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 . I N T R O D U C T I O N . . . . . . . . . 1 9 3

2 . M A T E R I A L A N D M E T H O D S . . . . . . . 1 9 4

3 . L I T E R A T U R E . . . . . . . . . 1 9 5

4 . G E N E R A L A C C O U N T . . . . . . . . 1 9 6

5 . N U C L E U S , N U C L E O L U S , A N D N U C L E O L A R E X T R U S I O N . . 2 0 0

6 . T H E M I T O C H O N D R I A . . . . . . . . 2 0 7

7 . Y O L K - F O R M A T I O N . . . . . . . . 2 1 1

8 . T H E G O L G I A P P A R A T U S . . . . . . . 2 1 3

9 . D I S C U S S I O N 2 1 5

1 0 . S U M M A R Y 2 1 8

1 1 . L I S T O F R E F E R E N C E S . . . . . . . . . 2 2 0

1 2 . E X P L A N A T I O N O F F I G U R E S . . . . . . . 2 2 0

INTRODUCTION.

THE present investigation was undertaken with a view toworking out the details of oogenesis in a Copepod, about whichcomparatively little is known from the modern cytologicalstandpoint. C a l a n u s f i n m a r c h i c u s was suggested byMrs. E. C. Bisbee, of the Department of Zoology, University ofLiverpool, as a suitable subject; it is easily obtainable in quantityand has relatively large eggs; moreover the nucleolus in its be-haviour throughout the growth of the egg presents certain veryinteresting features.

The work was carried out partly in the Department of Biology,University College of Swansea, and partly in the Department ofZoology in the University of Edinburgh.

194 IRENE F. HILTON

MATERIAL AND METHODS.

The specimens of C a l a n u s finmarchicus (Gunnerus,1770) used in the present investigation were obtained mainlyfrom Plymouth, but also from the Marine Laboratories at PortErin, Isle of Man, and at Millport, Buteshire.

The animals were examined at intervals of about fourteen daysduring the greater part of two years.

Some difficulty was experienced in obtaining satisfactoryfixation of the ovary. Dissecting out the ovary before fixationwas not found practicable but by cutting off the abdomen at thefirst joint before immersing in the fixing fluid, rapid penetrationof 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, haema-toxylin) generally gave the best results, but Bouin's fluid andcorrosive sublimate were found excellent as nuclear fixatives.The Champy-Kull method gave good results in the older oocytesapproaching 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 acidfollowed by iron haematoxylin was particularly successful inolder oocytes.

For the study of the Golgi apparatus Da Fano's method gavethe 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 wasbegun, to Professor J. H. Ashworth, of the University of Edin-burgh, to Professor W. J. Dakin, and Mrs. R. C. Bisbee, of theUniversity of Liverpool, and Mr. L. A. Harvey, of the Universityof Edinburgh, for much helpful advice, and to the Committeeof the Earl of Moray Endowment of the University of Edinburghfor a grant in aid of expenses.

OOGENESIS OF CALANUS 195

LITERATURE.

Although the C o p e p o d a a s a group have been widely studiedin the past, comparatively little is known of the details of theirgametogenesis from the modern cytological standpoint. Priorto the development of modern cytological methods attentionwas directed almost exclusively to the nucleus and its chromo-some content. The chromosome numbers for thirty-five speciesof Copepods are recorded in the 'Tabulae Biologicae 4' (1927).The majority of the numbers given are for members of thegenus Cyc lops but some species of G y m n o p l e a are in-cluded. Among the G y m n o p l e a , with the exception of aJapanese form D i a p t o m u s s p . Ishikawa (1891) and D ia -p t o m u s c o e r u l e u s (Amma, 1911) the haploid number ofchromosomes present in the female is either sixteen or seventeen,sixteen being the more usual number.

McClendon (1906, 1910) and Kornhauser (1915) both workingupon parasitic copepods described the formation of ring-shapeddouble chromosomes by parasyndesis in a way which appearssimilar to that recorded for C a l a n u s finmarchicus in thepresent investigation.

Matscheck (1910) in a paper upon growth and developmentof copepod eggs recorded fragmentation of the nucleolus priorto the maturation divisions of the egg. This author also foundformation of yolk in the half-grown oocytes and suggested apossible secretory function of the nucleolus. The more recentwork of Ludforcl (1922, 1924) upon the morphology and physi-ology of the nucleolus provides strong evidence in support ofthis view. Gardiner (1927) working upon L i m u l u s p o l y -p hem us has suggested a very specialized secretory functionfor the nucleolus in the transport of phosphorus to the cytosome.

The study of C a l a n u s finmarchicus affords strongevidence for suggesting that the mitochondria play an importantpart in yolk-formation. A close relationship between the mito-chondria and yolk-formation has been recorded in the eggs ofvarious 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 thealimentary canal. It extends about two-thirds of the entirelength of the thorax and tapers to a blunt point at its posteriorend. Prom the anterior end of the ovary paired oviducts ariseand run forward as wide thin walled tubes. These lie parallel toeach 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 alimen-tary canal. The two oviducts open together on the first ab-dominal segment close to the opening of the spermathecae. Itappears probable that fertilization takes place as the eggs leavethe oviducal opening.

Three distinct zones can be observed in the ovary; (Text-fig.l).

(1) A multiplication zone situated in the posterior part of theovary and consisting of small cells—oogonia—under-going mitosis. One or two cells at the tip of the ovaryare 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 endof the ovary and which contains oocytes in a progressiveseries of growth phases. The anterior end of the ovarypasses almost imperceptibly into the oviducts in whichthe later growth phases of the oocytes take place.

M u l t i p l i c a t i o n Zone .

The oogonia at the posterior end of the ovary are very closelypacked. Two or three nucleoli are present in the resting stageof the oogonial nucleus (figs. 1 and 2, PL 9). Owing to somedifficulty which was experienced in obtaining good fixation ofthis part of the ovary the nature of the oogonial nucleoli isnot perfectly clear. It was observed that one nucleolus is usuallyconspicuously larger than the others. After fixation by chrome-osmium technique followed by staining with iron haematoxylin,

OOGENBSIS OF CALANUS

TEXT-FIG. 1.

197

Diagram of longitudinal section through thorax showing zones ofovary, and arrangement of oocytes in the oviducts, m, multiplica-tion 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 smallerones. After Champy-Kull technique it stains a uniform ambercolour and after Mann's methyl blue eosin, bright pink. Itis a plasmosome and remains spherical in shape. The smallernucleoli are irregular in outline and stain more deeply with ironhaematoxylin. With Feulgen's ' Nuclealfarbung' method thesenucleoli give a deep pink reaction: they are karyosomes.

The final oogonial mitoses, which actually mark the initiationof the growth phases of the oocyte, are visible in fixed and stainedpreparations 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 chromatinnetwork resolves itself into a very fine spireme thread whichassumes a marked spiral arrangement within the nucleus. Thisthread breaks up into a number of long twisted chromosomeswhich shorten and thicken to form small rods. After the dis-appearance of the nuclear membrane these chromosomes, ofwhich there appear to be about thirty-four, arrange themselvesupon the equator of the spindle; metaphase, anaphase, andtelophase follow in rapid succession. The chromosomes of thisfinal oogonial mitosis swell in the telophase, forming a deeplystaining mass at each pole of the spindle. The details of theevents immediately following are rather obscured by this pro-cess, but the chromatin in the daughter cells formed by thedivision passes into the resting stage.

S y n a p s i s Zone .The resting stage of the nucleus of the early oocyte is of very

short duration. Soon after the formation of the oocytes thenuclear network aggregates into masses which finally becomedrawn out to form a thread similar in appearance to the spiremethread of the early prophase nucleus of the oogonia. This threadbreaks up into a large number of parts which become twistedand are extremely difficult to count in consequence. Thechromatin then contracts to one pole of the nucleus and thereforms a tangled, deeply staining knot. From this knot thicklooped 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 synizesisfigure in which the formation of bivalent chromosomes is takingplace (fig. 4, PI. 9). The bivalent chromosomes do not appearuntil a much later stage, the entire growth period of the oocyteintervening between the initiation and completion of the firstmaturation division. The synizesis knot, after a period ofcondensation in Avhich it appears as a mass of chromatin at onepole of the nucleus, finally separates out to form a thread whichclosely resembles a spireme. This thread is much twisted uponit itself and surrounds the nucleolus Avhen this latter re-forms.The oocytes remain in this condition throughout the subsequentgrowth stages.

Growth Zone .

In the very young oocytes at the beginning of the growthstage, a large central nucleus is present surrounded by a thinlayer of rather flocculent cytoplasm. One or two nucleoli arepresent, one of which is a large plasmosome. The karyosomesat this stage stain a decided purplish pink when treated byFeulgen's method.

In slightly older oocytes only one large nucleolus is presentwhich appears to be formed by the fusion of plasmosome andkaryosomes and is therefore an amphinucleolus. Preparationsmade by Feulgen's method at this stage show a large clear bodyin the centre of the nucleus with a smaller pinkish body deeplyembedded in its surface. This is interpreted as a stage in thefusion process of the plasmosome and karyosome. Preparationsstained by the Champy-Kull method show the compoundnucleolus as an amber-coloured body containing in its centre avarying number of highly refractive vesicles; these appear to bean intense bluish-green in colour. While this colour may be dueto refraction, and the blue stain of the Champy-Kull method isa somewhat capricious one and cannot be taken as proving thepresence of chromatin in the nucleolus, it appears probable fromits mode of origin that chromatin is present in it at this stage.

The time of egg-laying varies considerably in different localitiesand is prolonged over a considerable period. Copepods from thewest 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 fromthe Isle of Man were later and in some cases showed eggs ina very immature condition in mid-June.

THE NUCLEUS, NUCLEOLUS, AND NUCLEOLAR EXTRUSION.

Oogonia and young O o c y t e s .

In the oogonia and very young oocytes the nucleus, which iscentrally situated, occupies the greater part of the cell and a well-marked nuclear membrane is visible. The nucleoplasm stainsa pale uniform grey with iron haematoxylin. The chromatin isaggregated into a number of irregular, deeply staining masseswhich lie close to the nuclear membrane, thus leaving a clearspace in the centre of the nucleus in which the plasmosome lies.The karyosome is situated at one side of the nucleus in theoogonia and very young oocytes. It is frequently very irregularin shape and small, consequently it is easily confused with thechromatin masses round the periphery of the nucleus (fig. 3, PI. 9).In preparations which were overstained with haematoxylin itretained the black stain more deeply than the surroundingchromatin. In slightly older oocytes the karyosome takes up amore central position and approximates towards the plasmosome,with which it finally fuses. Whether the other karyosomes whenpresent fuse with the plasmosome or are used in chromosomeformation it is impossible to say from the evidence available.No preparations were seen in which more than one karyosomeappeared in the process of fusion with the plasmosome. In theolder oocytes in which nucleolar extrusion was beginning onlyone nucleolus is present; this stains a uniform dark grey withiron haematoxylin and amber yellow with one or two highlyrefractive blue-green vesicles in the centre with Champy-Kull.

Nuc leo l a r E x t r u s i o n .

Nucleolar extrusion takes place very rapidly throughout theearly growth phases of the oocyte in C a l a n u s . Portions ofnucleolar material varying in size from minute spheres no largerthan the mitochondria, to spheres which are as large as the fullyformed yolk-globules, are extruded from the surface of the

OOGBNESIS OF CALANUS 201

nucleolus over the whole of its area. Although large spheres areseen in many preparations lying upon the surface of the nucleolus(Text-fig. 2 a), the nucleolus is not distorted and there is noindication that these extrusions arise by being pinched off. Thenucleolar extrusions lying against the nuclear membrane areusually much smaller than those which are seen on or near thesurface of the nucleolus; this suggests that the larger extrusions

TEXT-FIG. 2.

a. Nucleus of half-grown oocyte showing nucleolar extrusions uponthe surface of the nuclear membrane in the form of small spheres,b. Nucleus of half-grown oocyte showing nucleolar extrusions uponthe surface of the membrane in the form of a network, c. Portionof the nuclear membrane showing coincidence of drops of nucleolarmaterial on the inside and outside, n ext, nucleolar extrusions;n r, nuclear reticulum.

break up in their passage across the nucleus to the nuclear mem-brane. Since the extrusions are almost certainly of a liquidnature it is possible on the other hand that the drops are merelyfixation effects, their size depending upon the concentration ofextruded material in the region where they occur.

In very young oocytes the material which is extruded fromthe nucleolus forms a deeply staining mass which appears as athick black line in the region of the nuclear membrane in pre-parations stained with iron haematoxylin (Text-fig. 4 a). Thisdeposit shows more clearly in slightly older oocytes. In sections

202 IRENE F. HILTON

3 to 5ju. thick of material fixed with Flemming-'without-aceticacid and stained by the long method in iron haematoxylin, aseries of small black dots is seen closely apposed to the surfaceof the nuclear membrane. In some preparations the extrudedmaterial appears as a darkly staining reticulum lying upon theouter surface of the nuclear membrane (Text-fig. 2 b). In sectionsfrom 8 to 10/x thick including portions of the surface of thenuclear membrane the extrusions sometimes appear as spherulesof variable size lying outside the membrane but closely apposedto it. These are obviously fixation effects which signify thepresence of a condensation of nucleolar material outside thenuclear membrane.

During the whole period of nucleolar extrusion there is presentin the cytoplasm a varying number of deeply staining sphereswhich are in every way identical with the nucleolar extrusionsinside the nuclear membrane. There can be no doubt that alarge part of the nucleolar material is transported to the cyto-plasm by the process of nucleolar emission. Although inyoung oocytes the majority of these spheres in the cytoplasm arefound surrounding the nucleus, in the later stages of the growthof the oocyte they are sometimes found near the periphery ofthe cell. All these spheres in the cytoplasm pass graduallyfrom a basophil to an acidophil condition, lose their stainingproperties and finally become invisible: presumably they aredissolved in the cytoplasm. In one or two preparations portionsof the extruded material from the nucleolus appeared as thoughpassing through the nuclear membrane into the cytoplasm(Text-fig. 2 c). While this apparently supports the view heldby some cytologists that nucleolar extrusions are passed throughthe nuclear membrane into the cytoplasm as individual bodies,a consideration of the whole process in C a l a n u s suggests thatthis does not take place. In any case it seems highly improbablethat bodies of the size of nucleolar extrusions could pass throughthe nuclear membrane without losing their identity or rupturingthe membrane. It is probable that the appearance of thesepreparations is due to the coincidence of drops of nucleolarmaterial closely apposed to the outside and the inside of thenuclear membrane at the moment of fixation. On the outer

OOGENESIS OF CALANUS 203

surface of the nucleus there must be a considerable confluenceof liquid nucleolar material which has passed through themembrane by a process of diffusion. From this semi-fluidperinuclear layer the nucleolar material appears to condenseout as drops which migrate outwards into the cytoplasm, andfinally become dissolved in its substance.

In the half-grown oocytes the nucleolus is no longer visibleas a homogeneous mass in the centre of the nucleus, but is seento consist of two distinct regions: a large central vacuole anda narrow outer rim. This appearance is constant with all fixa-tives and stains used, with the exception of Feulgen's method,which does not stain the nucleolus at this stage. In the youngeroocytes the rim stains a uniform dark grey with iron haema-toxylin, but in oocytes in which the process of nucleolar extru-sion has been proceeding for a longer period a number of smallvacuoles make their appearance in the rim (fig. 8, PI. 9). Thisindicates a reorganization of the nucleolar material followingnucleolar extrusion. Occasionally smaller vesicles can be seenin the core of the nucleolus which give it the appearance of analveolar structure, but more often a faint granulation is allthat is visible. This may be due to the coagulation of liquidsubstances in the central vacuole.

The C h r o m a t i n .

The nuclear reticulum of the half-grown oocytes is in the formof a fine network of much coiled interlacing threads. Thesethreads are not of uniform thickness, but in places show smallknots which probably represent aggregations of chromatin uponthe linin network (fig. 8, PI. 9). This network, although showingno trace of individual chromosomes, may be regarded as adiffuse and much modified diplotene stage. In all preparationsobserved the nuclear reticulum was seen contracted to a greateror lesser extent away from the edge of the nucleus and was mostmarked in the region immediately surrounding the nucleolus.This contraction does not necessarily indicate shrinkage dueto fixation but may be an actual condition representing a re-concentration of the chromatin. For a short period the chroma-tin in the older oocytes approaching maturation is in the form

204 IRENE F. HILTON

of a continuous thread. No indications of a double nature canbe seen in this thread.

The maturation divisions of the egg usually take place in theventral arms of the oviducts, although in some cases maturationappears to take place after the egg has left the oviduct. Theearly stages of the divisions of the ripe oocyte are very unstable,and the majority of the preparations examined during thematuration process showed either the metaphase or earlyanaphase position of the chromosomes upon the spindle. Whenthe first maturation division has reached the metaphase thereappears to be a distinct pause after which the final stages of thefirst division, the formation of the second spindle, and the com-pletion of the second division are accomplished with greatrapidity. It was impossible to say from the preparationsexamined whether the first polar body divides or not. The firstmaturation division, which is the true reduction division of theegg, enters upon its second phase with the breakdown of thechromatin reticulum to form bivalent chromosomes. Duringthe period which intervenes between the first phase of thereduction division and the second phase, the entire growth ofthe oocyte takes place; changes occur in the cytoplasm and thegreater part of the yolk is laid down. The nuclear membranebecomes constricted at one pole, the nucleus assuming anelongated pear shape as a result of the pressure of the eggs inthe oviduct. At this time the nucleolus, which is comparativelyquiescent during the later growth phases of the oocyte, entersupon a period of great activity. Quantities of nucleolar materialare given off from the nucleolus and pass across the nucleoplasminto the cytoplasm. In fixed preparations this material appearsin the form of spheres of considerable size which stain deeplywith all basic dyes. After a short time in the cytoplasm theemissions lose their staining properties and become invisible(fig. 6, PL 9).

The chromatin reticulum during this process undergoes asecond contraction into a tangled mass from which circular,bivalent chromosomes emerge. These are extremely small andaggregated together towards one side of the nucleus; theirstructure 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. Promthese bivalent ring-shaped chromosomes tetrads are formed bythe appearance of a transverse constriction in each half of thering; condensation takes place, and the tetrad is reduced to acompact body in which the four components are plainly visible.

The dissolution of the nuclear membrane begins at one poleof the nucleus and spreads rapidly. Immediately before itsfinal disappearance, the nucleolus, which by this time is reducedto a small sphere, breaks up and passes into the cytoplasm.Throughout the period of the formation of the chromosomes aprogressive loss in staining property is noticeable in the chroma-tin content of the cell. This is also true of the nucleolus, whichfinally shows a very similar staining reaction to the plasmosomeof the early oocyte nucleus. The ring-shaped chromosomes whenfirst formed stain faintly, but at a later stage Avhen they arearranged upon the spindle they stain deeply with all basic dyes.Just before the disappearance of the nuclear membrane thegroup of seventeen tetrads is seen situated close to the nuclearmembrane, usually at the opposite side of the nucleus to thedisintegrating nucleolus (Text-fig. 3). With the disappearanceof the membrane the tetrads pass out into the cytoplasm andtake up their position on the equator of the maturation spindle.

The M a t u r a t i o n S p i n d l e s .

The spindles are small truncated structures 10-13JU, in length(fig. 13, PI. 10). Astral rays were not visible in any of the pre-parations examined but the longitudinal fibres were clearlyvisible. The spindles appear to be constructed of dense proto-plasm which shows slight acidophil reactions. When arrangedupon the equator of the spindle the chromosomes are verydifficult to count owing to their small size and close proximity.When examined upon the equatorial plate the chromosomessometimes appear V-shaped, converging towards the centreof the spindle while their free ends point outwards. This isprobably due to tension at the point of attachment to thespindle fibres. After the halves of the tetrads have separatedthey move apart rapidly and the final stages of the divisionwhich was begun before the growth of the oocyte are completed.

206 IRENE F. HILTON

The spindle of the second maturation division is formed at rightangles to that of the first, and in rapid succession. Presumablya rotation of the second spindle occurs. Portions of first spindleare 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 mem-brane at a, tetrads {t) and final group of nucleolar extrusions.d, droplets; n ext, nucleolar extrusions; v, vacuoles.

monovalent chromosomes. Presumably the tetrads become soorientated upon the spindle that synaptic mates are separatedfrom each other in the first division. The behaviour of all thechromosomes is identical. The polar bodies may be observedflattened against the surface of the egg by the pressure of thewalls of the oviduct. They are very small, about 5 or 6/x indiameter.

The chromatin in the nucleus is in a very unstable conditionthroughout the growth phases of the oocyte. Tests were madefor chromatin by Feulgen's method, with the one modificationthat sections were left for two hours in the fuchsin sulphurousacid, and afterwards washed very quickly in two changes of

OOGENESIS OF CALANUS 207

S02 water before mounting. It was found that in the nuclei ofthe oogonia and very young oocytes the nuclear reticulumstained a distinct purplish pink. In at least two cases whichshowed the karyosome in process of fusion with the plasmosome,the karyosome alone was stained. Everything else in the cellwas colourless. Throughout the remainder of the growth stagesthe oocyte showed no trace of colour when treated by this method,and the nuclear network was invisible. In cells which wereundergoing maturation, however, the chromosomes upon thespindle Avere deeply stained.

The affinity of the early oocyte karyosome for the staincoupled with the results obtained with the Champy-Kull methodstrongly suggests the presence of chromatin in the amphi-nucleolus during the early part of its history. There is no positiveevidence for believing that chromatin is extruded from thenucleolus, but on the other hand the nucleoli of the more matureoocytes do not show any positive chromatin reaction. It ispossible that the nucleolus acts as a reservoir for nucleic acidduring the growth stages of the oocyte and that this is releasedprevious to the formation of the chromosomes.

THE MITOCHONDRIA.

It was found that in the stages oogonia to oocytes the mito-chondria were progressively more resistant to acetic acid andwere in no cases completely destroyed by it. A marked resis-tance to acetic acid is not uncommon in the mitochondria ofgerm cells. It has been recorded by Nath (1926) for the scorpionP a l a m n a e u s .

S t r u c t u r e and D i s t r i b u t i o n of t h e M i t o c h o n d r i a .The mitochondria are present in the oogonia and very young

oocytes in the form of a cap of mitochondrial material situatedat one pole of the cell and closely adpressed to the nuclearmembrane (fig. 2, PI. 9). The cap is small and compact, and hasclearly denned edges; it stains a dark uniform grey with ironhaematoxylin following Flemrning-without-acetic fixation, andbright pink with Champy-Kull. Occasionally the mitochondrial

208 IRENE F. HILTON

cap appears in the form of several isolated masses, generallysituated at one pole of the cell. It is probable that these slightvariations in number and form of the mitochondrial masses aredue 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 andgradually spreads out, moving as it does so away from thenuclear membrane into the cytoplasm. The amount of mito-chondrial material increases considerably during this spreadingout, but it is impossible to be certain whether the individualelements which compose the mass arise de novo in the cytoplasmor by the division of pre-existing mitochondria. One or twopreparations showed figures which might be interpreted asdivision stages of the mitochondria and this correlated with thefact that the new masses of mitochondria always arise near theolder ones makes it appear probable that multiplication doestake place in this way.

In oocytes measuring 20-30^, in diameter, the mitochondriain the form of small spheres surround the nucleus and aresituated about half way between the nuclear membrane andthe edge of the cytoplasm (Text-fig. 4 a). In the younger oocytesgaps occur in this hollow sphere of mitochondria but in the olderoocytes it is quite complete.

Champy-Kull preparations at this stage show the mitochon-drial masses to be composed of a large number of vesicles ofvarying sizes; the largest of these probably represent aggrega-tions of several mitochondrial elements. With iron haematoxylinfollowing Flemming-without-acetic, the elements which con-stitute the mass of mitochondrial material vary in size and shapefrom small spheres to filamentous structures which again prob-ably represent a degree of coalescence. In all iron haematoxylinpreparations observed the mitochondria appear to be arrangedin a zone of cytoplasm which is darker and rather more flocculentin appearance than the surrounding medium. This appearancewas observed to a lesser degree in preparations stained by theChampy-Kull method, but was not visible in material fixed inNassonov or Kolatschev solutions which are specific for themitochondria. While this cloud in the cytoplasm may be entirely

OOGENESIS OF CALANUS 209

due to imperfect fixation of the mitochondria, it is also possiblethat it represents an accumulation of other substances which arepresent in the cytoplasm and come into relation with the mito-chondria at this time. Nucleolar extrusion is very marked duringthe early growth phases of the oocyte and it is suggested that

TEXT-FIG. 4.

•n ext

a. Young oocytes showing mitochondrial ring and micleolar materiallying on the nuclear membrane, b. Oocytes after dispersal of mito-chondrial ring (magnification about half that of a), m, mitochon-dria ; n ext, nucleolar extrusions.

this cloud may represent a concentration of dissolved nucleolarmaterial in the region of the developing mitochondria.

Woltereck in his paper upon growth and development ofOstracod eggs (1898) observed in C y p r i s a cap of materialoutside the nuclear membrane in the young oocytes whichstained darkly with haematoxylin. This he described as a 'yolknucleus' which in later stages of its development spreads out inthe cytoplasm, sometimes appearing as small flocculent massesand at other times showing distinct granules of deeply stainingsubstance upon a uniformly grey background. These variousstages correspond so closely with the history of the developmentand gradual spreading of the mitochondrial ring in C a l a n u s

210 IRENE F. HILTON

finmarchicus that it seems probable the 'yolk nucleus' ofWoltereck's description and the mitochondrial cap are identicalstructures.

The individual mitochondria which are scattered in thecytoplasm at a later stage of development are much smaller thanthe vesicles and spheres, which probably represent groups offused mitochondria.

Before the oocytes are half grown the mitochondria begin tospread out through the cytoplasm. The spreading-out processby which the mitochondria pass from a stage when they areaggregated round the nucleus to a stage when they are more orless evenly distributed throughout the cytoplasm, begins at onepoint in the ring. The visible sign of this dispersal is the ap-pearance of a well-marked gap which is first seen in oocytesmeasuring about SQfj, in diameter. In slightly older oocytes thering is seen to have dispersed with the exception of certainaggregations of mitochondria which appear to be much morestable than the rest of the mitochondrial ring (Text-fig. 4 b).

Occasionally two or three smaller groups remain, but one isthe more usual condition. In half-grown oocytes traces of thislast aggregation of mitochondria are visible which stain as adark grey irregular mass with iron haematoxylin. It is significantthat in a number of preparations yolk-formation was seen to bein progress near the periphery of the cytoplasm in this region(fig. 5, PL 9).

In the older oocytes the individual mitochondria are visibleas minute spherical structures; these stain dark grey with ironhaematoxylin and pink with Champy-Kull; they are uniformlydistributed throughout the cytoplasm. There is a tendency forindividual 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-formationbegins. The mitochondria swell and lose some of their stainingproperties. In their place small yolk-droplets appear which areat first arranged in small groups but which finally becomescattered throughout the cytoplasm, where they enlarge. (Seesection on yolk-formation.)

OOGENESIS OF CALANUS 211

In the mature oocytes the entire cytoplasm is packed withyolk-droplets and no mitochondria are visible (fig. 7, PI. 9).

YOLK-FORMATION.

Although superficially the condition which is found inCa lanus finmarchicus appears to support the view thatyolk is formed by the direct chemical transformation of themitochondria, a consideration of the other processes observedin the cell during the period of vitellogenesis leads the author tofavour the view that a number of other factors are equallyinvolved.

The yolk which is present in the oocytes of Ca l anusfinmarchicus appears to be homogeneous and non-fatty incomposition. Generally speaking, yolk-formation is first ob-served in the half-grown oocytes before the final distribution ofthe mitochondria has taken place. The quantity of yolk presentat this time and its position in the cell is subject to slightvariation. It may take the form of a group of well-markeddroplets, situated at one side of the cell, or a few small dropletsirregularly scattered near the periphery. It is impossible to saywith certainty that this early formed yolk has any direct relationto 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 remainin the cytoplasm, the yolk-droplets are more numerous in theregion of the cytoplasm lying between this mitochondrial groupand the periphery of the cell (fig. 5, PI. 9).

At a later stage the mitochondria which are dispersed swelland stain much more deeply, finally becoming replaced inposition by yolk-droplets (fig. 10, PL 10). This replacement ofthe mitochondria by yolk-droplets proceeds from the peripheryof the cell inwards until the cytoplasm is packed with yolk.This is the condition found in the eggs which have undergonematuration and are situated near the posterior end of the ovi-ducts.

While it is obvious in this case that the mitochondria areintimately connected with yolk-formation', it is impossible tosay with certainty from the observed facts whether the scattered

212 IRENE F. HILTON

mitochondria are directly transformed into yolk by a chemicalchange, or whether they serve as reservoirs for materialsdeposited in them at this time.

The period of most rapid yolk-formation appears to be thatimmediately preceding the breaking down of the nuclear mem-brane in the ripe oocyte. This period coincides with a rapid andfinal activity on the part of the nucleolus. During the formationof the chromosomes this splits into fragments, the parts pass-ing out into the cytoplasm, where they are finally dissolved. Itis significant that the most rapid period of yolk-formation inthe egg of C a l a n u s should coincide with this final period ofnucleolar extrusion. If the nucleolar extrusions play any partin yolk-formation, and if, as was suggested, they come intorelation with the mitochondrial ring during its formation, whatappears to be a precocious formation of yolk in the region ofthe mitochondrial masses can be partially explained. In thesection on the mitochondria certain cases were described inwhich the mitochondrial masses appeared to be surrounded bya darkly staining cloud in the cytoplasm. It was suggested thatthis cloud might indicate the presence of an accumulation ofdissolved material in the region of the mitochondria and thatthis material is probably nucleolar in origin. In these casesyolk-droplets were frequently seen near the mitochondrial mass.

About the time when yolk makes its first appearance in thecell, changes were observed in the structure and staining reac-tions of the cytoplasm, which in the young oocytes is flocculentin appearance and oxyphil. Throughout the growth phases ofthe oocyte a gradual change from oxyphily to basophily has beenobserved, the cytoplasm at the same time becoming denser andmore granular in appearance. In the older oocytes a return toa condition of secondary oxyphily takes place and the cytoplasmloses its granular appearance, becoming at first flocculent andlater highly vacuolated. The beginning of vacuolation corre-sponds with the onset of yolk-formation (fig. 9, PL 9). Thevacuoles in the cytoplasm are filled with a watery fluid whichcondenses out in some preparations as large drops which staina greenish grey with iron haematoxylin and yellowish withChampy-Kull. When visible these drops are always seen in

OOGENESIS OF CALAMUS 218

association with yolk-droplets. It is possible that they representaccumulations of substances passing from the cytoplasm tothe mitochondria during the formation of yolk therein (fig. 9,PI. 9).

With the exception of one or two doubtful cases the Golgielements have, so far, not been observed in the younger oocytes,and it is therefore impossible to say whether or not they playany part in yolk-formation. In the older oocytes no visibleconnexion was observed between individual Golgi elements andyolk-droplets, nor was their position in any way correlated withthe region of yolk-formation in the cell.

It is suggested therefore, that yolk in C a l a n u s finmarchi-c u s is formed in the mitochondria by the transformation of partof their own substance and the deposition in them of substancesderived from the nucleolus and the cytoplasm.

THE GOLGI APPARATUS.

In the oogonia and very young oocytes fixed with Memmingand stained with iron haematoxylin, deeply-staining sphericalstructures were seen closely pressed against the nuclear mem-brane in some preparations. Although adjacent to it, thesestructures appeared quite separate from the mitochondrial cap(fig. 2, PI. 9). The close proximity of the mitochondrial cap inthe oogonia and the presence of nucleolar emissions upon thesurface of the membrane in the young oocytes render it difficultto be certain of the nature of these bodies. While they mayrepresent smaller aggregations of mitochondria which haveseparated from the cap and tend, owing to their small size, tobecome spherical there are indications of a non-staining chromo-phobe centre and a deeply-staining chromophilic rim in one ortwo cases. In some other cases where this rim is not visible theyare stained more deeply with iron haematoxylin than theadjacent mitochondrial masses. This staining reaction suggeststhe possibility that these structures are Golgi bodies. No traceof impregnation was found by silver nitrate or osmium tetroxidemethods in oogonia or young oocytes to substantiate this viewand all attempts to demonstrate the apparatus in the youngcell by any other method have so far been unsuccessful.

NO. 294 P

214 IRENE F. HILTON

The Golgi apparatus was first identified without doubt inoocytes measuring 40-50/* in diameter. By Da Pano's methodclear pictures were obtained which showed the apparatus in theform of black uneven granules of irregular shape, lying mainlytowards one pole of the cell (fig. 15, PL 10).

In one or two cases a very heavy impregnation occurred, butit is unlikely that the whole of this signifies the presence ofGolgi elements.

In half-grown oocytes the silver deposit is much lighter andthe individual elements are scattered over a much larger areawhile still being mainly concentrated towards one side of thenucleus. In the mature oocytes the silver nitrate method showsa uniform distribution of the Golgi elements throughout thecytoplasm in the form of small bodies of irregular shape butsmooth 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 withiron haematoxylin a structure of doubtful origin was seen lyingin the cytoplasm close to the nuclear membrane. This structure,which was spherical when examined in section, appeared toconsist of a clear chromophobe centre and a well-definedchromophile rim. Although lying close to the nucleus it had noconnexion with it. The cytoplasm immediately surrounding thisbody stained rather more lightly than the cytoplasm in the restof the cell. In staining reaction and in general appearance thisstructure was very like the spherical form of the Golgi apparatusseen in optical section when stained by this method, but it wasunusually large for a Golgi body and furthermore was not foundin any of the other oocytes of the same age treated by the samemethod. Its exact nature and origin remains a mystery (fig. 10,PI. 10).

In the older oocytes at the onset of yolk-formation one or twopreparations stained with iron haematoxylin showed the ring-shaped Golgi elements scattered in the cytoplasm. These weresmaller than those found in the younger oocytes (Text-fig. 5).

The last stage of the Golgi apparatus, in which it occurs asscattered granules distributed throughout the cytoplasm of theripe 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 ofthe cell.

Careful examination failed to reveal any connexion betweenthe developing yolk-droplets and the individual Golgi elements,nor was the position of the complex stage in the cell in any wayrelated to regions where yolk-formation was taking place. The

TEXT-FIG. 5.

Gb

Oocyte at the beginning of yolk-formation showing Golgi bodies,scattered mitochondria, yolk-droplets, and beginning of vacuola-tion in the cytoplasm. G b, Golgi bodies ; v, vacuoles; w ov, wall ofoviduct; y d, yolk-droplets.

function of the Golgi apparatus in Calanus remains an openquestion.

DISCUSSION.

In the present investigation several points arise for discussion.These fall into two main groups; questions concerning thebehaviour of the chromosomes and the process of nucleolaractivity. Associated with the latter is the question of yolk-formation in the oocyte and the part played in the process bynucleolar emissions. The following points are dealt with underthese headings.

The chromosomes. In the very young oocytes ofCalanus finmarchicusa typical synizesis figure is formed,but no bivalent chromosomes emerge from this although pre-sumably the bivalents are present in the synizesis knot. Thebivalents appear for the first time after the growth of the oocyte

216 IEENE F. HILTON

is completed and immediately prior to the first maturationdivision. During the entire growth phase no individual chromo-somes are visible but a much twisted thread surrounds thenucleolus. Among the Amphibia x it is known that the decon-centration of the chromosomes proceeds so far that many, orall of them, are indistinguishable during the growth phases ofthe egg. In these extreme cases the germinal vesicle shows onlyan oxyphilic lightly-staining meshwork surrounding one or twonucleoli which presumably contain the entire basophilic contentof the nucleus. In the disappearance of the individual chromo-somes and the presence of the twisted thread surrounding thenucleolus the condition found in O a l a n u s is comparable tothis, but there is no evidence for believing that the nucleoluscontains the entire basophilic content of the nucleus; on thecontrary the thread surrounding it stains with basic stains ifrather more lightly than the nucleolus. It appears probablethat this network surrounding the nucleolus is in reality a muchmodified spireme formed from the deconcentrated chromosomes,which lose their individuality during the growth phases of theoocyte and reappear as ring-shaped bivalents at maturation.

The ring-shaped form of the chromosomes is very constant forthe C o p e p o d a . Many of the earlier Avorkers who observedthem believed them to be formed from chromosomes which hadunited by telosyndesis during the period at which the chromatinis massed at one pole of the early oocyte nucleus. Kornhauser(1915) observed these rings in the C o p e p o d a but believedthem to be formed from chromosomes which had united byparasyndesis in the early oocyte nucleus. He claimed that eachof these chromosomes showed at an early stage a distincttransverse split which he supposed to be an integral part of thestructure of the chromosome. No trace of such a split wasvisible in any of the chromosomes of C a l a n u s finmarchicusand from the material examined it was impossible to say withany degree of certainty whether the bivalents were formed by

1 A n u r a OscarSchultze (1887), Carnoy andLebrun (1879), King (1908).U r o d e l a Born (1894), Carnoy and Lebrun (1878, 1879), Schmidt (1905),Jorgenssen (1913), Stieve (1920).

OOGENESIS OP CALANUS 217

telosyndesis or parasyndesis, though the latter appears moreprobable.

N u c l e o l a r e x t r u s i o n . Throughout the entire growthphase of the oocyte nucleolar activity is very marked in C a 1 a -n u s finmarchicus. The exact nature of the portions ofnucleolar material which pass out into the cytoplasm and theirultimate fate is difficult to determine. The majority of the earlierworkers upon the C o p e p o d a failed to establish the passageof the nucleolar extrusions into the cytoplasm although theyobserved them within the nuclear membrane. Moroff (1909)figured for P a r a c a l a n u s p a r v u s fragmentation of thenucleolus and the presence in the cytoplasm of granular massesof material which he believed to be nucleolar in origin. Thesefigures correspond so closely with those obtained for C a l a n u sf i n m a r c h i c u s after Flemming and iron haematoxylin thatthere is little doubt the granular masses in the cytoplasm weremitochondrial in nature: in no cases were masses of nucleolarmaterial seen in the cytoplasm in C a l a n u s , having oncecondensed out from the surface of the nuclear membrane theextrusions appeared as scattered drops moving towards theperiphery of the cell. There is absolutely no evidence that theypierce the membrane as whole bodies though this condition isreported by Nath and Mehta (1929) in the eggs of the Firefly.While it has not been possible to establish a definite periodicityin the behaviour of the nucleolus in C a l a n u s there is evidencefor believing that its activity is much more marked at certainstages of the growth of the oocyte than at others. In all theyoung oocytes examined, numerous nucleolar emissions of vary-ing sizes were seen inside the nucleus, while outside the nuclearmembrane an accumulation of nucleolar material was visible.This was much less marked in the older oocytes but immediatelybefore maturation a second period of marked activity on thepart of the nucleolus occurs. The first of these periods coincideswith the stage at which the mitochondria are arranged in a ringsurrounding the nucleus. It has already been suggested that thenucleolar emissions may come into relation with the developingmitochondria at this time and that the dark cloud in the cyto-plasm surrounding them which is seen in some preparations may

218 IRENE F. HILTON

consist of secretions from the cytoplasm and nucleolus. Thesecond period of marked activity coincides with the depositionof yolk in the oocyte, the breakdown of the nuclear membrane,and the formation of the chromosomes. That part of thenucleolar substance is used up in the formation of the chromo-somes is probable, but there is considerable evidence to showthat the emissions which pass into the cytoplasm are in someway connected with yolk-formation.

The method of yolk-formation in the egg has been the subject ofmuch recent research. Numerous records of the close relationshipbetween mitochondria and yolk, and between Golgi apparatusand yolk are to be found. Many of these are reviewed in a recentpaper by Hibbard (1928). Other cytologists have put forwardthe view that the nucleolus is directly concerned in yolk-forma-tion. Nath and Mehta (1929) and Gresson (1929) have recordedthe formation of yolk from nucleolar emissions in the cyto-plasm. The formation of albuminous yolk from nucleolaremissions has been described in the cockroach by Hogben (1920)and in S a c c o c i r r u s by Gatenby (1922); other examples mightbe cited. In a paper on the oogenesis of L i m u l u s p o l y -p h e m u s Gardiner (1927) proved the presence of substancesrich in phosphorus in the nucleolus and suggested that themechanism of nucleolar emission effects the transport ofphosphorus from the nucleus to the cytoplasm. It appearsprobable that in C a l a n u s one of the functions of nucleolaractivity is to provide a means of transport by which substancesused in the formation of yolk are passed from the nucleus intothe cytoplasm.

SUMMARY.

Three regions can be recognized in the ovary: a multiplica-tion zone containing oogonia undergoing mitosis, a synapsiszone containing the first formed oocytes in the prophases of thematuration division, and a growth zone containing oocytes ina series of growth phases with the nucleus in a ' resting condition'.

The oogonial nuclei contain two or three nucleoli—plasmo-some and karyosomes. In the oocytes a single nucleolus ispresent; this is formed by the fusion of the plasmosome and at

OOGENESIS OP CALANUS 219

least one karyosome and is therefore an amphinucleolus. Thechroma tin in the oogonia and young oocy tes is arranged round theperiphery of the nucleus andis aggregated in knots (pp. 196-200).

Nucleolar extrusion begins in the young oocyte and continuesthroughout the growth period. It is most marked in the youngoocytes and in oocytes about to undergo maturation (pp. 200-3).

In the older oocytes the chromatin is in the form of a tangledthread surrounding the nucleolus. Immediately before matura-tion this condenses and circular chromosomes emerge: theseform tetrads (pp. 203-5).

The mitochondria are present in the oogonia and very youngoocytes in the form of a cap lying upon the surface of the nuclearmembrane. The mitochondrial elements spread and multiplyuntil 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 issometimes earlier. The formation of yolk-droplets begins at theperiphery of the cell and proceeds inwards. It is suggested thatyolk is formed by transformation of the mitochondria and thedeposition in them of substances derived from the cytoplasmand the nucleolus. The cytoplasm is flocculent in the youngoocytes, granular in the half-grown oocytes, and filled withfluid vacuoles in mature oocytes. It passes from a primarycondition of oxyphily to basophily and finally back to a secondaryoxyphil condition in mature oocytes (pp .211-13).

In the young oocytes deeply-staining spherical structures wereseen adjacent to the mitochondrial cap. From their appearanceit is possible these bodies represent the Golgi apparatus, butDa Pano fixation failed to demonstrate them. In half-grownoocytes the apparatus was visible in the complex condition atone side of the cell. As growth proceeds it passes from a complexto a diffuse condition and in mature oocytes the Golgi elementsare uniformly distributed throughout the cytoplasm (pp. 213-15).

220 IRENE F. HILTON

LIST OF REFERENCES.

Amma, K. (1911).—"Ueber die DiSerenzierung der Keimbahnzellen bei denCopepoden", '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 certainTenthredinidae", ibid., vol. 73, p. 177.

Harvey, L. A. (1925).—"On the relation of the mitochondria and Golgiapparatus 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, deBiol.', 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 inDiaptomus 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-parasiticcopepod 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, I I I" ,

'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 FIGURESThe 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, nucleo-lar extrusions; n r, nuclear reticulum; p, plasmosome; sp, spindJe; spir,spireme; syn st, synapsis stage; t, tetrads; v, vacuole; W OF,wall ofoviduct; x, cytoplasmic body; y A, yolk-droplets; 1st TO sp, first matura-tion 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 restingstages. (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 andGolgi bodies. (E.w.a., I.H.).

Eig. 4.—Oocytes from the synapsis zone showing synapsis and leptotenestages and mitochondrial cap. (E.w.a., I.H.).

Eig. 5.—Half-grown oocyte showing mitochondrial masses, nucleolarextrusion 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 packedwith yolk-droplets. (F.w.a., I.H.).

Fig. 8.—Young oocyte showing vacuolation in tho rim of the nucleus andgranular core, nuclear reticulum, and nucleolar extrusion. (F.w.a., I.H.).

Eig. 9.—Part of the cytoplasm of an oocyte before maturation showingyolk-formation, vacuolation of the cytoplasm, and chromophobe dropletsat d. (E.w.a., I.H.).

PLATE 10.

Eig. 10.—Half-grown oocytes from anterior end of the oviduct showingnucleolar extrusion, beginning of yolk-formation and body of unknownorigin in the cytoplasm at x. (E.w.a., I.H.).

Fig. 11.—Oocyte during first maturation division showing equatorialplate and diffuse condition of Golgi apparatus. (Da E., E.H.).

Eig. 12.—Young oocyte showing large central vacuole of nucleolus andGolgi bodies surrounding nucleus on one side. (Da F., E.H.).

Eig. 13.—Oocyte during first maturation division showing spindle withtetrads 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 inrim of nucleolus, circular chromosomes, and Golgi apparatus in diffusecondition. (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.).

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