Electron-microscope observations on the differentiation of germ cells in Xenopus laevis have revealed that the Balbiani body, cytoplasmic nucleolus-like bodies and groups of mitochondria associated with granular material previously reported only in older amphibian oocytes, are also present in the primordial germ cells, oogonia and early meiotic (pre-diplotene) oocytes of this species.

Although there is considerable morphological reorganization of the gonad as a whole at the time of sex determination, little visible change in the ultrastructure of the primordial germ cells appears to take place during their transition to oogonia. Both primordial germ cells and oogonia have highly lobed nuclei and their cytoplasm contains a conspicuous, juxtanuclear organelle aggregate (consisting for the most part of mitochondria), which is considered to represent the precursor of the Balbiani body.

In marked contrast, the transition from oogonium to oocyte in Xenopus is characterized by a distinctive change in nuclear shape (from lobed to round) associated with the onset of meiosis.

During leptotene the oocyte chromatin becomes visibly organized into electron-dense axial elements (representing the single unpaired chromosomes) which are surrounded by a fibrillar network. Towards the end of leptotene, these axial elements become attached to the inner surface of the nuclear membrane in a localized region adjacent to the juxtanuclear mitochondrial aggregate. Zygotene is marked by the initiation of axial element pairing over short regions, resulting in the typical synaptonemal complex configuration of paired homologous chromosomes. The polarization of these tripartite ribbons within the nucleus becomes more pronounced in late zygotene, producing the familiar Bouquet arrangement. The synaptonemal complexes are more extensive as synapsis reaches a climax during pachytene, whereas the polarization is to some extent lost. The fine structure of synaptonemal complexes in the Xenopus oocyte is essentially the same as that described in numerous other plant and animal meiocytes. It is not until the beginning of the extended diplotene phase that any appreciable increase in cell diameter takes place. During early diplotene (oocyte diameter approximately 50/μm), the compact Balbiani body characteristic of the pre-vitellogenic anuran oocyte is formed by condensation of the juxtanuclear mitochondrial aggregate.

Electron-dense, granular material appears to pass between nucleus and cytoplasm via nuclear pores in all stages of Xenopus germ cell differentiation studied. There is a distinct similarity in electron density and granular content between this ‘nuage material’ associated with the nuclear pores and the cytoplasmic aggregates of granular material in association with mitochondria or in the form of nucleolus-like bodies.

The importance of oogenesis as a prelude to embryonic differentiation and the significance of it as a period of intense synthetic activity is now universally recognized (see Davidson, 1968, for review). However, to date, ultrastructural studies on the development of the amphibian oocyte have been almost exclusively confined to the extended period from the diplotene stage of the first meiotic prophase to the formation of the mature oocyte (e.g. Kemp, 1956; Wischnitzer, 1960; Wartenburg, 1962; Balinsky & Devis, 1963; Kessel, 1963, 1969; Hope, Humphries & Bourne, 1964; Takamoto, 1966; Massover, 1968). It appears, therefore, that there is a considerable gap in our present knowledge as to the fine structural changes that occur in amphibian germ cells immediately prior to, during and after sex determination. It is self-evident that an investigation of the ultrastructure of primordial germ cells, oogonia and very early oocytes is essential for a complete understanding of the events which lead to the production of the unfertilized egg.

As far as is known, the only observations undertaken on the early differentiation of amphibian germ cells are those made during classical studies more concerned with such problems as the origin of primordial germ cells (e.g. Humphrey, 1925; Nieuwkoop, 1946; Bounoure, 1934; Blackler, 1958) than changes in their cellular organization that lead to oocyte formation in the female. There appears to have been no work employing modern techniques of fixation and electron microscopy to visualize the ultrastructural detail of primordial germ cells and their immediate derivatives in the ovary of any amphibian. There have, however, been more recent studies at the fine structural level on the oogonia and early oocytes of other animals; notably the work of Anderson & Beams (1960), Franchi & Mandl (1962), Tsuda (1965) and Baker & Franchi (1967) on mammals, and Greenfield (1966) on the chicken.

The indifferent gonad and newly formed ovary of Xenopus laevis provide excellent material for the study of these initial stages of oogenesis. The primordial germ cells can be observed in the indifferent gonad of stage 48–52 (Nieuwkoop & Faber, 1967) tadpoles. Subsequent stages in tadpole development reveal the sequential appearance within the immature ovary, initially of oogonia (stage 54/55) and by stage 56, groups of oocytes at the primary stages of meiotic prophase. Moreover, it has proved possible to recognize the majority of stages in the adult ovary which correspond to those visible in the differentiation of the early tadpole ovary. However, minor differences in ultrastructure between oogonia found in the tadpole and those seen in adult ovary of Xenopus are apparent.

The aim of the present study is therefore threefold. First, to describe the ordered series of ultrastructural changes that occur in the formation of the young oocyte from the primordial germ cell, and so provide information about the earliest period of oogenesis which has so far received little or no attention in amphibians. Secondly, to illustrate that many of the associations between organelles characteristic of the older oocyte (e.g. the Balbiani body) clearly have their origins in the oogonia, if not earlier. Finally, to provide a means of identifying, largely on the basis of ultrastructural criteria, the stages observed in the formation of the early oocyte (up to diplotene of the first meiotic prophase) in the definitive ovary of Xenopus laevis.

The observations reported in this investigation were made on the indifferent gonad and newly formed ovarian tissue removed from larvae and mature ovarian material excised from adult females of Xenopus laevis. The premeta-morphic animals were staged according to the scheme proposed by Nieuwkoop & Faber (1967). Up to stage 56, the genital ridges of the larva are somewhat difficult to remove without damage. Therefore, the gonads of these tadpoles were fixed in situ attached to the larval mesonephros. From stage 57 onwards, the whole ovary was excised, as were small portions of adult ovary, immediately prior to fixation.

The fixative which has proved most satisfactory for Xenopus gonad in our experience is 1 % aqueous osmium tetroxide buffered with veronal acetate (pH 7·4) according to the technique of Palade (1952). Fixation was carried out at approximately 5 °C for . After dehydration in a graded ethanol series, the tissue was transferred to propylene oxide before infiltration and embedding in Araldite. Serial 1 μm thick and ultrathin sections were cut from the same blocks on a LKB Ultratome using glass knives. The 1 μm sections were mounted on glass slides, stained with 1 % toluidine blue in 1 % borax at 60 °C for 1–2 min and used for light-microscopical examination of the tissue. The ultrathin sections were mounted on uncoated grids, stained with 2% aqueous uranyl acetate and lead citrate (Reynolds, 1963) and examined in a Philips EM 300 electron microscope at 60 kV. All sections and measurements presented in this paper were made from tissue fixed by the Palade technique. Although considerable tissue shrinkage has been experienced when using a variety of alternative fixatives containing glutaraldehyde, they have served to substantiate the observations made on material fixed in osmium tetroxide alone.

Primordial germ cells

It has proved extremely difficult to locate primordial germ cells with any degree of certainty in the genital ridges of Xenopus larvae prior to stage 48. By this stage, despite the limited size of the gonadal ridges, sections 1 μm thick frequently reveal a single primordial germ cell surrounded by epithelial cells lying within the indifferent gonad (Fig. 1 A). The primordial germ cell is easily distinguished in these thick sections from the enveloping epithelial cells by its relative size (average diameter approximately 17 μm), highly lobed nucleus containing one or two large, compact nucleoli and somewhat diffuse chromatin (i.e. low toluidine blue affinity compared with that of the epithelial cell nuclei).

Figure 1.

(A) Light micrograph of a 1 μm Araldite cross-section of the indifferent gonads of a Xenopus larva at stage 48, showing a single primordial germ cell surrounded by epithelial cells.

(B) A cross-section of the indifferent gonads of a stage 51 larva. Note the larger gonads and increase in number of primordial germ cells compared with the stage 48 gonads shown in Fig. .1 A.

(C) A low-power electron micrograph of the indifferent gonad shown on the left of the dorsal mesentery in Fig. 1B, illustrating the detailed structure of the gonad at this stage. The primordial germ cells are easily distinguished from the surrounding epithelial cells by their relative size and characteristic irregularly shaped nucleus.

Figure 1.

(A) Light micrograph of a 1 μm Araldite cross-section of the indifferent gonads of a Xenopus larva at stage 48, showing a single primordial germ cell surrounded by epithelial cells.

(B) A cross-section of the indifferent gonads of a stage 51 larva. Note the larger gonads and increase in number of primordial germ cells compared with the stage 48 gonads shown in Fig. .1 A.

(C) A low-power electron micrograph of the indifferent gonad shown on the left of the dorsal mesentery in Fig. 1B, illustrating the detailed structure of the gonad at this stage. The primordial germ cells are easily distinguished from the surrounding epithelial cells by their relative size and characteristic irregularly shaped nucleus.

By stage 51 the indifferent gonads have enlarged considerably and appear conical in cross-section, being located laterally to the dorsal mesentery (Fig. 1B). The number of primordial germ cells has also increased, one section containing perhaps two or three germ cells situated within one gonad. A low-power electron micrograph of one of the gonads illustrated in Fig. 1B shows more of the detailed structure of the indifferent gonad at this stage (Fig. 1C). The more or less round primordial germ cells now measure about 20 μm in diameter.

The fine structural detail of a primordial germ cell in Xenopus is shown in the electron micrograph in Fig. 2A. As observed in the 1 μm sections, the primordial germ cell nucleus has a highly lobular profile, contains at least one large nucleolus and relatively diffuse chromatin. The double-layered nuclear envelope is perforated by numerous, irregularly spaced pores, closely associated with which are small fragments of electron-dense granular material (Fig. 2B). This granular material resembles the ‘nuage material’ reported by several authors to be found in the vicinity of nuclear pores in older amphibian oocytes (Swift, 1965; Kessel, 1966, 1968; Massover, 1968).

Figure 2.

(A) Electron micrograph of a primordial germ cell in the indifferent gonad of a stage 51 larva showing the juxtanuclear organelle aggregate and highly lobed nucleus containing a large, prominent nucleolus.

(B) High-power electron micrograph of part of the nuclear membrane of a primordial germ cell, illustrating the electron-dense, granular material associated with the nuclear pores (arrowed).

(C) The primordial germ cell organelle aggregate shown in Fig. 2 A at higher magnification. Note the presence of a nucleolus-like body, electron-dense granular material, some of which is associated with mitochondria within the aggregate (arrows), a small lipid body, and pigment granule.

(D) A high-power electron micrograph showing the association between electron-dense, granular material and mitochondria in the perinuclear cytoplasm of a primordial germ cell. There is a distinct similarity between the granular material in association with these mitochondria and the ‘nuage material’ adjacent to the nuclear pores (arrowed).

Figure 2.

(A) Electron micrograph of a primordial germ cell in the indifferent gonad of a stage 51 larva showing the juxtanuclear organelle aggregate and highly lobed nucleus containing a large, prominent nucleolus.

(B) High-power electron micrograph of part of the nuclear membrane of a primordial germ cell, illustrating the electron-dense, granular material associated with the nuclear pores (arrowed).

(C) The primordial germ cell organelle aggregate shown in Fig. 2 A at higher magnification. Note the presence of a nucleolus-like body, electron-dense granular material, some of which is associated with mitochondria within the aggregate (arrows), a small lipid body, and pigment granule.

(D) A high-power electron micrograph showing the association between electron-dense, granular material and mitochondria in the perinuclear cytoplasm of a primordial germ cell. There is a distinct similarity between the granular material in association with these mitochondria and the ‘nuage material’ adjacent to the nuclear pores (arrowed).

The primordial germ cell cytoplasm is rich in small vesicles but devoid of any conventional endoplasmic reticulum (Fig. 2C). The majority of the mitochondria appear to be filamentous (average dimensions in section approximately 2·5 × 0·4μm) and contain many transverse cristae (Fig. 2C, D). There is also a marked tendency for a large proportion of the mitochondria to be aggregated on one side of the nucleus (Figs. 1C, 2 A). Small patches of dense, granular material similar in appearance to that associated with the nuclear pores (Fig. 2B) are frequently interspersed between the mitochondria of this aggregate (Fig. 2C, D). Another consistent feature of the primordial germ cell cytoplasm is the presence of large, regularly shaped, electron-opaque bodies similar in size and appearance to nucleoli (Fig. 2C). These round bodies are readily visible in 1μm sections and display a basophilic staining reaction with toluidine blue not unlike that of the nucleoli. In many respects they are similar to the cytoplasmic bodies described by Kessel (1969) in Rana pipiens oocytes. The apparent resemblance of these bodies to nucleoli at both light-and electron-microscope levels prompted Kessel to refer to them as cytoplasmic nucleolus-like bodies. Our observations confirm the presence of these cytoplasmic nucleolus-like bodies in the female germ cells of Xenopus laevis, but at much earlier stages (namely primordial germ cells, oogonia and early oocytes) than has previously been reported in other anurans (Kessel, 1969). However, the nucleolus-like bodies in Xenopus germ cells differ from those of Rana oocytes in that they seldom have mitochondria or vesicles closely associated with them. Moreover, the granular content of the nucleolus-like bodies in Xenopus is distinctly finer than that of the nucleoli proper (see section on oogonia for Figs.).

In addition, the cytoplasm of primordial germ cells in Xenopus invariably contains large lipid bodies adjacent to the mitochondrial aggregate (Fig. 1C), and a small number of pigment granules (Figs. 1C, 2 A). However, throughout this investigation yolk material has not been encountered in the primordial germ cell cytoplasm of stage 48–52 Xenopus tadpoles.

Oogonia

The oogonia are readily detected in the newly formed ovary of Xenopus larvae at stage 54, presumably arising as a result of mitotic division of the primordial germ cells. The tadpole ovary is characterized by the appearance of an ovarian cavity lined with medullary cells (Fig. 3 A). Oogonia at this stage are arranged in the cortical region of the gonad and measure between 17 and 24 μm in diameter. Each oogonium is enveloped by a small number of flattened follicle cells which are closely applied to the oogonial cell membrane (Figs. 3A-C, 5 A). The overall appearance of the oogonia at both light-and electron-microscope levels is essentially similar to that of the primordial germ cell, the most characteristic feature being the highly lobed nucleus (Figs. 3B-D, 5 A, B, E).

Figure 3.

(A) A light micrograph of a 1 μm Araldite cross-section of a newly developed ovary of a Xenopus larva at stage 54, showing the distribution of the oogonia in the cortex of the gonad. The ovary is characterized by the appearance of an ovarian cavity lined with medullary cells.

(B) Two adjacent oogonia in the ovary of a stage 54 tadpole seen at higher magnification under the light microscope. The oogonia are surrounded by a number of follicle cells. The oogonium denoted by an arrow is undergoing mitosis.

(C) Electron micrograph of an oogonium from the ovary of a stage 54 larva illustrating the detailed structure of the oogonium. The irregularly shaped nucleus contains numerous micronucleoli in addition to the large prominent nucleolus.

(D) As in the primordial germ cell, the majority of the mitochondria within the oogonium are found in an aggregate on one side of the nucleus. A nucleolus-like body is clearly visible within the aggregate and numerous micronucleoli are arranged peripherally in the nucleus of this larval oogonium.

Figure 3.

(A) A light micrograph of a 1 μm Araldite cross-section of a newly developed ovary of a Xenopus larva at stage 54, showing the distribution of the oogonia in the cortex of the gonad. The ovary is characterized by the appearance of an ovarian cavity lined with medullary cells.

(B) Two adjacent oogonia in the ovary of a stage 54 tadpole seen at higher magnification under the light microscope. The oogonia are surrounded by a number of follicle cells. The oogonium denoted by an arrow is undergoing mitosis.

(C) Electron micrograph of an oogonium from the ovary of a stage 54 larva illustrating the detailed structure of the oogonium. The irregularly shaped nucleus contains numerous micronucleoli in addition to the large prominent nucleolus.

(D) As in the primordial germ cell, the majority of the mitochondria within the oogonium are found in an aggregate on one side of the nucleus. A nucleolus-like body is clearly visible within the aggregate and numerous micronucleoli are arranged peripherally in the nucleus of this larval oogonium.

Fig. 3B illustrates two adjacent larval oogonia as seen in 1μm section. One of these oogonia is undergoing mitosis, and the other shows the irregularly shaped nucleus and large, dense nucleoli so typical of these early stages in germ cell differentiation in Xenopus. In the adult, the oogonia are present as groups or ‘nests’ between older oocytes (Fig. 5A).

The majority of both adult and larval oogonia have an area of high toluidine blue affinity to one side of their nuclei (Figs. 3 A, 5 A). This region also has several more intensely basophilic foci easily visible in the light microscope (Fig. 5A). Ultrathin sections of the oogonia reveal these dense cytoplasmic regions to be organelle aggregates (Figs. 3D, 5B) consisting for the most part of mitochondria. The foci within these cytoplasmic regions are found either to consist of clumps of electron-dense granular material (with which some of the mitochondria are closely associated) or nucleolus-like bodies (Figs. 3D, 4B, C, 5 B, D). Particularly in tadpole oogonia, it is common to find many of the mitochondria not included in the juxtanuclear aggregate lying close to the nuclear membrane and enclosed within the folds and lobes of the nucleus (Fig. 3C). Oogonial mitochondria are somewhat shorter than those of the primordial germ cells (average dimensions in section approximately 1·5 × 0·4 μm) and have both transverse and longitudinal cristae. In addition, tadpole oogonia possess some aberrant septate mitochondria which have not been observed in adult oogonia. These mitochondria appear constricted and may be partially or completely divided by a transverse septum (Fig. 4D).

The close associations between dense, granular material and some mitochondria observed in primordial germ cells (Fig. 2D) are more numerous in the oogonia. Similar associations have been reported in the cytoplasm of a variety of mammalian oocytes (Blanchette, 1961; Adams & Hertig, 1964; Hope, 1965; Odor, 1965), and in the differentiating neoblasts of planarians (Morita, Best & Noel, 1969). This association between granular material and mitochondria is in some instances so intimate, that the structural integrity of the mitochondria is lost in those regions of contact between the two components of the complex (Figs. 4C, 5D). The appearance of such ‘incomplete mitochondria’ within.these complexes has led some authors to speculate that these associations represent sites of mitochondriogenesis (see Kessel, 1969). It seems likely, however, that any impressions of mitochondrial formation from such associations is merely an artifact of the sectioning plane.

There is evidence from our observations that the granular material that becomes associated with the mitochondria may be of nuclear origin. Many sections reveal ‘strands’ of the material running between nuclear pores and mitochondria adjacent to the nuclear envelope (Fig. 4A-C). More particularly, there are instances where the granular, ‘nuage material’ associated with the nuclear pores seems to arise directly from fragmentation of the nucleoli (Fig. 4B). However, close scrutiny of the granular composition of the nucleolus and the material associated with the mitochondria in the cytoplasm reveals that the latter is considerably more finely granular than the nucleolonema (Fig. 4B, C). Fragmentation of the main nucleoli is a common feature of oogonia, and to a lesser extent primordial germ cells. It seems likely that this process gives rise to the numerous micronucleoli present within the oogonial nucleus (Fig. 3C, D).

Figure 4.

(A) Electron micrograph of part of a larval oogonium showing the possible nuclear origin of the dense, granular material associated with mitochondria in the perinuclear cytoplasm. The granular material associated with the nuclear pores (arrowed) has a similar electron density to that associated with the mitochondria in Fig. 4B and C.

(B) Fragmentation of the nucleoli as seen in a larval oogonium. There is a mass of granular material associated with several mitochondria close to the nuclear membrane (arrowed). Note that the granular content of this mitochondria-associated material is considerably finer than that of the nucleolus.

(C) Electron micrograph of part of a larval oogonium showing a cytoplasmic nucleolus-like body and an association between granular material and mito-chondria (arrowed). The difference in granule size between these cytoplasmic masses and the nucleolus is clearly visible.

(D) A high-power electron micrograph showing one of the aberrant mitochondria encountered in the cytoplasm of larval oogonia. The mitochondrion is completely divided by a transverse septum in a constricted region of the organelle (arrowed).

Figure 4.

(A) Electron micrograph of part of a larval oogonium showing the possible nuclear origin of the dense, granular material associated with mitochondria in the perinuclear cytoplasm. The granular material associated with the nuclear pores (arrowed) has a similar electron density to that associated with the mitochondria in Fig. 4B and C.

(B) Fragmentation of the nucleoli as seen in a larval oogonium. There is a mass of granular material associated with several mitochondria close to the nuclear membrane (arrowed). Note that the granular content of this mitochondria-associated material is considerably finer than that of the nucleolus.

(C) Electron micrograph of part of a larval oogonium showing a cytoplasmic nucleolus-like body and an association between granular material and mito-chondria (arrowed). The difference in granule size between these cytoplasmic masses and the nucleolus is clearly visible.

(D) A high-power electron micrograph showing one of the aberrant mitochondria encountered in the cytoplasm of larval oogonia. The mitochondrion is completely divided by a transverse septum in a constricted region of the organelle (arrowed).

The nucleolus-like bodies described in Xenopus primordial germ cells are also found in the cytoplasm of oogonia (Figs. 3D, 4C, 5A), but unlike primordial germ cells, pigment granules have not been detected in the oogonial cytoplasm. The majority of the cytoplasmic organelles tend to be concentrated in the region of the mitochondrial aggregate in oogonia. These organelles include large numbers of small vesicles (Fig. 5F), Golgi bodies (Fig. 5F), centrioles (Fig. 5F), lamellated bodies with granular centres (Fig. 5 E) and lipid bodies, all of which contribute to the juxtanuclear aggregate. In addition, systems of annulate lamellae have been observed in the cytoplasm of adult oogonia but not in the cytoplasm of larval oogonia (Fig. 5E).

A distinctive feature of oogonia is the presence of fine, unimembranous, tubular vesicles within the nucleus in close association or in some cases clearly continuous with the inner nuclear membrane. Similar vesicles can be seen in both transverse and longitudinal section adjacent to the outer nuclear membrane (Fig. 5C), and resemble those described by Hsu (1963) in the developing oocytes of the tunicate Boltenia villosa.

Figure 5.

(A) Light micrograph of a 1 μm section of a group of oogonia in the ovary of an adult Xenopus. These ‘nests’ of oogonia are found lying between older oocytes. The areas of high toluidine blue affinity within the oogonial cytoplasm are denoted by arrows. Note the intensely basophilic foci within these areas.

(B) Low-power electron micrograph of one of the oogonia shown in Fig. 5 A. The adult oogonium, like its larval counterpart, has a highly lobed nucleus containing several micronucleoli, a cytoplasmic mitochondrial aggregate and associated granular material.

(C) Part of an adult oogonial nucleus showing the presence of unimembranous, tubular vesicles in association with the inner and outer nuclear membranes.

(D) High-power electron micrograph of associations between electron-dense, granular material and mitochondria in the perinuclear cytoplasm of an adult oogonium.

(E) Low-power electron micrograph of an adult oogonium illustrating the cytoplasmic lamellated bodies with granular centres and annulate lamellae.

(F) Some of the components of the cytoplasmic organelle aggregate in an adult oogonium. In addition to mitochondria, the aggregate contains numerous small vesicles, Golgi bodies and a centriole.

Figure 5.

(A) Light micrograph of a 1 μm section of a group of oogonia in the ovary of an adult Xenopus. These ‘nests’ of oogonia are found lying between older oocytes. The areas of high toluidine blue affinity within the oogonial cytoplasm are denoted by arrows. Note the intensely basophilic foci within these areas.

(B) Low-power electron micrograph of one of the oogonia shown in Fig. 5 A. The adult oogonium, like its larval counterpart, has a highly lobed nucleus containing several micronucleoli, a cytoplasmic mitochondrial aggregate and associated granular material.

(C) Part of an adult oogonial nucleus showing the presence of unimembranous, tubular vesicles in association with the inner and outer nuclear membranes.

(D) High-power electron micrograph of associations between electron-dense, granular material and mitochondria in the perinuclear cytoplasm of an adult oogonium.

(E) Low-power electron micrograph of an adult oogonium illustrating the cytoplasmic lamellated bodies with granular centres and annulate lamellae.

(F) Some of the components of the cytoplasmic organelle aggregate in an adult oogonium. In addition to mitochondria, the aggregate contains numerous small vesicles, Golgi bodies and a centriole.

Early oocytes

The oocytes with which this study is primarily concerned are those in the initial stages of the first meiotic prophase (i.e. up to early diplotene). After the completion of mitosis, the oogonia are transformed into oocytes. The characteristic sequence of chromatin transformations that takes place in the initial stages of meiosis within the oocyte nucleus has been referred to as a ‘premeiotic phenomenon’ (Raven, 1961). It is mainly the oocytes in this premeiotic or generative phase of oogenesis which fall within the scope of this investigation. The observations reported here were made on Xenopus oocytes up to about 50 μm in diameter, and therefore include some remarks on the early diplotene phase (i.e. oocytes that have commenced the growth phase). However, since a comprehensive study at the fine structural level has already been undertaken on the differentiation of Xenopus oocytes from about 50μm upwards (Balinsky & Devis, 1963), it was our intention to study the stages in germ cell differentiation prior to those covered by these previous workers. The observations on the early diplotene oocyte in the present study are conveniently terminated at about the 50 μm stage when the characteristic Balbiani body has been fully formed.

All the stages in early oocyte differentiation described were recognizable in both larval and adult ovary, although the nuclear changes throughout early prophase were most conveniently studied as a succession in developing tadpole ovaries. For descriptive purposes, the meiotic prophase is conventionally divided into several substages. However, it is as well to remember that this subdivision is merely a convenient way of labelling a continuous process, and consequently transitionary phases between substages are invariably encountered.

Preleptotene

The first signs of oocyte formation are the appearance in stage 55 tadpole ovaries of groups of cells (average diameter approximately 18 μm) with large, round nuclei and vesiculated nucleoli (Fig. 6 A). It is not possible to detect any changes in the nature of the chromatin at this stage. The highly lobed nucleus so characteristic of the oogonium has been lost and it is concluded that this cell type represents the first visible transition to oocyte formation. It is possible that these cells are either in the pre-meiotic interphase or in the so-called preleptotene stage. These ‘preleptotene’ oocytes are also seen in the ovaries of larvae at later stages of development and to some extent in the adult ovary. The nucleus fills the majority of the cell at this stage and the mitochondria appear to be distributed throughout the cytoplasm, there being little or no sign of the juxtanuclear aggregate present in the oogonium. However, the aggregate would need to be dissociated during oogonial mitosis if the mitochondria were to be segregated equally between the daughter cells, therefore lending support to the view that these cells represent an immediately post-mitotic stage.

Figure 6.

(A) Light micrograph of a 1 μm cross-section of one lobe of the ovary from a stage 55 Xenopus larva, illustrating the appearance of groups of early meiotic oocytes in the cortex of the ovary. Note the regular shape of the oocyte nucleus compared with that of the oogonial nucleus.

(B) A group of early meiotic oocytes from a stage 56 larva seen under higher magnification in the light microscope. Oocytes at leptotene, zygotene or pachytene may be found within a single group. Oocytes at the Bouquet stage (zygotene) are usually distinguishable in light micrographs (arrowed). The polarization of the chromosomes with respect to the basophilic cytoplasmic area (the organelle aggregate) is quite apparent in these oocytes.

(C) Electron micrograph of two adjacent oocytes from a group similar to that shown in Fig. 6B. These oocytes are at an early leptotene stage, when the chromosomes have contracted and thickened just sufficiently to be resolved (arrowed). Note the reticulate appearance of the nucleolus and the undulating nuclear membrane.

(D) Part of the nucleus of a leptotene oocyte showing two axial elements in longitudinal section and surrounded by a fibrillar network. Numerous dark ‘granules’ (arrowed) probably represent axial elements in cross-section.

Figure 6.

(A) Light micrograph of a 1 μm cross-section of one lobe of the ovary from a stage 55 Xenopus larva, illustrating the appearance of groups of early meiotic oocytes in the cortex of the ovary. Note the regular shape of the oocyte nucleus compared with that of the oogonial nucleus.

(B) A group of early meiotic oocytes from a stage 56 larva seen under higher magnification in the light microscope. Oocytes at leptotene, zygotene or pachytene may be found within a single group. Oocytes at the Bouquet stage (zygotene) are usually distinguishable in light micrographs (arrowed). The polarization of the chromosomes with respect to the basophilic cytoplasmic area (the organelle aggregate) is quite apparent in these oocytes.

(C) Electron micrograph of two adjacent oocytes from a group similar to that shown in Fig. 6B. These oocytes are at an early leptotene stage, when the chromosomes have contracted and thickened just sufficiently to be resolved (arrowed). Note the reticulate appearance of the nucleolus and the undulating nuclear membrane.

(D) Part of the nucleus of a leptotene oocyte showing two axial elements in longitudinal section and surrounded by a fibrillar network. Numerous dark ‘granules’ (arrowed) probably represent axial elements in cross-section.

Leptotene

By stage 56 the ovary contains clusters of oocytes which may be in leptotene, zygotene or pachytene stages (Fig. 6B). During leptotene, the chromosomes shorten and thicken sufficiently to be resolved under the electron microscope. The leptotene chromosomes appear as single axial elements surrounded by a fibrillar network (Fig. 6D). Towards the end of leptotene, the ends of the axial elements become attached to the inner surface of the nuclear membrane prior to synapsis (Fig. 7A). The nuclear membrane at this stage is somewhat undulating and becomes noticeably more electron-opaque at the points of attachment of the axial elements (Fig. 7 A). The nucleolus of the leptotene oocyte is normally reticulate (Fig. 6C). An interesting feature of the attachment of the axial elements to the nuclear membrane is the consistent proximity of these points of attachment to the juxtanuclear mitochondrial aggregate (Fig. 7 A). During leptotene the mitochondrial aggregate has reformed and becomes situated close to the nuclear membrane, frequently forming a ‘cap’ over one end of the nucleus (Figs. 6C, 7A).

Figure 7.

(A) Electron micrograph of part of an oocyte at the leptotene/zygotene stage illustrating the attachment of axial elements to the inner surface of the nuclear membrane immediately prior to pairing of homologues. Note the proximity of the cytoplasmic organelle aggregate in relation to the region of attachment of the axial elements.

(B) A zygotene oocyte showing the appearance of the Bouquet arrangement as seen under the electron microscope. The polarization with respect to the organelle aggregate is again quite noticeable. The nucleolus during pre-diplotene stages is often fragmented or reticulate. Axial elements have commenced synapsis so forming the synaptonemal complex configuration over short regions with a pronounced array of lateral, fibrillar projections.

(C) Under higher magnification, the tripartite ribbon structure of the synaptonemal complexes at zygotene is readily distinguished when seen in longitudinal section. There is an increase in electron density of the nuclear membrane at the point of lateral element attachment, and the lateral elements become noticeably thicker in this region (arrowed). As in Fig. 7A and B, the synaptonemal complexes are polarized with respect to the organelle aggregate.

(D) High-power electron micrograph of a part of a synaptonemal complex at pachytene, illustrating in more detail the structure of the complex as seen in longitudinal section. The thick, lateral elements are clearly visible with the much finer central element running between them. Note also the spiral twisting of the synaptonemal complex, the associated fibrillar network, and the appearance of the complex when viewed in cross-section (arrowed).

Figure 7.

(A) Electron micrograph of part of an oocyte at the leptotene/zygotene stage illustrating the attachment of axial elements to the inner surface of the nuclear membrane immediately prior to pairing of homologues. Note the proximity of the cytoplasmic organelle aggregate in relation to the region of attachment of the axial elements.

(B) A zygotene oocyte showing the appearance of the Bouquet arrangement as seen under the electron microscope. The polarization with respect to the organelle aggregate is again quite noticeable. The nucleolus during pre-diplotene stages is often fragmented or reticulate. Axial elements have commenced synapsis so forming the synaptonemal complex configuration over short regions with a pronounced array of lateral, fibrillar projections.

(C) Under higher magnification, the tripartite ribbon structure of the synaptonemal complexes at zygotene is readily distinguished when seen in longitudinal section. There is an increase in electron density of the nuclear membrane at the point of lateral element attachment, and the lateral elements become noticeably thicker in this region (arrowed). As in Fig. 7A and B, the synaptonemal complexes are polarized with respect to the organelle aggregate.

(D) High-power electron micrograph of a part of a synaptonemal complex at pachytene, illustrating in more detail the structure of the complex as seen in longitudinal section. The thick, lateral elements are clearly visible with the much finer central element running between them. Note also the spiral twisting of the synaptonemal complex, the associated fibrillar network, and the appearance of the complex when viewed in cross-section (arrowed).

Zygotene

Complete polarization of the chromosomes towards one end of the nucleus (adjacent to the mitochondrial aggregate) results in the typical Bouquet arrangement of zygotene (Fig. 7B). The axial elements become paired over short regions, particularly close to their points of attachment to the nuclear membrane (Fig. 7C). Once homologous axial elements have paired, they are referred to as lateral elements. Under higher magnification the chromosome structure in these paired regions is recognizably of the synaptonemal complex configuration, which has been described in a wide variety of animal and plant meiocytes (see Discussion). The now familiar tripartite structure is clearly visible, each synaptonemal complex consisting in longitudinal section of two thick, lateral elements and a fine, central element running between them (Fig. 7C, D). The lateral elements are distinctly thicker towards their point of attachment with the nuclear membrane (Fig. 7C). The width of a tripartite ribbon is approximately 1250Â, each lateral element varying in width from 290 to 480 Â and the central element measuring about 100Â in thickness. The outer edges of the lateral elements are surrounded by fibrillar material (Fig. 7B, D). The same structure can also be seen from cross-sectional views of the complex, the two lateral elements appearing as large ‘granules’ on either side of a smaller ‘granule’ representing the central element (Fig. 7D). As in leptotene, the nuclear membrane is undulating, perforated by regularly spaced pores with associated ‘nuage material’, and appears more electron-opaque at the sites of lateral element attachment (Fig. 7 C). The nucleolus is usually reticulate (Fig. 7B) and often fragmented, resulting in the formation of micronucleoli similar to those seen in the oogonia and primordial germ cells. The oocyte mitochondria throughout early prophase are longer and thinner than those in the oogonia (average dimensions in section approximately 2·0 ×0·2 μm), but are often found in association with fine, granular material in the same way as some of the mitochondria of primordial germ cells and oogonia.

Pachytene

Synapsis is completed during early pachytene but it is difficult to distinguish in thin sections from late zygotene unless complete pairing of homologous chromosomes is confirmed by serial sections. However, the disappearance of the polarization characteristic of zygotene and the dispersal of the synaptonemal complexes throughout the nucleus may give an indication that the pachytene stage has been reached. Also the synaptonemal complexes frequently show signs of spiralling over considerable lengths as they approach the fully synapsed condition (Fig. 7D). The appearance of the nucleolus, nuclear membrane, mitochondrial aggregate and associated organelles are unaltered from that seen in the leptotene and zygotene stages.

Early diplotene

Once the oocyte enters diplotene it commences its growth phase. At the early stages, when the oocyte is about 45 μm in diameter, the mitochondrial aggregate still appears as a ‘cap’ over one end of the nucleus. Gradually, however, as the oocyte and its nucleus enlarge, the mitochondrial aggregate appears to become more compact and spherical. It is the transformation to a more spherical aggregate which marks the appearance of the Balbiani body that has been described in many anuran pre-vitellogenic oocytes. It seems therefore that the Balbiani body has its origins in the mitochondrial aggregate as far back as the primordial germ cell, since the aggregate is a common feature of all the stages (with the possible exception of the immediately post-mitotic oocyte) in Xenopus germ cell differentiation.

Despite the wealth of information that has now accumulated on the complex morphological (see Norrevang, 1968, for review) and biochemical (see Davidson, 1968, for review) changes that take place during oogenesis, little attention appears to have been paid to the ultrastructure of primordial germ cells, oogonia and early meiocytes in amphibia.

Although Balinsky & Devis (1963) concluded from their work on Xenopus that the Balbiani body was absent from oocytes of less than 90 μm diameter, our observations on Xenopus germ cells suggest that a precursor of this body exists in the form of a cytoplasmic organelle aggregate as early as the primordial germ cell stage. In this respect it is noteworthy that Czolowska (1969) has reported there to be a considerable aggregation of mitochondria within the ‘germinal cytoplasm’ before it becomes clearly visible within Xenopus primordial germ cells. This aggregation of mitochondria is retained throughout the pre-vitellogenic stages of germ cell differentiation in Xenopus and would strongly suggest a localized mitochondriogenesis. This is in contrast to the situation observed in the axolotl (Al-Mukhtar, 1970), where the mitochondria are more evenly distributed throughout the germ cell cytoplasm. Furthermore, it seems likely that the Balbiani body is a highly dynamic structure that varies in its degree of compactness and number of mitochondria during the course of germ cell differentiation.

In terms of spatial arrangement there would appear to be a distinct correlation between the site of axial element attachment to the nuclear membrane during leptotene and the position of the juxtanuclear, mitochondrial aggregate. This cellular polarization is continued throughout zygotene and early pachytene, there being a consistent relationship between the distribution of the synaptonemal complexes and the mitochondrial aggregate. It is possible that the aggregation of mitochondria in this region reflects a physiological energy requirement during pairing of homologous chromosomes, since synapsis is thought to occur after the axial elements have attached to the inside of the nuclear membrane during late leptotene (Moens, 1969). Moreover, at the onset of vitellogenesis Balinsky & Devis (1963) maintain that the Balbiani body fragments to provide the subcortical layer with mitochondria. It is in this region that yolk is initially deposited; a process which it seems reasonable to assume also has a high energy requirement. Alternatively, the proximity of axial elements and mitochondria may be coincidental, the true association being between chromosomes and centrioles. The centrioles have been consistently observed adjacent to or within the mitochondrial aggregate of Xenopus oocytes (see Fig. 7C) and a similar situation has been reported in locust spermatocytes (Moens, 1969). The presence of mitochondria around the centriole would result from the polar accumulation of these organelles following their segregation at mitosis.

The first indication of the transformation of oogonium to oocyte and the commencement of meiosis is a change in nuclear outline. Such a nuclear transformation from lobate to round is probably symptomatic of the nuclear swelling one normally associates with the onset of cell division. An increase in nuclear volume is also indicative of the series of changes in chromosome ultrastructure that follow during the first meiotic prophase. The fine structural changes in the nucleus of amphibian oocytes at this stage do not appear to have been described previously, but they resemble closely those reported by other authors to take place in the oocytes (Franchi & Mandl, 1962; Tsuda, 1965; Greenfield, 1966; Baker & Franchi, 1967) and spermatocytes (see Moses, 1968, for review) of a wide variety of animals. The presence of the same basic structure in yet another meiocyte lends support to the view that the synaptonemal complex is of widespread occurrence within these cells in both plants and animals. Until recently, the generally accepted interpretation of the tripartite structure seen in zygotene and pachytene meiocytes appears to have been that the dense, lateral elements running parallel to each other in longitudinal section represent the synapsed homologous axial elements and the finer, central element between the lateral elements represents the pairing surface of the homologous chromosomes. However, recent work by Comings & Okada (1970) indicates that this interpretation may not be altogether correct. These authors concluded from their investigation of the enzyme sensitivity of synaptonemal complexes in a variety of spermatocytes that the structure visualized in the electron microscope is the proteinaceous ‘backbone’ on which the pairing of homologous chromosomes takes place. The synaptonemal complex functions therefore merely to bring homologues into close approximation so that precise base-base pairing can occur over relatively short distances.

The appearance of the fibrillar network projecting from the axial and lateral elements is somewhat reminiscent of the lateral loop system extending from the lampbrush chromosomes seen in the amphibian diplotene oocyte. Similar lateral fibres in spermatocytes have proved to be composed of chromatin, indicating that the lampbrush-type chromosome is not exclusively confined to the oocyte (Comings & Okada, 1970). It has been generally assumed that the lampbrush stage in amphibian oogenesis commences at the onset of diplotene and that lateral loop formation is a visual expression of genomic function. Much of our knowledge concerning the stockpiling of long-lived RNAs during amphibian oogenesis comes from investigations undertaken based upon these assumptions (e.g. Davidson, Allfrey & Mirsky, 1964; Crippa, Davidson & Mirsky, 1967). If the fibrillar network associated with the pre-diplotene chromosomes in Xenopus were shown to be DNase sensitive, it could prove interesting to attempt an analysis of gene products in these much younger oocytes.

Our observations support the view of previous workers on older amphibian oocytes (Ornstein, 1956; Balinsky & Devis, 1963; Takamoto, 1966; Massover, 1968 ; Kessel, 1969) that the cytoplasmic, granular material intimately associated with some mitochondria may have a nuclear origin and be extruded into the perinuclear region of the ooplasm via nuclear pores. In the light of these previous observations and those that have emerged from the present study on Xenopus, it now seems likely that this process may represent a prominent feature of the whole of the pre-vitellogenic stage of amphibian oogenesis. However, we are forced to record that on the basis of our observations there is at present no way of eliminating the possibility that the passage of this granular material is from cytoplasm to nucleus.

Although there are several reports in the literature of nucleoli being extruded from the nucleus of certain echinoderm oocytes (Kessel & Beams, 1963) and there is some evidence that nucleolar fragmentation may contribute to the formation of micronucleoli lying close to the nuclear membrane in Xenopus germ cells (see Fig. 4B), nucleolar extrusion has not been observed during the course of this investigation. It has been suggested by a number of authors (Ornstein, 1956; Swift, 1965; Kessel & Beams, 1968; Kessel, 1969) that nucleolar material undergoes some type of conformational change into fibrillo-granular material in the vicinity of the nuclear pores before it passes into the perinuclear cytoplasm in the form of ‘nuage material’. Here it may either become associated with mitochondria or condense to form nucleolus-like bodies. The significance of these cytoplasmic aggregates of granular material is not yet clear, but there has been speculation (see Kessel, 1969) that they contain DNA, RNA or RNP and are possibly involved in some form of informational transfer to mitochondria or in mitochondriogenesis. Preliminary investigations using high-resolution autoradiography and enzyme digestion have so far proved unsuccessful in demonstrating any nucleic acid content in either the mitochondria-associated material or nucleolus-like bodies, although both [3H]thymidine (Al-Mukhtar, 1970) and [3H]uridine (Webb, unpublished results) become incorporated into the nucleoli of Xenopus oogonia and early oocytes.

The authors wish to express their sincere gratitude to Dr F. S. Billett for his guidance throughout the investigation and helpful criticism of the manuscript. Our thanks are also due to Mr T. Courtenay for his invaluable technical assistance with the electron microscopy. K. Al-M. would like to record her appreciation to the University of Baghdad, Iraq, for the award of a Gulbenkian Foundation Scholarship. A. C. W. gratefully acknowledges the award of a Science Research Council Studentship.

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  • ae

    axial elements;

  •  
  • al

    annulate lamellae;

  •  
  • c

    cytoplasm;

  •  
  • ce

    central element;

  •  
  • chm

    chromosomes;

  •  
  • cn

    centriole;

  •  
  • dm

    dorsal mesentery;

  •  
  • ep

    epithelial ceil ;

  •  
  • f

    follicle cell;

  •  
  • fn

    fibrillar network;

  •  
  • g

    Golgi body;

  •  
  • gm

    granular material;

  •  
  • lb

    lamellated bodies;

  •  
  • le

    lateral elements;

  •  
  • lp

    lipid bodies;

  •  
  • m

    mitochondrion;

  •  
  • ma

    mitochondrial aggregate;

  •  
  • md

    medullary cells;

  •  
  • mn

    micronucleoli;

  •  
  • n

    nucleus;

  •  
  • nm

    nuclear membrane;

  •  
  • no

    nucleolus;

  •  
  • nob

    nucleolus-like body;

  •  
  • np

    nuclear pores;

  •  
  • oc

    oocytes;

  •  
  • og

    oogonium;

  •  
  • ov

    ovarian cavity;

  •  
  • p

    pigment granule;

  •  
  • pgc

    primordial germ cell;

  •  
  • sc

    synaptonemal complex;

  •  
  • tv

    tubular vesicle;

  •  
  • v

    vesicles.