A Study of the Cytoplasmic Inclusions and Nucleolar Phenomena during the Oogenesis of the Mouse

The present investigations were undertaken in order to determine the history of the cytoplasmic inclusions and the behaviour of the nucleolus during the growth of the oocyte of the mouse (Mus musculus). As the mouse ovum contains but little yolk it was thought that an investigation of the ovarian oocyte and the early stages of cleavage, using silver and osmic techniques, might throw further light on the behaviour of the Golgi bodies and mitochondria of the vertebrate egg.

Kingery (18) records that in the mouse ovary there is an embryonic proliferation of cells from the germinal epithelium and a second proliferation commencing in the young animal of about one to two days old and continuing almost to sexual maturity. The cells from the first proliferation degenerate and are resorbed, while those of the second form the definitive ova.

The development of the oocyte is divided into three stages: Stage A includes the very early oocytes derived from the germinal epithelium. Stage B is distinguished by a slight increase in the size of both cell and nucleus. Stage C is marked by further increase in the size of the oocyte: a follicle wall is usually present. The cell remains in this stage until ready for maturation.

According to Kingery (op. cit.) ovaries fixed in Helly’s, Zenker’s, and Benda’s fluid and mordanted in potassium bichromate, or fixed in Benda’s fluid and subsequently stained in copper-haematoxylin, revealed the mitochondria as granules situated round the nuclei of the germinal epithelial cells. As the oocytes increase in size the mitochondria come to lie in one end of the cell, forming a crescent-shaped mass; at the beginning of Stage C they are distributed, as granules, uniformly throughout the ooplasm. The mitochondria of the follicle-cells are described as threads, rods, and granules. Kingery does not deal with yolk-formation.

Lams and Doorme (21) do not deal with the early stages of oogenesis of the mouse; consequently the youngest ovum to be described and figured is of considerable size and is surrounded by follicle-cells. The mitochondria are revealed as (osmic fixation followed by iron-haematoxylin) large granules collected in groups or scattered singly through the ooplasm, and also as fine granules many of which form short chains. In a later ovum, which is undergoing maturation in the oviduct, the majority of the mitochondria are figured as collected into fairly compact masses distributed through the ooplasm; the individual granules, however, do not appear to be as large as in the earlier oocytes.

Globules occur at the periphery of the nearly fully formed oocytes; these are faintly brown-black after osmic acid, but appear as clear spaces after corrosive-acetic fixation. They are identified as fat-globules. In ova, undergoing maturation in the oviduct, plastic and deutoplasmic zones are present; the fat-globules and the majority of the mitochondria are situated in the latter. After the entrance of the sperm the mitochondria, although still in preponderance in the deutoplasmic zone, tend to be more evenly distributed in the cytoplasm.

In an egg containing a sperm nucleus a body coloured red by safranin and deep blue by haematoxylin is figured. This body is identified as a ‘pseudochromosome’ such as is described by Van der Stricht (27) as giving rise to mitochondria in the egg of the bat. In a slightly more advanced mouse ovum numerous small bodies, similar in staining reactions to the large ‘pseudochromosome’ of the earlier egg, are present. Lams and Doorme consider that the ‘pseudochromosomes ‘result from a condensation of mitochondria, and that at a later stage they break up to form mitochondria again.

Branca (5), referring to the ooplasm of the mouse egg, mentions that his observations agree with those of Lams and Doorme. His figures of oocytes in atretic follicles show granules and bodies which, apparently, correspond to the mitochondria and fat-globules respectively of Lams and Doorme.

Kingery (19), dealing with follicular atresia in the ovary of the mouse, states that the cytoplasm of degenerating eggs stains more deeply than in the normal, and that numerous fatglobules are present. He does not describe Golgi bodies or mitochondria.

From the above account it will be seen that although certain aspects of mouse oogenesis have been worked out in detail no work has been carried out on the Golgi apparatus or on the details of yolk-formation. Thus Lams and Doorme give a detailed account of oogenesis in the mouse, but owing to the fact that their researches were carried out at a time when work on the Golgi apparatus had not been greatly developed, there remains room for further inquiry into the form and behaviour of the cytoplasmic inclusions. Kingery’s papers, although of more recent date, are concerned chiefly with the growth of the oocyte, the nucleus and related phenomena, and with atresia. Consequently, he does not appear to have employed technique suitable for the demonstration of the Golgi apparatus.

For the study of the Golgi apparatus and mitochondria, ovaries were fixed according to the methods of Cajal, Da Fano, Mann-Kopsch, Kolatchev, and Flemming (without acetic).

The investigation of the nucleolar phenomena and of yolkformation was carried out in material fixed in Flemming, Carnoy, and corrosive-acetic fixatives. Certain ovaries were treated according to Ciaccio’s method for the identification of fats. As a further test for the presence of fats an ovary, fixed in Bouin’s picro-formol, was treated with ether and subsequently stained in iron-haematoxylin and counter-stained with eosin.

Certain mice were killed at short intervals after pairing, and the upper part of the oviducts fixed in Cajal, Mann-Kopsch, Bouin, and Carnoy fixatives.

In all cases the material was dissected out as speedily as possible and immediately placed in the fixing fluid. For the study of the Golgi apparatus and mitochondria, sections were cut 3 μ, and 5 μ. in thickness; the other material was cut in sections 5μ. and 8μ, in thickness.

The greater part of the present work was carried out in the Department of Natural History, University College, Dundee, while the investigations on the tubal eggs and a certain part of the work on the Golgi apparatus and nucleolar phenomena was worked out in the Department of Zoology, University of Edinburgh. I wish, therefore, to express my thanks for research facilities granted in these Departments. That part of the work carried out at Edinburgh University was aided by a grant from the Earl of Moray Endowment of the University of Edinburgh.

For the demonstration of the Golgi apparatus and mitochondria Cajal’s uranium nitrate and silver nitrate technique was found to be most satisfactory. In material treated by this method both Golgi bodies and mitochondria could be identified with ease. Consequently, the following account, unless where otherwise stated, refers to tissue treated according to Cajal’s technique.

In the early oocytes, which are situated in and below the germinal epithelium and have not yet acquired a definite follicle wall, a dark mass of material occurs at one pole of the nucleus, while smaller masses of similar appearance are scattered through the ooplasm (fig. 3, Pl. 39). Dark masses occupy similar positions in preparations treated by the Da Fano, Mann-Kopsch, and Kolatchev methods. Owing to the structure and disposition of these bodies, and to their reaction to silver and osmic techniques, they are identified as the Golgi apparatus.

The juxta-nuclear Golgi apparatus of the young oocytes consists of rods and granules closely massed together, so that in many cases part of the apparatus appears as a solid body. This solid appearance, however, is, in all probability, a fixation effect. Most of the smaller masses of Golgi material scattered through the ooplasm are closely similar to the larger, while a few seem to consist of single rods (fig. 3, Pl. 39).

The mitochondria are revealed, in untoned sections, as small granules of a golden-brown colour distributed in the ooplasm but especially numerous round the large mass of Golgi material (fig. 3, Pl. 39).

In the older oocytes, which have acquired a follicle wall consisting of a single layer of cells, the Golgi material is clumped at one pole of the nucleus so as to form a juxta-nuclear body similar in structure to that of the earlier oocytes (fig. 5, Pl. 39). Only a few rods and granules remain outside this mass. On comparing this condition with that of the younger cells described above, it is seen that the majority of the smaller masses of Golgi material have joined the larger, thus forming a dense juxta-nuclear body.

At this stage the mitochondria are scattered through the ooplasm but appear to be more numerous in the vicinity of the nucleus and Golgi apparatus.

In slightly older oocytes the Golgi apparatus begins to break up into the individual rods and granules of which it is composed. These become distributed through the ooplasm (figs. 6 and 7, Pl. 39), although in certain cases they remain more numerous, until a later stage of oogenesis, in that part of the ooplasm originally occupied by the juxta-nuclear body.

The mitochondria are now fairly evenly distributed in the ooplasm except at the periphery, where they are less numerous.

In the next stage the Golgi bodies are distributed through the ooplasm in the form of large granules or thick rods, while most of the mitochondria are collected into clumps (fig. 8, Pl. 39). In late oocytes, with a follicle wall of several cell-layers, the mitochondria are gathered into clumps, leaving the intervening ooplasm practically free of these bodies. The Golgi bodies occur distributed through the cell and on and around the mitochondrial clumps (fig. 9, Pl. 39). In the later oocytes in mature follicles this tendency of the Golgi material to collect round the groups of mitochondria is more marked; in many cases the mitochondria are difficult to observe owing to the number of the Golgi bodies around the clumps. In these oocytes the Golgi bodies appear to be more numerous than in the earlier cells (fig. 9, Pl. 39).

Additional evidence in favour of the above findings was produced by an examination of the material treated by the Da Pano and osmic techniques. The Da Pano preparations revealed oocytes at different growth stages, in which the form and disposition of the Golgi bodies and mitochondria agreed closely with the above account. In Mann-Kopsch and Kolatchev material the mitochondria were not so well shown as by the silver methods. They demonstrated, however, that while the mitochondria of the early oocytes are scattered through the ooplasm, those of the older cells occur in clumps. The Golgi material was revealed as deeply osmophil masses in close association with the nucleus of the very young cell, but in the later oocytes distributed as granules or rods occupying similar positions to those of the silver preparations.

In ovaries fixed in Flemming’s fixative (Gatenby’s modification) and subsequently stained in iron-haematoxylin and light green, the ooplasm of many of the young oocytes, situated below the germinal epithelium, was filled with small granules, while in certain parts of the cell, chiefly at one pole of the nucleus, denser clumps of larger granules or rods occurred. The small granules are undoubtedly mitochondria, while the larger ones, owing to their size and disposition, are identified as the Golgi bodies. The latter, due to the technique employed, are imperfectly preserved.

In young oocytes, with a single follicle layer, masses of darkly stained material occupy similar positions to the Golgi apparatus of the silver and osmio preparations. Mitochondria in the form of fine granules occur distributed through the ooplasm.

In the more advanced oocytes numerous dark granules are scattered through the cell. It was not possible to differentiate with certainty between Golgi bodies and mitochondria; this is due to the clumping of the mitochondria and to the imperfect preservation of the Golgi material. It should be noted that in many of the oocytes fixed in Flemming’s fixative no cytoplasmic granules were observed.

In ovaries treated according to Ciaccio’s method for the identification of fats, the fats in the corpora lútea and stroma stain brightly with Sudan 111. An examination of the early oocytes revealed bodies in the ooplasm which, although stained with Sudan 111, were not of the same shade as the fats mentioned above. These bodies were of various sizes and shapes, and stained orange-brown. A large number of oocytes at this stage of growth were examined and in all cases the orange-brown bodies occupied similar positions to the Golgi apparatus of the osmic and silver preparations (fig. 4, Pl. 39). Moreover, their subsequent behaviour is similar to that of the Golgi bodies, for, in the young oocytes, they form a mass at one pole of the nucleus and at a later stage become distributed in the ooplasm. In the older oocytes they are more difficult to distinguish than in the earlier cells. There can be no doubt that these are the Golgi bodies which give a reaction with Ciaccio’s technique, but do not stain brightly like the fats of the corpora lútea and stroma.

The oviducts treated by Cajal’s technique did not contain many ova, nor were those present as well preserved as the ovarian ova described above. This material, however, showed that the Golgi bodies are smaller in size and are more numerous than in the late ovarian ova, and that the mitochondrial clumps have increased in number (fig. 10, Pl. 40). An examination of preparations treated by osmio methods confirmed these findings and showed that the Golgi bodies and mitochondria tended to be more numerous towards one pole of the egg. The earliest tubal eggs observed in these preparations had already undergone maturation.

The Golgi apparatus of the cells of the germinal epithelium consists of a dark mass of material situated at one pole of the nucleus.

The mitochondria occur scattered through the cytoplasm (fig. 2, Pl. 39).

In Cajal preparations a dark juxta-nuclear body is situated at one pole of the follicle-cell nuclei, and is identified as the Golgi apparatus (figs. 5, 6, 7, and 8, Pl. 39). It appears to consist of a very close network, or possibly of rods and granules massed together as in the young oocytes. In the older follicles the Golgi apparatus is similar in appearance (fig. 9, Pl. 39).

The mitochondria, in the form of rods and granules, occur scattered through the cytoplasm, but in the cells of the young follicles are more numerous in the part of the cell adjoining the oocyte (figs. 5, 6, 7, and 8, Pl. 39).

It is worthy of note that the Golgi apparatus in follicles consisting of a single layer of cells is situated in the cytoplasm between the nucleus and the surface of the cell bordering on the oocyte (figs. 5, 6, 7, and 8, Pl. 39); in follicles consisting of several layers of cells the Golgi material, in the majority of cases, is localized at the pole of the nucleus situated towards the oocyte. The mitochondria are evenly distributed throughout the cytoplasm (fig. 9, Pl. 39). An examination of cells, in late follicles, surrounding the follicular cavity revealed the Golgi apparatus of many cells as situated between the nucleus and the surface of the cell directed towards the cavity; in other cases the position of the Golgi material varies from cell to cell. The probable significance of this phenomenon is discussed later (p. 715).

In material treated by Ciaccio’s method for the identification of fat, masses of material stain orange-brown with Sudan 111; they occupy similar positions to the Golgi apparatus of the follicle-cells as shown in silver and osmic preparations.

The Golgi apparatus of the theca-cells is in the form of a network or mass of closely applied rods and granules occupying a juxta-nuclear position. The mitochondria are distributed throughout the cell (fig. 1, Pl. 89).

The following investigations on the oocyte nucleolus and nucleolar emissions were carried out chiefly on material fixed in Carnoy, Flemming, and corrosive-acetic fixatives, and subsequently stained in iron-haematoxylin. The basophility of the nucleoli and nucleolar emissions was determined by preparations fixed in corrosive-acetic and stained in Mann’s methyl-blue eosin.

In the early oocytes, situated below the germinal epithelium, one to three nucleoli are usually present; in certain oocytes, however, three to five small nucleoli were observed (Text-fig. 1). In haematoxylin preparations the nucleoli are deeply chromophil, and as revealed by sections stained in Mann’s methyl-blue eosin, are basophil.

In young oocytes, with a single layer of follicle-cells, one to three basophil nucleoli are present. In sections stained in iron-haematoxylin the nucleoli are deeply stained homogeneous bodies (Text-fig. 2).

The nuclei of the slightly older oocytes (with about two layers of follicle-cells) contain numerous small homogeneous basophil bodies, many of which are in contact with the single nucleolus, while others are scattered through the nucleoplasm (Textfig. 8). The position and staining reactions of these bodies point to their nucleolar origin. In certain oocytes a second nucleolus was observed; in most cases the latter contained small vacuoles (Text-fig. 4). Both types of nucleoli and the nucleolar emissions are basophil.

The older oocytes, surrounded by several layers of folliclecells, usually contain a single deeply-stained nucleolus; in certain oocytes, however, a second vacuolated nucleolus was observed.

The nucleolar emissions are stained deeply by iron-haematoxylin and, as described for the previous stage, are basophil.

The nucleoli appear to be more faintly basophil than in the young oocytes (Text-fig. 5).

In the late oocytes, situated in mature follicles, the nucleolus has lost its spherical shape and, in many cases, appears to be giving rise to numerous nucleolar buds or emissions (Text-fig. 6). In some cases the central part of the nucleolus stained more faintly than the periphery. Both the nucleolus and the nucleolar emissions are basophil. It is of interest to note that a second nucleolus was present in certain of the late oocyte nuclei examined; in these cases both nucleoli stained in the usual manner.

The presence in the older oocytes of nucleolar buds or emissions close to, and in contact with, the inside of the nuclear membrane suggests that these bodies are extruded to the ooplasm. An examination of the late oocytes, surrounded by several layers of follicle-cells, produced further evidence in favour of this view; for numerous small bodies occur on the outside of the nuclear membrane and scattered through the ooplasm in the vicinity of the nucleus and towards the periphery of the cell (Text-figs. 5 and 6). These bodies are closely similar in shape and staining properties to the nucleolar emissions situated in the nucleoplasm; in size they resemble the smaller nucleolar buds. These extrusions were particularly well shown in preparations fixed in Carnoy and in corrosive-acetic.

In no case were nucleolar extrusions observed passing through the nuclear membrane, although in a few oocytes the nuclear membrane seemed to be pushed outward by the presence of emissions on its inner surface. This appearance was probably produced during fixation or the subsequent manipulation of the sections.

Owing to the presence of nucleolar buds in the nucleoplasm and to the occurrence of the same type of structure on both sides of the nuclear membrane, there appears to be little doubt that the bodies situated in the ooplasm are of nucleolar origin. The nucleolar extrusions are, in all probability, passed through the nuclear membrane in solution and become condensed on reaching the ooplasm.

In some of the late oocytes, in fully-formed follicles, many of the nucleolar extrusions are more lightly stained than those of the younger oocytes, while numerous small faintly-stained granules occur scattered through the ooplasm (Text-fig. 6). The appearance and position of these granules suggest that they may have been formed by the fragmentation of the less deeply-stained nucleolar extrusions. No direct evidence in favour of this view was produced.

In young oocytes, with about two layers of follicle-cells, a number of bodies make their appearance in the ooplasm; they are situated, in the majority of oocytes, towards the periphery but may also occur in the vicinity of the nucleus. These bodies are roughly spherical or somewhat egg-shaped; they stain deeply with iron-haematoxylin except the central portion, which, in many cases, stains but lightly, thus giving to the bodies a vacuolated appearance (Text-figs. 3 and 4).

In order to determine the nature of the cytoplasmic bodies ovaries were treated according to Ciaccio’s method for the identification of fat. The fat present in the corpora lútea and in the stroma was deeply stained by Sudan 111, while the cytoplasmic bodies failed to give the correct reaction. Consequently, it was assumed that these bodies were non-fatty. As a further test sections from an ovary fixed in Bouin’s picro-formol were treated with ether in order to extract any fats which might be present; these sections were subsequently stained in iron-haematoxylin and counter-stained in eosin. An examination of this material showed that the cytoplasmic bodies stained with eosin and were unaffected by the ether. A further proof of the non-fatty nature of these bodies was revealed by the fact that they did not blacken in ovaries fixed by osmic methods. As the result of these tests there can be no doubt as to the non-fatty nature of the cytoplasmic bodies; consequently, they are identified as protein yolk-spheres.

The method of yolk-formation could not be determined with certainty. In the young oocytes, before the nucleolar extrusions make their appearance in the ooplasm, a few yolk-globules are present; in the late oocytes the yolk-globules are slightly more numerous, and in the majority of cases they have increased in size (Text-figs. 5 and 6). It seems likely that the nucleolar extrusions fragment into granules and that the latter become dissolved in the ooplasm, their substance, in all probability, being added to the yolk-spheres already present. This matter is discussed further on p. 717.

The amount of yolk present in all stages of the growth of the oocyte is scanty; even the late oocytes contain few more yolkspheres than are present in the young cells (Text-figs. 3, 4, 5, and 6).

An examination of sections of the upper part of oviducts, fixed in Bouin’s fixative, showed that the yolk-globules in the unsegmented ova are situated towards one pole of the cell (Text-fig. 7). Yolk-globules were identified in sections of the two-cell stage; they appear to be fairly evenly distributed between the two cells (Text-fig. 8).

As atresia in the mouse has been described by several workers the following account is confined to the description of the cytoplasmic inclusions. For a summary of previous work on atresia see Branca (5).

An examination of oocytes, which, although situated in atretic follicles, had not yet undergone degenerative fragmentation, revealed the majority of the mitochondria (Cajal preparation) as clumped together, forming large masses, while a few occurred scattered through the cell. The Golgi bodies are distributed in small groups or are closely applied to the mitochondrial masses.

Certain of the Flemming material contained a greater number of atretic follicles than were present in the silver preparations; for this reason and also because the corresponding nuclear changes could be followed, thus enabling the stages of degeneration to be determined, the behaviour of the cytoplasmic inclusions was carefully studied in that material. A comparison with the other preparations enabled the Golgi bodies and mitochondria to be identified.

In eggs containing the first spindle the mitochondria occur in clumps and are also scattered through the cell. The Golgi bodies are situated chiefly towards the pole opposite the spindle (fig. 11, Pl. 40).

At a later stage the mitochondrial clumps are larger and denser, and the Golgi bodies are, for the most part, situated towards one pole of the egg (fig. 12, Pl. 40).

In eggs which have fragmented into several pieces the majority of the mitochondria are still clumped and the Golgi bodies distributed unevenly through the cytoplasm. Large vacuoles may be present (fig. 13, Pl. 40).

The number and disposition of the yolk-globules in the early stages of atresia appear to be the same as in the normal oocyte. In the later stages, however, yolk could not be identified. During atresia the cytoplasm becomes filled with numerous fatglobules, and in some cases ‘crystalloid bodies’ were also observed.

In material treated according to Ciaccio’s technique irregularly-shaped granular masses were observed in the cytoplasm; these stain faintly with Sudan 111 and correspond in position to the masses of mitochondria and Golgi elements revealed by silver and osmic methods. It is of interest to note that in most cases fat-globules are more numerous in the vicinity of these granules than in other parts of the cell (Text-fig. 9).

The above account of the position and behaviour of the juxta-nuclear Golgi apparatus appears to agree fairly closely with the findings of other workers on the Golgi material of the mammalian ovum. The actual structure of the Golgi apparatus of the oocyte has been the subject of a certain amount of disagreement; consequently, before discussing the present findings it is necessary briefly to refer to certain papers on vertebrate oogenesis.

The first observation on the Golgi apparatus in eggs was carried out by Sjovall (26), working on the ovum of Cavia, who states that the Golgi apparatus of the young oocyte is in the form of a hollow sphere situated at one pole of the nucleus. Later, the sphere breaks up and the fragments pass to the periphery. Weigl (Nihoul, 23) and Kulesch’s findings (20) agree with those of Sjôvall.

According to Rio Hortega (25) the Golgi apparatus of the early oocyte of the guinea-pig and rabbit is in the form of a network occupying a juxta-nuclear position. The Golgi elements of later oocytes, in primary follicles, are figured as scattered through the ooplasm in the form of a loose mesh-work or collections of threads united by short extensions. In the older eggs the Golgi material occurs as a loose network of thick threads situated towards the periphery so that a clear space is left surrounding the nucleus.

Gatenby and Woodger (10) believe that the mammalian Golgi apparatus ‘consists of numerous semi-lunar plates or rods and not of branched straight bodies as drawn by Hortega’; the appearance of branched rods, they state, is possibly due to distortion caused by formalin fixation.

Cattaneo (6) states that the Golgi apparatus of the young oocyte of the bat, guinea-pig, and rabbit is in the form of a network situated at one pole of the nucleus. In the older oocytes the apparatus increases in size, breaks up, and passes to the periphery, where it forms a sort of fenestrated membrane in the neighbourhood of the pellucid zone.

Henneguy (16), in a short note, states that the Golgi apparatus of the young oocyte of the guinea-pig occurs as ‘quelques amas irréguliers de petits cordons granuleux’. They are disposed without order in the ooplasm, but in the late oocyte, surrounded by a follicle with a follicular cavity, they are situated at the periphery.

Nihoul (23) believes that the Golgi apparatus of the young oocyte of the rabbit consists of grains or bâtonnets forming a compact mass at one pole of the nucleus. Later, the Golgi apparatus fragments into several masses which pass to the periphery of the cell. With the growth of the egg it is probable that the substance of the Golgi apparatus increases. In silver preparations of eggs at this stage the Golgi apparatus is constituted ‘par une série de travées sans structure, ou présentant une structure finement granuleuse, et anastomosée’. A tangential section of an egg which has just reached this stage gives the impression of a fenestrated membrane similar to that described by Cattaneo. In sections treated according to Weighs method the Golgi material seems to be formed of filaments which appear as closely entangled and compact masses. The filaments are sometimes curved and give the impression of vesicles of which the wall is strongly coloured and the contents uncoloured. Nihoul believes that the appearance of the Golgi material in silver preparations is due to precipitation on and between the filaments.

The present findings for the mouse agree with those mentioned above in that the Golgi apparatus of the young oocyte is at first in the localized condition and later breaks up and becomes scattered through the ooplasm. In the mouse ovum, however, the Golgi bodies are fairly evenly distributed throughout the cell; furthermore, the individual Golgi elements appear as rods and granules and do not form a loose mesh-work or masses of entangled filaments, as described for the bat, guinea-pig, and rabbit. The present writer believes that the juxta-nuclear Golgi apparatus of the early oocyte is composed of rods and granules which have come together to form a compact mass; this agrees with Nihoul’s description of the young oocyte of the rabbit.

It is of interest to note that the localized Golgi apparatus of the early oocytes of certain other vertebrates has recently been described as consisting of collections of individual Golgi elements. Thus Brambell (4) has shown that the Golgi material of the young oocyte of the fowl consists of rods and granules surrounding the centrosphere. Bhattacharya and Lal (2) have figured the Golgi elements in the young oocyte of the tortoise, Kachuga, as spherical or granular bodies forming a fairly compact mass beside the nucleus; while more recently Nath (22) states that, in unstained Champy preparations, the Golgi material of the young oocytes of Rana tigrina is present as granules occupying a juxta-nuclear position. In Da Fano and Kolatchev material ‘they tend to appear as one compact body’. Later, they increase in size and finally become distributed throughout the ooplasm.

As previously stated (p. 698) Lams and Doorme described two types of mitochondria in the oocyte of the mouse—large granules collected into groups or scattered singly through the cell, and fine granules which in some cases were arranged in short chains. In the opinion of the present writer the large granules of Lams and Doorme are Golgi bodies which, owing to the methods employed, were confused with the mitochondria. It has been shown, in the present paper, that the Golgi elements in the diffuse state tend to collect on the clumps of mitochondria and that imperfectly preserved Golgi bodies are present in certain of the Flemming preparations. It is evident, therefore, that without the aid of osmic and silver methods, suitable for the demonstration of the Golgi material, it would be impossible to distinguish with certainty between the two types of cytoplasmic inclusions.

In an ovum undergoing maturation Lams and Doorme figure the majority of the mitochondria as collected into fairly compact masses; the larger granules appear to be smaller than is the case in the young oocytes. This grouping of the mitochondria agrees with the present findings; furthermore, the Golgi granules and rods of the late ova are slightly smaller than those of the ovarian oocytes and apparently correspond to the large type of mitochondria of Lams and Doorme.

The mitochondria of the early oocyte are more numerous in the neighbourhood of the localized Golgi material; this agrees with Kingery’s observation (18) that as the oocyte increases in size the mitochondria come to lie at one end of the cell. The present findings do not agree with Kingery’s statement that the mitochondria form a crescent-shaped mass at one end of the cell. According to Kingery the mitochondria later become evenly scattered throughout the cell; the present writer, however, finds that they are less numerous at the periphery, and that in the older oocytes they become collected into clumps.

In view of previous work it is of interest to note that the Golgi material stained faintly with Sudan 111. Bowen (3) has pointed out that, although it is impossible to arrive at any definite opinion as to the chemical nature of the-Golgi apparatus, Ciaccio (7) found that the area of the Golgi apparatus of the testis of the mouse stained with Sudan 111, while Karpova (17), and Parat and Painlevé (24) obtained positive results with the male germ-cells of Helix, and Weiner (Bowen, 3), with a modification of Ciaccio’s technique, with the cells of the intestinal epithelium of the mouse.

Henneguy (16) states that in the young follicle of the guineapig, when it consists of a single layer of cells, the Golgi apparatus is situated between the nucleus and the surface of the cell in contact with the oocyte. In follicles in which the granulosa consists of several layers the situation of the Golgi apparatus varies from cell to cell. In the cells of the discus proligerus the Golgi material is situated in the part of the cell directed towards the follicular cavity. Henneguy believes that the follicle-cells which surround the young oocyte secrete a substance which is absorbed by the latter, and that with the appearance of the follicular liquid, which is the product of the cells, an inversion in the situation of the Golgi apparatus takes place.

The position of the Golgi apparatus in the follicle-cells of the mouse, described in the present contribution, is closely similar to that of the Golgi apparatus in the follicle-cells of the guineapig previously recorded by Henneguy. In the opinion of the writer the situation of the Golgi material in the young follicle of the mouse strongly suggests that it takes part in the formation of a secretion which is utilized by the growing oocyte. In appearance it closely resembles the polarized Golgi apparatus of gland-cells; the latter, however, is so well known as not to require discussion in this paper. The inversion in the position of many of the Golgi bodies in older follicles would seem to support Henneguy’s suggestion that the Golgi material plays some part in the formation of the follicular liquid.

As the mitochondria of the young follicle-cells are more numerous in the part of the cell next to the oocyte, it is reasonable to suppose that the mitochondria, as well as the Golgi apparatus, may take part in the formation of a secretion which is absorbed by the oocyte.

Attention has already been directed to the occurrence, in degenerating ova, of granules of varying size which take on a faint coloration, and to large fat-globules which stain brightly with Sudan 111 after treatment according to Ciaccio’s method for the identification of fats. The masses of smaller granules correspond in position to the clumps of mitochondria and Golgi elements of osmic and silver preparations. The larger granules are apparently Golgi bodies which stain more deeply with Sudan 111 than do those of the normal oocyte. Their staining reaction, together with the occurrence of granules intermediate in size between the smaller granules and the fat-globules, suggests that the Golgi elements, in degenerating eggs, give rise to fat.

Bell (1) has recently described the origin of neutral fats from the Golgi bodies of the spermatid of the dog, while the origin of fatty yolk from the Golgi element of the oocyte has been recorded by several workers. Reference to recent work on fatty yolk-formation has been made in previous contributions by the present writer (11 and 12).

As many of the granules are extremely small and as the surrounding cytoplasm appears to stain faintly with Sudan 111, thus forming a faintly granular background to the larger granules, it is possible that the mitochondria, as well as the Golgi elements, may give rise to fat-globules.

The number of nucleoli observed during the present investigation agrees with Kingery’s findings (18); the occurrence of nucleolar extrusions, however, does not appear to have been previously recorded for the mouse ovum. It is of interest to note that although Lams and Doorme (21) do not mention nucleolar emissions they figure what seem to be nucleolar buds in an oocyte nucleus.

The occurrence of nucleolar emissions on the inside of the nuclear membrane, and of closely similar bodies outside the nuclear membrane and scattered through the ooplasm, offers strong evidence in favour of the view that nucleolar material is extruded from the nucleus. It is highly probable that this material is passed through the nuclear membrane in solution and is condensed into granular form on reaching the ooplasm. This view of nucleolar extrusion has been put forward by Harvey for Carcinus (13) and for Antedon (15).

The presence of small granules in the ooplasm suggests that the nucleolar extrusions finally fragment in a somewhat similar manner to that recently described by the writer (12) for Periplaneta.

The yolk-globules present in all stages of the mouse oocyte are not numerous; they appear chiefly towards the periphery of the oocyte, but a few may be situated in the neighbourhood of the nucleus. They were not observed to arise in relation to the cytoplasmic inclusions.

Harvey (13 and 14) has recently stated his belief that protein yolk arises under the influence of the mitochondria and Golgi bodies from material derived from the plasmosome, ground cytoplasm, and from external sources. If this be true it is probable that the mitochondria, Golgi elements, or both, play some part in the formation of the scanty yolk of the mouse ovum. Material derived from the nucleolar extrusions may be added to the yolk-globules, as suggested by Harvey (13) for the protein yolk of Carcinus. Owing to the large number of nucleolar extrusions present, and to the scanty amount of yolk, it is probable that much of the nucleolar material remains dissolved in the ooplasm, possibly in the form of nutriment which is utilized at a later stage.

Lams and Doorme (21) state that fat-globules are present at the periphery of the mouse oocyte and that they are faintly brown-black after osmic acid and appear as clear spaces after corrosive-acetic fixation. The present investigation shows that these globules, as demonstrated by Ciaccio’s method, by the action of ether, acetic and osmic acid, are non-fatty in nature. The only ooplasmic fat-globules present are those of the oocytes situated in atretic follicles. These fats stain (Ciaccio’s method) in a similar manner to those of the corpora lútea and stroma cells; the latter have recently been described by Deanesly (8 and 9).

Fat-globules in degenerating eggs of the mouse have previously been recorded by Kingery (19) and by Branca (5). The ‘crystalloid bodies’ described by Kingery were observed during the present investigation.

1. The Golgi apparatus of the germinal epithelium consists of a dark mass of material situated at one pole of the nucleus. The mitochondria occur scattered throughout the cytoplasm.

2. The Golgi material of the very early oocyte consists of rods and granules clumped together to form a large body at one pole of the nucleus; smaller masses of Golgi material may also be present.

3. In the young oocyte, surrounded by a follicle wall, a single juxta-nuclear body is present; at a later stage the individual Golgi elements break away from the juxta-nuclear body and become distributed throughout the ooplasm.

4. In the late oocytes the Golgi elements occur in close association with the mitochondrial clumps and also scattered through the ooplasm. In tubal eggs the Golgi bodies are smaller in size and more numerous than in the ovarian ova.

5. It is concluded that the large mitochondria of Lams and Doorme correspond to the oocyte Golgi elements of the present contribution. The behaviour of the Golgi material during the growth of the ovum resembles that of the eggs of other mammals. The present findings on the structure of the juxta-nuclear Golgi material agrees with Nihoul’s account for the rabbit.

6. The mitochondria of the young oocytes occur scattered through the ooplasm, but are more numerous in the vicinity of the nucleus and Golgi material. Later, the majority of the mitochondria become collected into clumps; in the tubal eggs the mitochondrial clumps are more numerous.

7. The Golgi apparatus of young follicles is situated between the follicle-cell nucleus and the pole of the cell directed towards the oocyte; in follicles consisting of several layers the position of the Golgi apparatus varies, while in fully-formed follicles the Golgi material of many of the cells surrounding the follicular cavity are directed towards the cavity. This agrees with Henneguy’s findings for the Golgi apparatus of the follicle-cells of the guinea-pig. The mitochondria of the follicle-cells occur scattered through the cytoplasm but are more numerous towards the pole of the cell adjoining the oocyte.

8. The number of nucleoli present in the early oocyte varies from one to five; the majority of the older oocytes contain a single nucleolus but two may be present. Extrusion into the ooplasm of nucleolar material takes place; the nucleoli and the nucleolar extrusions are basophil (Mann’s methyl-blue eosin).

9. Fatty yolk is not present in the mouse ovum. It is suggested that the Golgi elements and mitochondria play some part in yolk-formation, and that some of the granules formed by the fragmentation of the nucleolar extrusions are added to the yolkglobules already present. The yolk-globules of unsegmented tubal eggs are situated towards one pole of the cell; at the twocell stage they appear to be evenly distributed between the two cells.

10. In degenerating eggs the mitochondria are clumped; the Golgi bodies occur in small groups or are closely applied to the mitochondrial clumps. In eggs which have undergone fragmentation the Golgi bodies occur in groups, while the majority of the mitochondria are clumped. The fat-globules, previously recorded by Kingery in degenerating eggs, were identified. In material treated by Ciaccio’s method for the identification of fats, appearances suggest that the Golgi elements, and possibly the mitochondria, give rise to fat. Yolk-globules could not be distinguished in the late stages of these eggs.

Lettering

F.G, follicle-cell; G, localized Golgi material; G.E, Golgi element;

M, mitochondria; M.C, mitochondrial clump; N, nucleus; Nu, nucleolus; V, vacuole.

PLATE 39.

Fig. 1.—Theca-cells showing Golgi apparatus and mitochondria. Cajal.

Fig. 2.—Germinal epithelium showing Golgi apparatus and mitochondria. Cajal.

Fig. 3.—Early oocyte; follicle wall not formed. Cajal.

Fig. 4.—Oocyte at same stage as fig. 3. Ciaccio.

Fig. 5.—Oocyte with single layer of follicle-cells. Cajal.

Fig. 6.—Slightly later stage showing Golgi elements breaking away from juxta-nuclear clump. Cajal.

Fig. 7.—Later stage; most of the Golgi elements have become distributed throughout the ooplasm. Cajal.

Fig. 8.—Older oocyte with single layer of follicle-cells; the majority of the mitochondria are collected into clumps; the Golgi elements are distributed throughout the ooplasm. Cajal.

Fig. 9.—Late oocyte; Golgi bodies and mitochondria clumped in ooplasm; the position of the Golgi apparatus of the follicle-cells varies from cell to cell. Cajal.

PLATE 40.

Fig. 10.—Part of ovum from upper part of oviduct. Cajal.

Fig. 11.—Oocyte from atretic follicle showing arrangement of Golgi elements and mitochondria. Flemming.

Fig. 12.—Later stage of degeneration. Cajal.

Fig. 13.—Egg which has fragmented into several pieces. Flemming.