The Oogenesis of Calanus Finmarchicus. by Irene F

The Oogenesis of Calanus finmarchicus. By Irene F. Hilton. M.Sc, Lecturer in Zoology, University of Edinburgh.

With Plates 9 and 10, and 5 Text-figures.

CONTENTS. PAGE

1. INTRODUCTION ...... 193 2. MATERIAL AND METHODS ...... 194 3. LITERATURE ...... 195 4. GENERAL ACCOUNT ...... 196 5. NUCLEUS, NUCLEOLUS, AND NUCLEOLAR EXTRUSION . . 200 6. THE MITOCHONDRIA ...... 207 7. YOLK-FORMATION ...... 211 8. THE GOLGI APPARATUS ...... 213 9. DISCUSSION 215 10. SUMMARY 218 11. LIST OF REFERENCES...... 220 12. EXPLANATION OF FIGURES ...... 220

INTRODUCTION. THE present investigation was undertaken with a view to working out the details of oogenesis in a Copepod, about which comparatively little is known from the modern cytological standpoint. Calanus finmarchicus was suggested by Mrs. E. C. Bisbee, of the Department of Zoology, University of Liverpool, as a suitable subject; it is easily obtainable in quantity and has relatively large eggs; moreover the nucleolus in its behaviour throughout the growth of the egg presents certain very interesting features. The work was carried out partly in the Department of Biology, University College of Swansea, and partly in the Department of Zoology in the University of Edinburgh. 194 IRENE F. HILTON

MATERIAL AND METHODS. The specimens of Calanus finmarchicus (Gunnerus, 1770) used in the present investigation were obtained mainly from Plymouth, but also from the Marine Laboratories at Port Erin, Isle of Man, and at Millport, Buteshire. The animals were examined at intervals of about fourteen days during the greater part of two years. Some difficulty was experienced in obtaining satisfactory fixation of the ovary. Dissecting out the ovary before fixation was not found practicable but by cutting off the abdomen at the first joint before immersing in the fixing fluid, rapid penetration of the fixative was obtained with improved fixation of the deli- cate posterior end of the ovary. Memming-without-acetic acid followed by iron haematoxylin (4 or 5 hours in iron alum and overnight in 0-5 per cent, haematoxylin) generally gave the best results, but Bouin's fluid and corrosive sublimate were found excellent as nuclear fixatives. The Champy-Kull method gave good results in the older oocytes approaching maturation. For the study of the mitochondria the methods of Kolatschev, Nassonov ('Microtomist's Vade-Mecum', 9th edition, page 345), and Champy-Kull were used. Flemming-without-acetic acid followed by iron haematoxylin was particularly successful in older oocytes. For the study of the Golgi apparatus Da Fano's method gave the best results. The methods of Nassonov, Kolatschev.. Kopsch. and Mann-Kopsch were tried without success. The author acknowledges her indebtedness to Dr. F. A. Mocke- ridge of the University College, Swansea, where the work was begun, to Professor J. H. Ashworth, of the University of Edin- burgh, to Professor W. J. Dakin, and Mrs. R. C. Bisbee, of the University of Liverpool, and Mr. L. A. Harvey, of the University of Edinburgh, for much helpful advice, and to the Committee of the Earl of Moray Endowment of the University of Edinburgh for a grant in aid of expenses. OOGENESIS OF CALANUS 195

LITERATURE. Although the Copepodaasa group have been widely studied in the past, comparatively little is known of the details of their gametogenesis from the modern cytological standpoint. Prior to the development of modern cytological methods attention was directed almost exclusively to the nucleus and its chromosome content. The chromosome numbers for thirty-five species of Copepods are recorded in the 'Tabulae Biologicae 4' (1927). The majority of the numbers given are for members of the genus Cyclops but some species of Gymnoplea are in- cluded. Among the Gymnoplea, with the exception of a Japanese form Diaptomus sp. Ishikawa (1891) and Dia- ptomus coeruleus (Amma, 1911) the haploid number of chromosomes present in the female is either sixteen or seventeen, sixteen being the more usual number. McClendon (1906, 1910) and Kornhauser (1915) both working upon parasitic copepods described the formation of ring-shaped double chromosomes by parasyndesis in a way which appears similar to that recorded for Calanus finmarchicus in the present investigation. Matscheck (1910) in a paper upon growth and development of copepod eggs recorded fragmentation of the nucleolus prior to the maturation divisions of the egg. This author also found formation of yolk in the half-grown oocytes and suggested a possible secretory function of the nucleolus. The more recent work of Ludforcl (1922, 1924) upon the morphology and physiology of the nucleolus provides strong evidence in support of this view. Gardiner (1927) working upon Limulus poly- p hem us has suggested a very specialized secretory function for the nucleolus in the transport of phosphorus to the cytosome. The study of Calanus finmarchicus affords strong evidence for suggesting that the mitochondria play an important part in yolk-formation. A close relationship between the mitochondria and yolk-formation has been recorded in the eggs of various animals by recent workers (Harvey, 1926; King, 1926; and Gardiner, 1917, and others). 196 IRENE F. HILTON

GENERAL ACCOUNT. The ovary is single and median and is situated dorsal to the alimentary canal. It extends about two-thirds of the entire length of the thorax and tapers to a blunt point at its posterior end. Prom the anterior end of the ovary paired oviducts arise and run forward as wide thin walled tubes. These lie parallel to each other, dorsal to the alimentary canal after leaving the ovary, but at the anterior end each oviduct bends ventrally and later- ally from its fellow and runs posteriorly, ventral to the alimentary canal. The two oviducts open together on the first ab- dominal segment close to the opening of the spermathecae. It appears probable that fertilization takes place as the eggs leave the oviducal opening. Three distinct zones can be observed in the ovary; (Text- fig.l). (1) A multiplication zone situated in the posterior part of the ovary and consisting of small cells—oogonia—undergoing mitosis. One or two cells at the tip of the ovary are larger than the other cells of the multiplication zone, these are probably primordial germ cells. (2) A narrow zone in which many of the cells show leptotene, pachytene, and synapsis stages in division. (3) A broad zone which occupies the whole of the anterior end of the ovary and which contains oocytes in a progressive series of growth phases. The anterior end of the ovary passes almost imperceptibly into the oviducts in which the later growth phases of the oocytes take place.

Multiplication Zone. The oogonia at the posterior end of the ovary are very closely packed. Two or three nucleoli are present in the resting stage of the oogonial nucleus (figs. 1 and 2, PL 9). Owing to some difficulty which was experienced in obtaining good fixation of this part of the ovary the nature of the oogonial nucleoli is not perfectly clear. It was observed that one nucleolus is usually conspicuously larger than the others. After fixation by chrome- osmium technique followed by staining with iron haematoxylin, OOGENBSIS OF CALANUS 197

TEXT-FIG. 1.

Diagram of longitudinal section through thorax showing zones of ovary, and arrangement of oocytes in the oviducts, m, multiplication zone; s, synapsis zone; g, growth zone; a, half-grown oocytes; 6, older oocytes approaching maturation. NO. 294 O 198 IRENE F. HILTON this larger nucleolus stains rather more lightly than the smaller ones. After Champy-Kull technique it stains a uniform amber colour and after Mann's methyl blue eosin, bright pink. It is a plasmosome and remains spherical in shape. The smaller nucleoli are irregular in outline and stain more deeply with iron haematoxylin. With Feulgen's ' Nuclealfarbung' method these nucleoli give a deep pink reaction: they are karyosomes. The final oogonial mitoses, which actually mark the initiation of the growth phases of the oocyte, are visible in fixed and stained preparations immediately behind the synapsis zone of the ovary. The nucleoli disappear, or at least lose their staining properties, before the breakdown of the nuclear membrane: the chromatin network resolves itself into a very fine spireme thread which assumes a marked spiral arrangement within the nucleus. This thread breaks up into a number of long twisted chromosomes which shorten and thicken to form small rods. After the disappearance of the nuclear membrane these chromosomes, of which there appear to be about thirty-four, arrange themselves upon the equator of the spindle; metaphase, anaphase, and telophase follow in rapid succession. The chromosomes of this final oogonial mitosis swell in the telophase, forming a deeply staining mass at each pole of the spindle. The details of the events immediately following are rather obscured by this process, but the chromatin in the daughter cells formed by the division passes into the resting stage.

Synapsis Zone. The resting stage of the nucleus of the early oocyte is of very short duration. Soon after the formation of the oocytes the nuclear network aggregates into masses which finally become drawn out to form a thread similar in appearance to the spireme thread of the early prophase nucleus of the oogonia. This thread breaks up into a large number of parts which become twisted and are extremely difficult to count in consequence. The chromatin then contracts to one pole of the nucleus and there forms a tangled, deeply staining knot. From this knot thick looped threads are seen projecting into the centre of the nucleus. Although no doubling of the threads was observed in the OOGENESIS OF CALANUS 199 leptotene stage, presumably this knot represents a synizesis figure in which the formation of bivalent chromosomes is taking place (fig. 4, PI. 9). The bivalent chromosomes do not appear until a much later stage, the entire growth period of the oocyte intervening between the initiation and completion of the first maturation division. The synizesis knot, after a period of condensation in Avhich it appears as a mass of chromatin at one pole of the nucleus, finally separates out to form a thread which closely resembles a spireme. This thread is much twisted upon it itself and surrounds the nucleolus Avhen this latter re-forms. The oocytes remain in this condition throughout the subsequent growth stages. Growth Zone. In the very young oocytes at the beginning of the growth stage, a large central nucleus is present surrounded by a thin layer of rather flocculent cytoplasm. One or two nucleoli are present, one of which is a large plasmosome. The karyosomes at this stage stain a decided purplish pink when treated by Feulgen's method. In slightly older oocytes only one large nucleolus is present which appears to be formed by the fusion of plasmosome and karyosomes and is therefore an amphinucleolus. Preparations made by Feulgen's method at this stage show a large clear body in the centre of the nucleus with a smaller pinkish body deeply embedded in its surface. This is interpreted as a stage in the fusion process of the plasmosome and karyosome. Preparations stained by the Champy-Kull method show the compound nucleolus as an amber-coloured body containing in its centre a varying number of highly refractive vesicles; these appear to be an intense bluish-green in colour. While this colour may be due to refraction, and the blue stain of the Champy-Kull method is a somewhat capricious one and cannot be taken as proving the presence of chromatin in the nucleolus, it appears probable from its mode of origin that chromatin is present in it at this stage. The time of egg-laying varies considerably in different localities and is prolonged over a considerable period. Copepods from the west coast of Scotland had shed their eggs in most cases by the o 2 200 IRENE F. HILTON beginning of June. Those from the south of England and from the Isle of Man were later and in some cases showed eggs in a very immature condition in mid-June.

THE NUCLEUS, NUCLEOLUS, AND NUCLEOLAR EXTRUSION. Oogonia and young Oocytes. In the oogonia and very young oocytes the nucleus, which is centrally situated, occupies the greater part of the cell and a well- marked nuclear membrane is visible. The nucleoplasm stains a pale uniform grey with iron haematoxylin. The chromatin is aggregated into a number of irregular, deeply staining masses which lie close to the nuclear membrane, thus leaving a clear space in the centre of the nucleus in which the plasmosome lies. The karyosome is situated at one side of the nucleus in the oogonia and very young oocytes. It is frequently very irregular in shape and small, consequently it is easily confused with the chromatin masses round the periphery of the nucleus (fig. 3, PI. 9). In preparations which were overstained with haematoxylin it retained the black stain more deeply than the surrounding chromatin. In slightly older oocytes the karyosome takes up a more central position and approximates towards the plasmosome, with which it finally fuses. Whether the other karyosomes when present fuse with the plasmosome or are used in chromosome formation it is impossible to say from the evidence available. No preparations were seen in which more than one karyosome appeared in the process of fusion with the plasmosome. In the older oocytes in which nucleolar extrusion was beginning only one nucleolus is present; this stains a uniform dark grey with iron haematoxylin and amber yellow with one or two highly refractive blue-green vesicles in the centre with Champy-Kull.

Nucleolar Extrusion. Nucleolar extrusion takes place very rapidly throughout the early growth phases of the oocyte in Calanus. Portions of nucleolar material varying in size from minute spheres no larger than the mitochondria, to spheres which are as large as the fully formed yolk-globules, are extruded from the surface of the OOGBNESIS OF CALANUS 201 nucleolus over the whole of its area. Although large spheres are seen in many preparations lying upon the surface of the nucleolus (Text-fig. 2 a), the nucleolus is not distorted and there is no indication that these extrusions arise by being pinched off. The nucleolar extrusions lying against the nuclear membrane are usually much smaller than those which are seen on or near the surface of the nucleolus; this suggests that the larger extrusions

TEXT-FIG. 2.

a. Nucleus of half-grown oocyte showing nucleolar extrusions upon the surface of the nuclear membrane in the form of small spheres, b. Nucleus of half-grown oocyte showing nucleolar extrusions upon the surface of the membrane in the form of a network, c. Portion of the nuclear membrane showing coincidence of drops of nucleolar material on the inside and outside, n ext, nucleolar extrusions; n r, nuclear reticulum. break up in their passage across the nucleus to the nuclear membrane. Since the extrusions are almost certainly of a liquid nature it is possible on the other hand that the drops are merely fixation effects, their size depending upon the concentration of extruded material in the region where they occur. In very young oocytes the material which is extruded from the nucleolus forms a deeply staining mass which appears as a thick black line in the region of the nuclear membrane in preparations stained with iron haematoxylin (Text-fig. 4 a). This deposit shows more clearly in slightly older oocytes. In sections 202 IRENE F. HILTON 3 to 5ju. thick of material fixed with Flemming-'without-acetic acid and stained by the long method in iron haematoxylin, a series of small black dots is seen closely apposed to the surface of the nuclear membrane. In some preparations the extruded material appears as a darkly staining reticulum lying upon the outer surface of the nuclear membrane (Text-fig. 2 b). In sections from 8 to 10/x thick including portions of the surface of the nuclear membrane the extrusions sometimes appear as spherules of variable size lying outside the membrane but closely apposed to it. These are obviously fixation effects which signify the presence of a condensation of nucleolar material outside the nuclear membrane. During the whole period of nucleolar extrusion there is present in the cytoplasm a varying number of deeply staining spheres which are in every way identical with the nucleolar extrusions inside the nuclear membrane. There can be no doubt that a large part of the nucleolar material is transported to the cytoplasm by the process of nucleolar emission. Although in young oocytes the majority of these spheres in the cytoplasm are found surrounding the nucleus, in the later stages of the growth of the oocyte they are sometimes found near the periphery of the cell. All these spheres in the cytoplasm pass gradually from a basophil to an acidophil condition, lose their staining properties and finally become invisible: presumably they are dissolved in the cytoplasm. In one or two preparations portions of the extruded material from the nucleolus appeared as though passing through the nuclear membrane into the cytoplasm (Text-fig. 2 c). While this apparently supports the view held by some cytologists that nucleolar extrusions are passed through the nuclear membrane into the cytoplasm as individual bodies, a consideration of the whole process in Calanus suggests that this does not take place. In any case it seems highly improbable that bodies of the size of nucleolar extrusions could pass through the nuclear membrane without losing their identity or rupturing the membrane. It is probable that the appearance of these preparations is due to the coincidence of drops of nucleolar material closely apposed to the outside and the inside of the nuclear membrane at the moment of fixation. On the outer OOGENESIS OF CALANUS 203 surface of the nucleus there must be a considerable confluence of liquid nucleolar material which has passed through the membrane by a process of diffusion. From this semi-fluid perinuclear layer the nucleolar material appears to condense out as drops which migrate outwards into the cytoplasm, and finally become dissolved in its substance. In the half-grown oocytes the nucleolus is no longer visible as a homogeneous mass in the centre of the nucleus, but is seen to consist of two distinct regions: a large central vacuole and a narrow outer rim. This appearance is constant with all fixatives and stains used, with the exception of Feulgen's method, which does not stain the nucleolus at this stage. In the younger oocytes the rim stains a uniform dark grey with iron haematoxylin, but in oocytes in which the process of nucleolar extrusion has been proceeding for a longer period a number of small vacuoles make their appearance in the rim (fig. 8, PI. 9). This indicates a reorganization of the nucleolar material following nucleolar extrusion. Occasionally smaller vesicles can be seen in the core of the nucleolus which give it the appearance of an alveolar structure, but more often a faint granulation is all that is visible. This may be due to the coagulation of liquid substances in the central vacuole.

The Chromatin. The nuclear reticulum of the half-grown oocytes is in the form of a fine network of much coiled interlacing threads. These threads are not of uniform thickness, but in places show small knots which probably represent aggregations of chromatin upon the linin network (fig. 8, PI. 9). This network, although showing no trace of individual chromosomes, may be regarded as a diffuse and much modified diplotene stage. In all preparations observed the nuclear reticulum was seen contracted to a greater or lesser extent away from the edge of the nucleus and was most marked in the region immediately surrounding the nucleolus. This contraction does not necessarily indicate shrinkage due to fixation but may be an actual condition representing a re- concentration of the chromatin. For a short period the chromatin in the older oocytes approaching maturation is in the form 204 IRENE F. HILTON of a continuous thread. No indications of a double nature can be seen in this thread. The maturation divisions of the egg usually take place in the ventral arms of the oviducts, although in some cases maturation appears to take place after the egg has left the oviduct. The early stages of the divisions of the ripe oocyte are very unstable, and the majority of the preparations examined during the maturation process showed either the metaphase or early anaphase position of the chromosomes upon the spindle. When the first maturation division has reached the metaphase there appears to be a distinct pause after which the final stages of the first division, the formation of the second spindle, and the completion of the second division are accomplished with great rapidity. It was impossible to say from the preparations examined whether the first polar body divides or not. The first maturation division, which is the true reduction division of the egg, enters upon its second phase with the breakdown of the chromatin reticulum to form bivalent chromosomes. During the period which intervenes between the first phase of the reduction division and the second phase, the entire growth of the oocyte takes place; changes occur in the cytoplasm and the greater part of the yolk is laid down. The nuclear membrane becomes constricted at one pole, the nucleus assuming an elongated pear shape as a result of the pressure of the eggs in the oviduct. At this time the nucleolus, which is comparatively quiescent during the later growth phases of the oocyte, enters upon a period of great activity. Quantities of nucleolar material are given off from the nucleolus and pass across the nucleoplasm into the cytoplasm. In fixed preparations this material appears in the form of spheres of considerable size which stain deeply with all basic dyes. After a short time in the cytoplasm the emissions lose their staining properties and become invisible (fig. 6, PL 9). The chromatin reticulum during this process undergoes a second contraction into a tangled mass from which circular, bivalent chromosomes emerge. These are extremely small and aggregated together towards one side of the nucleus; their structure is by no means clear but in the earlier stages the ring OOGENESIS OF CALANUS 205 forms are seen to be deeply indented at opposite poles. Prom these bivalent ring-shaped chromosomes tetrads are formed by the appearance of a transverse constriction in each half of the ring; condensation takes place, and the tetrad is reduced to a compact body in which the four components are plainly visible. The dissolution of the nuclear membrane begins at one pole of the nucleus and spreads rapidly. Immediately before its final disappearance, the nucleolus, which by this time is reduced to a small sphere, breaks up and passes into the cytoplasm. Throughout the period of the formation of the chromosomes a progressive loss in staining property is noticeable in the chromatin content of the cell. This is also true of the nucleolus, which finally shows a very similar staining reaction to the plasmosome of the early oocyte nucleus. The ring-shaped chromosomes when first formed stain faintly, but at a later stage Avhen they are arranged upon the spindle they stain deeply with all basic dyes. Just before the disappearance of the nuclear membrane the group of seventeen tetrads is seen situated close to the nuclear membrane, usually at the opposite side of the nucleus to the disintegrating nucleolus (Text-fig. 3). With the disappearance of the membrane the tetrads pass out into the cytoplasm and take up their position on the equator of the maturation spindle.

The Maturation Spindles. The spindles are small truncated structures 10-13JU, in length (fig. 13, PI. 10). Astral rays were not visible in any of the preparations examined but the longitudinal fibres were clearly visible. The spindles appear to be constructed of dense proto- plasm which shows slight acidophil reactions. When arranged upon the equator of the spindle the chromosomes are very difficult to count owing to their small size and close proximity. When examined upon the equatorial plate the chromosomes sometimes appear V-shaped, converging towards the centre of the spindle while their free ends point outwards. This is probably due to tension at the point of attachment to the spindle fibres. After the halves of the tetrads have separated they move apart rapidly and the final stages of the division which was begun before the growth of the oocyte are completed. 206 IRENE F. HILTON The spindle of the second maturation division is formed at right angles to that of the first, and in rapid succession. Presumably a rotation of the second spindle occurs. Portions of first spindle are occasionally seen in the cytoplasm during the second division. The second division is mitotic and separates the halves of the

TEXT-FIG. 3.

Nucleus of full-grown oocyte showing breakdown of nuclear membrane at a, tetrads {t) and final group of nucleolar extrusions. d, droplets; n ext, nucleolar extrusions; v, vacuoles. monovalent chromosomes. Presumably the tetrads become so orientated upon the spindle that synaptic mates are separated from each other in the first division. The behaviour of all the chromosomes is identical. The polar bodies may be observed flattened against the surface of the egg by the pressure of the walls of the oviduct. They are very small, about 5 or 6/x in diameter. The chromatin in the nucleus is in a very unstable condition throughout the growth phases of the oocyte. Tests were made for chromatin by Feulgen's method, with the one modification that sections were left for two hours in the fuchsin sulphurous acid, and afterwards washed very quickly in two changes of OOGENESIS OF CALANUS 207

S02 water before mounting. It was found that in the nuclei of the oogonia and very young oocytes the nuclear reticulum stained a distinct purplish pink. In at least two cases which showed the karyosome in process of fusion with the plasmosome, the karyosome alone was stained. Everything else in the cell was colourless. Throughout the remainder of the growth stages the oocyte showed no trace of colour when treated by this method, and the nuclear network was invisible. In cells which were undergoing maturation, however, the chromosomes upon the spindle Avere deeply stained. The affinity of the early oocyte karyosome for the stain coupled with the results obtained with the Champy-Kull method strongly suggests the presence of chromatin in the amphinucleolus during the early part of its history. There is no positive evidence for believing that chromatin is extruded from the nucleolus, but on the other hand the nucleoli of the more mature oocytes do not show any positive chromatin reaction. It is possible that the nucleolus acts as a reservoir for nucleic acid during the growth stages of the oocyte and that this is released previous to the formation of the chromosomes.

THE MITOCHONDRIA. It was found that in the stages oogonia to oocytes the mitochondria were progressively more resistant to acetic acid and were in no cases completely destroyed by it. A marked resis- tance to acetic acid is not uncommon in the mitochondria of germ cells. It has been recorded by Nath (1926) for the scorpion Palamnaeus. Structure and Distribution of the Mitochondria. The mitochondria are present in the oogonia and very young oocytes in the form of a cap of mitochondrial material situated at one pole of the cell and closely adpressed to the nuclear membrane (fig. 2, PI. 9). The cap is small and compact, and has clearly denned edges; it stains a dark uniform grey with iron haematoxylin following Flemrning-without-acetic fixation, and bright pink with Champy-Kull. Occasionally the mitochondrial 208 IRENE F. HILTON cap appears in the form of several isolated masses, generally situated at one pole of the cell. It is probable that these slight variations in number and form of the mitochondrial masses are due to varying degrees of coalescence either before or at fixation. No individual mitochondria are visible at this stage. As growth of the oocyte proceeds the cap breaks up and gradually spreads out, moving as it does so away from the nuclear membrane into the cytoplasm. The amount of mitochondrial material increases considerably during this spreading out, but it is impossible to be certain whether the individual elements which compose the mass arise de novo in the cytoplasm or by the division of pre-existing mitochondria. One or two preparations showed figures which might be interpreted as division stages of the mitochondria and this correlated with the fact that the new masses of mitochondria always arise near the older ones makes it appear probable that multiplication does take place in this way. In oocytes measuring 20-30^, in diameter, the mitochondria in the form of small spheres surround the nucleus and are situated about half way between the nuclear membrane and the edge of the cytoplasm (Text-fig. 4 a). In the younger oocytes gaps occur in this hollow sphere of mitochondria but in the older oocytes it is quite complete. Champy-Kull preparations at this stage show the mitochondrial masses to be composed of a large number of vesicles of varying sizes; the largest of these probably represent aggregations of several mitochondrial elements. With iron haematoxylin following Flemming-without-acetic, the elements which con- stitute the mass of mitochondrial material vary in size and shape from small spheres to filamentous structures which again probably represent a degree of coalescence. In all iron haematoxylin preparations observed the mitochondria appear to be arranged in a zone of cytoplasm which is darker and rather more flocculent in appearance than the surrounding medium. This appearance was observed to a lesser degree in preparations stained by the Champy-Kull method, but was not visible in material fixed in Nassonov or Kolatschev solutions which are specific for the mitochondria. While this cloud in the cytoplasm may be entirely OOGENESIS OF CALANUS 209 due to imperfect fixation of the mitochondria, it is also possible that it represents an accumulation of other substances which are present in the cytoplasm and come into relation with the mitochondria at this time. Nucleolar extrusion is very marked during the early growth phases of the oocyte and it is suggested that

TEXT-FIG. 4.

•n ext

a. Young oocytes showing mitochondrial ring and micleolar material lying on the nuclear membrane, b. Oocytes after dispersal of mitochondrial ring (magnification about half that of a), m, mitochondria ; n ext, nucleolar extrusions. this cloud may represent a concentration of dissolved nucleolar material in the region of the developing mitochondria. Woltereck in his paper upon growth and development of Ostracod eggs (1898) observed in Cypris a cap of material outside the nuclear membrane in the young oocytes which stained darkly with haematoxylin. This he described as a 'yolk nucleus' which in later stages of its development spreads out in the cytoplasm, sometimes appearing as small flocculent masses and at other times showing distinct granules of deeply staining substance upon a uniformly grey background. These various stages correspond so closely with the history of the development and gradual spreading of the mitochondrial ring in Calanus 210 IRENE F. HILTON finmarchicus that it seems probable the 'yolk nucleus' of Woltereck's description and the mitochondrial cap are identical structures. The individual mitochondria which are scattered in the cytoplasm at a later stage of development are much smaller than the vesicles and spheres, which probably represent groups of fused mitochondria. Before the oocytes are half grown the mitochondria begin to spread out through the cytoplasm. The spreading-out process by which the mitochondria pass from a stage when they are aggregated round the nucleus to a stage when they are more or less evenly distributed throughout the cytoplasm, begins at one point in the ring. The visible sign of this dispersal is the appearance of a well-marked gap which is first seen in oocytes measuring about SQfj, in diameter. In slightly older oocytes the ring is seen to have dispersed with the exception of certain aggregations of mitochondria which appear to be much more stable than the rest of the mitochondrial ring (Text-fig. 4 b). Occasionally two or three smaller groups remain, but one is the more usual condition. In half-grown oocytes traces of this last aggregation of mitochondria are visible which stain as a dark grey irregular mass with iron haematoxylin. It is significant that in a number of preparations yolk-formation was seen to be in progress near the periphery of the cytoplasm in this region (fig. 5, PL 9). In the older oocytes the individual mitochondria are visible as minute spherical structures; these stain dark grey with iron haematoxylin and pink with Champy-Kull; they are uniformly distributed throughout the cytoplasm. There is a tendency for individual mitochondria to aggregate in groups of four and five, but no fusion takes place in the dispersed condition (fig. 10, PL 10). Shortly after the dispersal of the mitochondria yolk-formation begins. The mitochondria swell and lose some of their staining properties. In their place small yolk-droplets appear which are at first arranged in small groups but which finally become scattered throughout the cytoplasm, where they enlarge. (See section on yolk-formation.) OOGENESIS OF CALANUS 211 In the mature oocytes the entire cytoplasm is packed with yolk-droplets and no mitochondria are visible (fig. 7, PI. 9).

YOLK-FORMATION. Although superficially the condition which is found in Calanus finmarchicus appears to support the view that yolk is formed by the direct chemical transformation of the mitochondria, a consideration of the other processes observed in the cell during the period of vitellogenesis leads the author to favour the view that a number of other factors are equally involved. The yolk which is present in the oocytes of Calanus finmarchicus appears to be homogeneous and non-fatty in composition. Generally speaking, yolk-formation is first observed in the half-grown oocytes before the final distribution of the mitochondria has taken place. The quantity of yolk present at this time and its position in the cell is subject to slight variation. It may take the form of a group of well-marked droplets, situated at one side of the cell, or a few small droplets irregularly scattered near the periphery. It is impossible to say with certainty that this early formed yolk has any direct relation to the mitochondrial masses still visible in the young oocyte, but subsequent events suggest that this is the case; furthermore, it has been observed that where a group of mitochondria remain in the cytoplasm, the yolk-droplets are more numerous in the region of the cytoplasm lying between this mitochondrial group and the periphery of the cell (fig. 5, PI. 9). At a later stage the mitochondria which are dispersed swell and stain much more deeply, finally becoming replaced in position by yolk-droplets (fig. 10, PL 10). This replacement of the mitochondria by yolk-droplets proceeds from the periphery of the cell inwards until the cytoplasm is packed with yolk. This is the condition found in the eggs which have undergone maturation and are situated near the posterior end of the oviducts. While it is obvious in this case that the mitochondria are intimately connected with yolk-formation', it is impossible to say with certainty from the observed facts whether the scattered 212 IRENE F. HILTON mitochondria are directly transformed into yolk by a chemical change, or whether they serve as reservoirs for materials deposited in them at this time. The period of most rapid yolk-formation appears to be that immediately preceding the breaking down of the nuclear membrane in the ripe oocyte. This period coincides with a rapid and final activity on the part of the nucleolus. During the formation of the chromosomes this splits into fragments, the parts passing out into the cytoplasm, where they are finally dissolved. It is significant that the most rapid period of yolk-formation in the egg of Calanus should coincide with this final period of nucleolar extrusion. If the nucleolar extrusions play any part in yolk-formation, and if, as was suggested, they come into relation with the mitochondrial ring during its formation, what appears to be a precocious formation of yolk in the region of the mitochondrial masses can be partially explained. In the section on the mitochondria certain cases were described in which the mitochondrial masses appeared to be surrounded by a darkly staining cloud in the cytoplasm. It was suggested that this cloud might indicate the presence of an accumulation of dissolved material in the region of the mitochondria and that this material is probably nucleolar in origin. In these cases yolk-droplets were frequently seen near the mitochondrial mass. About the time when yolk makes its first appearance in the cell, changes were observed in the structure and staining reactions of the cytoplasm, which in the young oocytes is flocculent in appearance and oxyphil. Throughout the growth phases of the oocyte a gradual change from oxyphily to basophily has been observed, the cytoplasm at the same time becoming denser and more granular in appearance. In the older oocytes a return to a condition of secondary oxyphily takes place and the cytoplasm loses its granular appearance, becoming at first flocculent and later highly vacuolated. The beginning of vacuolation corre- sponds with the onset of yolk-formation (fig. 9, PL 9). The vacuoles in the cytoplasm are filled with a watery fluid which condenses out in some preparations as large drops which stain a greenish grey with iron haematoxylin and yellowish with Champy-Kull. When visible these drops are always seen in OOGENESIS OF CALAMUS 218 association with yolk-droplets. It is possible that they represent accumulations of substances passing from the cytoplasm to the mitochondria during the formation of yolk therein (fig. 9, PI. 9). With the exception of one or two doubtful cases the Golgi elements have, so far, not been observed in the younger oocytes, and it is therefore impossible to say whether or not they play any part in yolk-formation. In the older oocytes no visible connexion was observed between individual Golgi elements and yolk-droplets, nor was their position in any way correlated with the region of yolk-formation in the cell. It is suggested therefore, that yolk in Calanus finmarchi- c u s is formed in the mitochondria by the transformation of part of their own substance and the deposition in them of substances derived from the nucleolus and the cytoplasm.

THE GOLGI APPARATUS. In the oogonia and very young oocytes fixed with Memming and stained with iron haematoxylin, deeply-staining spherical structures were seen closely pressed against the nuclear membrane in some preparations. Although adjacent to it, these structures appeared quite separate from the mitochondrial cap (fig. 2, PI. 9). The close proximity of the mitochondrial cap in the oogonia and the presence of nucleolar emissions upon the surface of the membrane in the young oocytes render it difficult to be certain of the nature of these bodies. While they may represent smaller aggregations of mitochondria which have separated from the cap and tend, owing to their small size, to become spherical there are indications of a non-staining chromophobe centre and a deeply-staining chromophilic rim in one or two cases. In some other cases where this rim is not visible they are stained more deeply with iron haematoxylin than the adjacent mitochondrial masses. This staining reaction suggests the possibility that these structures are Golgi bodies. No trace of impregnation was found by silver nitrate or osmium tetroxide methods in oogonia or young oocytes to substantiate this view and all attempts to demonstrate the apparatus in the young cell by any other method have so far been unsuccessful.

NO. 294 P 214 IRENE F. HILTON The Golgi apparatus was first identified without doubt in oocytes measuring 40-50/* in diameter. By Da Pano's method clear pictures were obtained which showed the apparatus in the form of black uneven granules of irregular shape, lying mainly towards one pole of the cell (fig. 15, PL 10). In one or two cases a very heavy impregnation occurred, but it is unlikely that the whole of this signifies the presence of Golgi elements. In half-grown oocytes the silver deposit is much lighter and the individual elements are scattered over a much larger area while still being mainly concentrated towards one side of the nucleus. In the mature oocytes the silver nitrate method shows a uniform distribution of the Golgi elements throughout the cytoplasm in the form of small bodies of irregular shape but smooth outline. No trace of a network could be distinguished (figs. 11 and 13, PL 10). In two half-grown oocytes fixed in Flemming and stained with iron haematoxylin a structure of doubtful origin was seen lying in the cytoplasm close to the nuclear membrane. This structure, which was spherical when examined in section, appeared to consist of a clear chromophobe centre and a well-defined chromophile rim. Although lying close to the nucleus it had no connexion with it. The cytoplasm immediately surrounding this body stained rather more lightly than the cytoplasm in the rest of the cell. In staining reaction and in general appearance this structure was very like the spherical form of the Golgi apparatus seen in optical section when stained by this method, but it was unusually large for a Golgi body and furthermore was not found in any of the other oocytes of the same age treated by the same method. Its exact nature and origin remains a mystery (fig. 10, PI. 10). In the older oocytes at the onset of yolk-formation one or two preparations stained with iron haematoxylin showed the ring- shaped Golgi elements scattered in the cytoplasm. These were smaller than those found in the younger oocytes (Text-fig. 5). The last stage of the Golgi apparatus, in which it occurs as scattered granules distributed throughout the cytoplasm of the ripe oocytes, may be termed the ' diffuse stage' in contrast to the OOGENBSIS OF OALANUS 215 earlier ' complex stage' where it is concentrated in one part of the cell. Careful examination failed to reveal any connexion between the developing yolk-droplets and the individual Golgi elements, nor was the position of the complex stage in the cell in any way related to regions where yolk-formation was taking place. The TEXT-FIG. 5.

Oocyte at the beginning of yolk-formation showing Golgi bodies, scattered mitochondria, yolk-droplets, and beginning of vacuolation in the cytoplasm. G b, Golgi bodies ; v, vacuoles; w ov, wall of oviduct; y d, yolk-droplets. function of the Golgi apparatus in Calanus remains an open question. DISCUSSION. In the present investigation several points arise for discussion. These fall into two main groups; questions concerning the behaviour of the chromosomes and the process of nucleolar activity. Associated with the latter is the question of yolk- formation in the oocyte and the part played in the process by nucleolar emissions. The following points are dealt with under these headings. The chromosomes. In the very young oocytes of Calanus finmarchicusa typical synizesis figure is formed, but no bivalent chromosomes emerge from this although presumably the bivalents are present in the synizesis knot. The bivalents appear for the first time after the growth of the oocyte 216 IEENE F. HILTON is completed and immediately prior to the first maturation division. During the entire growth phase no individual chromosomes are visible but a much twisted thread surrounds the nucleolus. Among the Amphibia x it is known that the decon- centration of the chromosomes proceeds so far that many, or all of them, are indistinguishable during the growth phases of the egg. In these extreme cases the germinal vesicle shows only an oxyphilic lightly-staining meshwork surrounding one or two nucleoli which presumably contain the entire basophilic content of the nucleus. In the disappearance of the individual chromosomes and the presence of the twisted thread surrounding the nucleolus the condition found in Oalanus is comparable to this, but there is no evidence for believing that the nucleolus contains the entire basophilic content of the nucleus; on the contrary the thread surrounding it stains with basic stains if rather more lightly than the nucleolus. It appears probable that this network surrounding the nucleolus is in reality a much modified spireme formed from the deconcentrated chromosomes, which lose their individuality during the growth phases of the oocyte and reappear as ring-shaped bivalents at maturation. The ring-shaped form of the chromosomes is very constant for the Copepoda. Many of the earlier Avorkers who observed them believed them to be formed from chromosomes which had united by telosyndesis during the period at which the chromatin is massed at one pole of the early oocyte nucleus. Kornhauser (1915) observed these rings in the Copepoda but believed them to be formed from chromosomes which had united by parasyndesis in the early oocyte nucleus. He claimed that each of these chromosomes showed at an early stage a distinct transverse split which he supposed to be an integral part of the structure of the chromosome. No trace of such a split was visible in any of the chromosomes of Calanus finmarchicus and from the material examined it was impossible to say with any degree of certainty whether the bivalents were formed by

1 Anura OscarSchultze (1887), Carnoy andLebrun (1879), King (1908). Urodela Born (1894), Carnoy and Lebrun (1878, 1879), Schmidt (1905), Jorgenssen (1913), Stieve (1920). OOGENESIS OP CALANUS 217 telosyndesis or parasyndesis, though the latter appears more probable. Nucleolar extrusion. Throughout the entire growth phase of the oocyte nucleolar activity is very marked in C a 1 a - nus finmarchicus. The exact nature of the portions of nucleolar material which pass out into the cytoplasm and their ultimate fate is difficult to determine. The majority of the earlier workers upon the Copepoda failed to establish the passage of the nucleolar extrusions into the cytoplasm although they observed them within the nuclear membrane. Moroff (1909) figured for Paracalanus parvus fragmentation of the nucleolus and the presence in the cytoplasm of granular masses of material which he believed to be nucleolar in origin. These figures correspond so closely with those obtained for Calanus finmarchicus after Flemming and iron haematoxylin that there is little doubt the granular masses in the cytoplasm were mitochondrial in nature: in no cases were masses of nucleolar material seen in the cytoplasm in Calanus, having once condensed out from the surface of the nuclear membrane the extrusions appeared as scattered drops moving towards the periphery of the cell. There is absolutely no evidence that they pierce the membrane as whole bodies though this condition is reported by Nath and Mehta (1929) in the eggs of the Firefly. While it has not been possible to establish a definite periodicity in the behaviour of the nucleolus in Calanus there is evidence for believing that its activity is much more marked at certain stages of the growth of the oocyte than at others. In all the young oocytes examined, numerous nucleolar emissions of varying sizes were seen inside the nucleus, while outside the nuclear membrane an accumulation of nucleolar material was visible. This was much less marked in the older oocytes but immediately before maturation a second period of marked activity on the part of the nucleolus occurs. The first of these periods coincides with the stage at which the mitochondria are arranged in a ring surrounding the nucleus. It has already been suggested that the nucleolar emissions may come into relation with the developing mitochondria at this time and that the dark cloud in the cytoplasm surrounding them which is seen in some preparations may 218 IRENE F. HILTON consist of secretions from the cytoplasm and nucleolus. The second period of marked activity coincides with the deposition of yolk in the oocyte, the breakdown of the nuclear membrane, and the formation of the chromosomes. That part of the nucleolar substance is used up in the formation of the chromosomes is probable, but there is considerable evidence to show that the emissions which pass into the cytoplasm are in some way connected with yolk-formation. The method of yolk-formation in the egg has been the subject of much recent research. Numerous records of the close relationship between mitochondria and yolk, and between Golgi apparatus and yolk are to be found. Many of these are reviewed in a recent paper by Hibbard (1928). Other cytologists have put forward the view that the nucleolus is directly concerned in yolk-formation. Nath and Mehta (1929) and Gresson (1929) have recorded the formation of yolk from nucleolar emissions in the cytoplasm. The formation of albuminous yolk from nucleolar emissions has been described in the cockroach by Hogben (1920) and in Saccocirrus by Gatenby (1922); other examples might be cited. In a paper on the oogenesis of Limulus polyphemus Gardiner (1927) proved the presence of substances rich in phosphorus in the nucleolus and suggested that the mechanism of nucleolar emission effects the transport of phosphorus from the nucleus to the cytoplasm. It appears probable that in Calanus one of the functions of nucleolar activity is to provide a means of transport by which substances used in the formation of yolk are passed from the nucleus into the cytoplasm.

SUMMARY. Three regions can be recognized in the ovary: a multiplication zone containing oogonia undergoing mitosis, a synapsis zone containing the first formed oocytes in the prophases of the maturation division, and a growth zone containing oocytes in a series of growth phases with the nucleus in a' resting condition'. The oogonial nuclei contain two or three nucleoli—plasmosome and karyosomes. In the oocytes a single nucleolus is present; this is formed by the fusion of the plasmosome and at OOGENESIS OP CALANUS 219 least one karyosome and is therefore an amphinucleolus. The chroma tin in the oogonia and young oocy tes is arranged round the periphery of the nucleus andis aggregated in knots (pp. 196-200). Nucleolar extrusion begins in the young oocyte and continues throughout the growth period. It is most marked in the young oocytes and in oocytes about to undergo maturation (pp. 200-3). In the older oocytes the chromatin is in the form of a tangled thread surrounding the nucleolus. Immediately before maturation this condenses and circular chromosomes emerge: these form tetrads (pp. 203-5). The mitochondria are present in the oogonia and very young oocytes in the form of a cap lying upon the surface of the nuclear membrane. The mitochondrial elements spread and multiply until they surround the nucleus as a ring; afterwards they dis- perse and are distributed evenly throughout the cytoplasm. They swell up and finally yolk-droplets appear in their place (pp. 207-11). Yolk-formation usually begins in half-grown oocytes, but is sometimes earlier. The formation of yolk-droplets begins at the periphery of the cell and proceeds inwards. It is suggested that yolk is formed by transformation of the mitochondria and the deposition in them of substances derived from the cytoplasm and the nucleolus. The cytoplasm is flocculent in the young oocytes, granular in the half-grown oocytes, and filled with fluid vacuoles in mature oocytes. It passes from a primary condition of oxyphily to basophily and finally back to a secondary oxyphil condition in mature oocytes (pp .211-13). In the young oocytes deeply-staining spherical structures were seen adjacent to the mitochondrial cap. From their appearance it is possible these bodies represent the Golgi apparatus, but Da Pano fixation failed to demonstrate them. In half-grown oocytes the apparatus was visible in the complex condition at one side of the cell. As growth proceeds it passes from a complex to a diffuse condition and in mature oocytes the Golgi elements are uniformly distributed throughout the cytoplasm (pp. 213-15). 220 IRENE F. HILTON

LIST OF REFERENCES. Amma, K. (1911).—"Ueber die DiSerenzierung der Keimbahnzellen bei den Copepoden", 'Arch. Zellforsch.', Bd. VI, p. 497. Gardiner, M. S. (1927).—"Oogenesis in Limulus polyphemus". 'Journ. Morph.', vol. 44, p. 217. Gatenby, J. B. (1922).—"The gametogenesis of Saccocirrus", 'Quart. Journ. Micr. Sci.', vol. 66, p. 1. Gresson, A. R. (1929).—"Nucleolar phenomena during oogenesis in certain Tenthredinidae", ibid., vol. 73, p. 177. Harvey, L. A. (1925).—"On the relation of the mitochondria and Golgi apparatus to yolk-formation in the eggs of the common earthworm, Lumbricus terrestris ", ibid., vol. 69, p. 292. Hibbard, H. (1928).—"Contribution a 1'etudedel'ovogenese, delafeconda- tion, et de l'histogenese chez Discoglossus pictus Otth.", 'Arch, de Biol.', vol. 38, p. 251. Hogben, L. T. (1920).—"Oogenesis in the Hymenoptera", 'Proc. Roy. Soc.', Series B, vol. 91, p. 268. Ishikawa, C. (1891).—" Spermatogenesis, oogenesis and fertilization in Diaptomus sp.", 'Journ. Coll. Soc. Imp. Univ. Japan', vol. v, Pt. I. King, S. D. (1926).—"Oogenesis in Oniscus asellus", 'Proc. Boy. Soc.', Series B, vol. 100, p. 1. Kornhauser, S. T. (1915).—"A cytological study of the semi-parasitic copepod Hirsilia apodiformis", 'Arch. ZeUforsch.', Bd. XIII, p. 399. Ludford, R. J. (1922).—"The morphology and physiology of the nucleolus", 'Journ. Roy. Micr. Soc.', p. 113. (1924).—•" Nuclear activity during melanosis", ibid., p. 13. Matscheck, H. (1910).—"Ueber Eireifung und Eiablage bei Copepoden", 'Arch. Zellforsch.1, Bd. V, p. 36. McClendon, J. F. (1910).—"Further studies on the gametogenesis of Pan- darus sinuatus ", ibid., Heft 2, p. 229. Moroff, T. (1909).—"Oogenetische Studien", ibid., Bd. II, Heft 3, p. 432. Nath, V. (1926).—"On the present position of the mitochondria and the Golgi apparatus", 'Biol. Reviews', vol. ii, No. 1. Nath, V. and Mehta, D. R. (1929).—"Studies in the origin of yolk, III", 'Quart. Journ. Micr. Sci.', vol. 73, Pt. I, p. 7. Woltereck, R. (1898).—"Zur Bildung und Entwicklung Ostrakoden-Eies. Cypriden", 'Zeitschr. wiss. Zool.', Bd. 64, p. 596.

EXPLANATION OF FIGURES The figures are magnified about 500 diameters except where otherwise stated. OOGENESIS OF CALANUS 221

KEY TO LETTERING. a, fusion of plasmosome and karyosome; ch m, chromatin mass; chr, chromatin; cJirom, chromosomes; d, droplets; e p, equatorial plate ; 0 6, Golgi bodies; h, karyosome; lep st, leptotene stage; in, mitochondria; m c, mitochondrial cap; n nucleus; n b, nuclear membrane; n ext, nucleolar extrusions; n r, nuclear reticulum; p, plasmosome; sp, spindJe; spir, spireme; syn st, synapsis stage; t, tetrads; v, vacuole; W OF,wall of oviduct; x, cytoplasmic body; y A, yolk-droplets; 1st TO sp, first maturation spindle. (In description of Plate, below) C.K., Champy-Kull; Da E., Da Fano; E.H., Ehrlich's haematoxylin; E., Elemming; F.w.a., Flemming- without-acetic acid; I.H., Iron haematoxylin.

PLATE 9. Pig. 1.—O o g o n i a from the tip of the ovary showing mitosis and resting stages. (Bouin, I.H.). Eig. 2.—0 o g o ni a showing mitochondrial cap and possible Golgi bodies. (Nassonov, I.H.). Eig. 3.—Young oocytes showing spreading of the mitochondrial cap and Golgi bodies. (E.w.a., I.H.). Eig. 4.—Oocytes from the synapsis zone showing synapsis and leptotene stages and mitochondrial cap. (E.w.a., I.H.). Eig. 5.—Half-grown oocyte showing mitochondrial masses, nucleolar extrusion and precocious yolk-formation. (F.w.a., I.H.). Eig. 6.—Nucleus of oocyte about to undergo maturation, showing ring- shaped chromosomes and final breaking up of the nucleolus. (C.K.). Eig. 7.—Oocyte from posterior end of oviduct showing cytoplasm packed with yolk-droplets. (F.w.a., I.H.). Fig. 8.—Young oocyte showing vacuolation in tho rim of the nucleus and granular core, nuclear reticulum, and nucleolar extrusion. (F.w.a., I.H.). Eig. 9.—Part of the cytoplasm of an oocyte before maturation showing yolk-formation, vacuolation of the cytoplasm, and chromophobe droplets at d. (E.w.a., I.H.).

PLATE 10. Eig. 10.—Half-grown oocytes from anterior end of the oviduct showing nucleolar extrusion, beginning of yolk-formation and body of unknown origin in the cytoplasm at x. (E.w.a., I.H.). Fig. 11.—Oocyte during first maturation division showing equatorial plate and diffuse condition of Golgi apparatus. (Da E., E.H.). Eig. 12.—Young oocyte showing large central vacuole of nucleolus and Golgi bodies surrounding nucleus on one side. (Da F., E.H.). Eig. 13.—Oocyte during first maturation division showing spindle with tetrads at metaphase, and diffuse condition of Golgi apparatus. (Da F., E.H.). 222 IRENE F. HILTON Fig. 14.—Oocyte about to undergo maturation showing vacuolation in rim of nucleolus, circular chromosomes, and Golgi apparatus in diffuse condition. (Da F., E.H.). Fig. 15.—Young oocyte showing Golgi apparatus in complex condition. (Da F., E.H.). Fig. 16.—Equatorial plate of first maturation division showing tetrads (magnification 1000 diameters). (F., I.H.). Fig. 17.—Telophase of second maturation division. (F.w.a., I.H.). Fig. 18.—Equatorial plate of second maturation division (magnification 1000 diameters). (F.w.a., I.H.). Quart. Journ. Micr. Sci Vol. 74, N. 8., PL 9

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