochemical Techniques. Third edition. London: Butterworth & Co. GURAYA, S. S. (1964). Histochemical studies on the yolk nucleus in the oogenesis of mammals. Armerican Journal of Anatomy 114, 283-291.

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GURAYA, S. S. (1968a). Histochemical study of developing ovarian oocyte of the American opossum. Acta embryologiae et morphologiae experimentalis 10, 181-191. GURAYA, S. S. (1968b). Further morphological and histochemical studies on the yolk nucleus and associated cell components in the developing oocytes of the Indian wall lizard. Journal of Morphology 124, 283-293. GURAYA, S. S. (1974). Morphology, histochemistry and biochemistry of human oogenesis and ovulation. International Review of Cytology 37, 121-151. HADEK, R. (1965). The structure of the mammalian egg. International Review of Cytology 18, 29-71. HERTIG, A. T. (1968). The primary oocyte: some observations on the fine structure of Balbiani's vitelline body and the origin of the annulate lamellae. American Journal of Anatomy 122, 107-138. HERTIG, A. T. & ADAMS, E. C. (1967). Studies on the human oocyte and its follicle. I. Ultrastructural and histochemical observations on the primordial follicle stage. Journal of Cell Biology 34, 647-675. KARASAKI, S. (1963). Studies on amphibian yolk. I. The ultrastructure of the yolk platelet. Journal of Cell Biology 18, 135-151. LINTERN-MOORE, S., MOORE, G. M. P., TYNDALE-BISCOE, C. H. & POOLE, W. E. (1976). The growth of the oocyte and follicle in the ovaries of monotremes and marsupials. Anatomical Record 185, 325-332. LYNE, A. G. (1976). Early embryology of the marsupials Isoodon macrourus and Perameles nasuta. Australian Journal of Zoology 24, 361-382. MORGAN, C. F. (1943). The normal development of the ovary of the opossum from birth to maturity and its reaction to sex hormones. Journal of Morphology 72, 27-85. N0RREVANG, A. (1968). Electron microscopic morphology of oogenesis. International Review of Cytology 23, 113-186. ODOR, D. L. (1965). The ultrastructure of unilaminar follicles of the hamster ovary. American Journal of Anatomy 116, 493-522. PAULSON, J. L. & ROSENBERG, M. D. (1974). Formation of lining bodies and oocyte bodies during avian oogenesis. Developmental Biology 40, 366-371. RAVEN, C. P. (1961). Oogenesis: The Storage of Developmental Information. Oxford: Pergamon Press. SOTELO, J. R. (1959). An electron microscopic study on the cytoplasmic and nuclear components of rat primary oocytes. Zeitschrift far Zellforschung und mikroskopische Anatomie 50, 749-765. SRIVASTAVA, H. D. (1965). Cytoplasmic inclusions in oogenesis. International Review of Cytology 18, 73-98. ULLMANN, S. L. (1977). Studies on the ooplasm of the bandicoot Isoodon macrourus. Journal of Anatomy 124, 528. WEAKLEY, B. S. (1966). Electron microscopy of the oocyte and granulosa cells in the developing ovarian follicles of the golden hamster Mesocricetus auratus. Journal of Anatomy 100, 503-534. ZAMBONI, L. (1972). Comparative studies on the ultrastructure of mammalian oocytes. In Oogenesis (ed. Biggers and Schuetz), pp. 5-45. Baltimore: University Park Press. ZAMBONI, L. & MASTROIANNI, L. (1966). Electron microscopic studies on rabbit ova. I. The follicular oocyte. Journal of Ultrastructure Research 14, 95-117. ABBREVIATIONS L af,8yA bodies within the paranuclear complex M ATC aggregate of tubular cisternae MT BM basement membrane MV C caveoli N DB dense body NU vesicle extension E PNC ER endoplasmic reticulum 0 F fibrous lamella S FC follicle cell T G Golgi apparatus V GV germinal vesicle IM 'intermitochondrial substance' VMC

lipid droplet mitochondrion microtubule microvillus nucleus nucleolus paranuclear complex oolemma stroma

aggregate of tubular cisternae vesicle vesicle-microtubule complex