A Cytological Study of the Urodele Obcyte With Special Reference to the "Lambrush" Chromosome.

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r

T FORDHAM G

raduate

Scho ol

U N I V E R S IT Y of

A

rts a n d

S c ie n c e s

_______ February 1,

19J>0

This dissertation prepared under my direction by

Mario A, Fontanella

entitled

A Cytological^Study of the IJrodele Obcyte with Special

Reference to the "Lampbrush” Chromosome,

has been accepted in partial fulfilment o f the requirements for the

Degree of

Charles A. Berger

,

S,J«

( Faculty Adviser)

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A CYTOLOGICAL STUDY OF THE URODELE OOCYTE WITH SPECIAL REFERENCE TO THE "LAMPBRUSH" CHROMOSOME

BY MARIO AUSTIN FONTANELLA B.S., New York University, *37 M.A., Columbia University, *40

DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF BIOLOGY AT FORDHAM UNIVERSITY NEW YORK 1950

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ProQuest N um ber: 10992991

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TABLE OF CONTENTS

LIST OF TABLES ..............................

Page iv

LIST OF FIGURES .............................

v

ACKNOWLEDGEMENT .............................

ix

I.

INTRODUCTION...........................

1

II. MATERIAL AND METHODS ....................

5

III. OBSERVATIONS AND RESULTS ................

8

Origin of the germ cells ................ Larva ............................... Adult ............................ The membranes .......................... Structure of the oocyte ................. Cytoplasm and y o l k ..................... The nucleoli ........................... The chromosomes ................... Premeiotic resting stage ............. Preleptotene stages ................. Leptotene stage ..................... Zygotene stage ......... Pachytene stage ................ Diplotene stage ................ Early "lampbrush” stage .............. Continuous "lampbrush" stage ......... Discontinuous "lampbrush" stage ...... Reappearance of meiotic bivalents ..... IV. DISCUSSION .............................

V.

8 8 8 9 10 13 16 39 39 40 40 41 41 42 43 44 44 45 77

Cytoplasm and yolk ..................... The nucleoli ......... The spirallization cycle ................ The "lampbrush" as a polyploid structure .. General considerations .............

77 81 83 85 89

SUMMARY ................................

93

BIBLIOGRAPHY

96

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A CYTOLOGICAL STUDY OF THE URODELE OOCYTE WITH SPECIAL REFERENCE TO THE "LAMPBRUSH" CHROMOSOME

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LIST OF TABLES

Table I,

II.

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Page Measurement of the diameters of serial sections of one oocyte and its germinal vesicle. Eft stage of Triturus viridescens viridescens ....

18

Correlation of -prophasic progress with the growth of the germinal vesicle and oocyte. Necturus maculosus maculosus ......

48

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LIST OF FIGURES Figure !•

Triturus viridescens viridescens. Genital ridge of 1 cm. larva ..............

20

2. Desmognathus fuscus fuscus. Genital ridge of 2.5 cm. larva ....................

20

3. Desmognathus fuscus fuscus. Cyst of oogonia and o8cytes in the cyst wall epithelium ...............................

22

4. Desmognathus fuscus fuscus. Cyst of oogonia and oocytes, showing telophase of last mitotic divisionbefore meiosis .... 5. Desmognathus fuscus fuscus. Portion of a cyst containing cells in different stages of development .................... 6.

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12. 13. l

Page

22

24

Necturus maculosus maculosus. Portion of a cyst containing cells in different stases of development ...................

24

Triturus viridescens viridescens. A cyst of obcytes in the eft stage .........

26

Triturus viridescens viridescens. Part of a cyst of the eft stase to show the vitelline areas .........................

26

Necturus maculosus maculosus. Late oocyte stained with Feulgen-reaction .....

28

Triturus viridescens viridescens. Section of large obcyte to show peri­ pheral migration of germinal vesicle .....

28

Triturus viridescens viridescens. An isolated section showing large basophilic bodies in the cytoplasm ...........

30

Triturus viridescens viridescens. Vitelline areas in adjacent o&cytes ......

30

Triturus viridescens viridescens. Large obcyte to show depos it ion of yolk ....

32 _i

Figure

Page

14. Desmognathus fuscus fuscus. Section showing degenerative changes in large oocyte ................................

32

15. Desmognathus fuscus fuscus. Section to show the role of follicular cells in a degenerating oocyte ....................

34

16. Desmognathus fuscus fuscus. Section to show the role of follicular cells in a degenerating oocyte ............... 17. Triturus viridescens viridescens♦ Section to show structure of germinal vesicle, ......... cytoplasm, and yolk spherule 18. Necturus maculosus maculosus. Section of whole obcyte showing nucleoli in the cvtoplasm ........................

34

36

36

I9* Necturus maculosus maculosus. Section to show nucleoli andlampbrush" loops stained with methyl-green-pyronine ..............

38

20 • Triturus viridescens viridescens. Over­ destained nucleoli in the eft stage .....

38

21. Necturus maculosus maculosus. Oogonial resting stage ..........................

50

22. Necturus maculosus maculosus. Early preleptotene stage ...............

50

23. Necturus maculosus maculosus. A later preleptotene stage .....................

52

24. Necturus maculosus maculosus. Leptotene stage ........................

52

25. Necturus maculosus maculosus. Zygotene stage ................................

54

25. Necturus maculosus maculosus. Pachytene stage .................................

54

27. Necturus maculosus maculosus. Early diplotene stage characterized by the opening-out of bivalents ...............

56

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Figure 28.

29.

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31.

32.

33.

34.

35.

33. 37. 38.

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Page Necturus maculosus maculosus. Later diplotene stage with chromosomes that are beginning to show the "lampbrush” characteristics ..............

56

Necturus maculosus maculosus. Section of whole nucleus in the continuous ”lampbrush” stage .......................

58

Necturus maculosus maculosus. The continuous "lampbrush” stage showing the structure with a definite central thread or "backbone” .....................

58

Necturus maculosus maculosus. Section of whole nucleus in the discontinuous "lampbrush” stage .......................

60

Necturus maculosus maculosus. The discontinuous "lampbrush" stage showing series of side-loops attached to discrete chromomeres ...................

60

Necturus maculosus maculosus. Crosssectional view of the "larapbrush" structure ...........................

62

Necturus maculosus maculosus. Section of whole nucleus showing disintegration of "lampbrush" structures and reappearance of bivalents ....

62

Necturus maculosus maculosus. Section to show a conventional bivalent that has appeared among the disintegrating "lamp­ brush" structures ..................

64

Necturus maculosus maculosus. Section to show newly-appearedbivalent ..........

64

Necturus maculosus maculosus. Section to show condensed nucleolarexudates ......

66

Necturus maculosus maculosus. Section to show newly-appeared bivalent in a stage of greater condensation than in previous figures .......................

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Figure 39.

40. 41.

42 •

43 •

44•

45•

46.

47•

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Page Necturus maculosus maculosus. Section to show newly-appeared bivalent with a visible coil and greater degree of condensation ...........................

68

Necturus maculosus maculosus. Another appearance of the bivalent structure ....

68

Necturus maculosus maculosus. A bi­ valent in which the degree of con­ densation is greater than in preceding figures ...............................

70

Necturus maculosus maculosus. A newlyappeared bivalent with the appearance of typical early diakinesis .............

70

Necturus maculosus maculosus. A newlyappeared bivalent similar to the one in previous figure ........................

72

Necturus maculosus maculosus. A newlyappeared bivalent in the configuration of early diakinesis .......

72

Necturus maculosus maculosus. Condensed bivalent in the configuration of early diakinesis .............................

74

Necturus maculosus maculosus. A typical diakinesis bivalent with a prominent chiasma ..............................

74

Necturus maculosus maculosus. Bivalent showing maximum degree of condensation ....

76

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ACKNOWLEDGEMENT

The author is indebted to Dr. Charles A. Berger, S. J., whose patience and help made possible the completion of this work

1

I.

INTRODUCTION

Beginning with the classical paper of 0. Schultze (1887) which described the development and fertilization of the amphibian oocyte, many accounts were published, each describing in some detail a part of the maturation cycle undergone by the female germ cells of amphibians, Roux (1885, 1887) contributed an accurate account of the route of the pronucleus in the egg of the frog. Born (1892, 1894) was concerned with the chromatin filaments in the germinal vesicle of Triton. Jordan (1893), following the lead of Born, reported further observations on the chromatin filaments and contributed an accurate account of the formation of polar bodies in Diemyctylus. Michaelis (1897) described fertili­ zation in Triton. Carnoy and Lebrun (1897, 1898, 1899, and 1900) took up the problem of the germinal vesicle and its contents.

Using the oocytes of amphibians,

elasmobranchs, and other fishes, they reported that, as the chromosomal structures became oxyphilic, the nucleoli became basophilic and assumed the shape of "spireme threads".

They concluded that the materials

of the chromosomes and the nucleoli were interchangeable. This conclusion cast doubt on the view that chromosomes maintained continuity throughout the meiotic cycle. L

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However, the work of Marechal (1906), Loyez (1906), Jorgensen (1913), and others demonstrated the individu­ ality and continuity of the chromosomes throughout the process of maturation.

Smith (1912) made a thorough

study of maturation and fertilization in Cryptobranchus. Makino (1934) reported a cytological investigation on the maturation and fertilization of the oocyte of Hynobius. Of all the early studies, only the paper of Makino (1934) is written in the light of modern cyto­ logical findings, but his paper is concerned chiefly with the process of fertilization, rather than with the history of the egg before fertilization.

The latter

development is the main concern of the present study. It seems odd that such authors as Smith (1912) and Makino (1934) noted the appearance of the multiplenucleolus and the "lampbrush” structure in the oocyte but did not attempt an explanation of these phenomena. In recent years, inspired by a more complete knowledge of the cytological and genetical implications of meiosis, many authors have used the chromosomes of gametogenesis as material for diverse studies.

Repre­

sentative animals from all phyla have been used, and, with few exceptions, male germ cells have been preferred.

The

choice of the male seems to be guided by the availability cf all stages of the meiotic cycle in one testis and by L

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the ready use of the smear method of study* germ cells have not proven as favorable.

Female

Material is

not as readily available, for the later stages of meiosis, beginning with Metaphase-I, are found only when the oocytes are admitted into the oviducts in the brief period preceding fertilization.

Detailed studies of

chromosomal structures in oocytes are hampered because the large deposits of yolk and unusually large amounts of nucleoplasm make the use of the smear method impracti­ cable.

For the same reason, it is difficult to make

an accurate chromosome count in the oocyte.

Difficulty

is encountered even in the preparation of sections, since the ordinary fixatives of Histology and Cytology do not lend themselves easily to the fixation of heavilyyolked eggs. Any work with telolecithal eggs of vertebrates will of necessity deal with the peculiar problem of the large germinal vesicle and the changes that occur within it. The chromosomes undergo profound changes in the prophase of the first meiotic division, assuming the character­ istic "lampbrush" formation, a condition accurately described by Ruckert (1892).

Because of the unusual

behavior exhibited by the chromosomes of telolecithal eggs of vertebrates, several recent studies not directly concerned with oogenesis have been made on amphibian

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oocytes.

These studies were undertaken with the view

of studying either some morphological peculiarity (Duryee 1941, Ris 1945) or some chemical affinity of the chromosomes (Dodson 1949). The present study is concerned with the development of the ovarian oocyte in several species of urodeles, beginning with the appearance of the genital ridge in the larva and ending with the diakinesis stage which just precedes metaphase-I.

It is felt that there is

need to re-interpret some of the older findings and correlate them with more recent cytological and chemical data, especially with .regard to the "lampbrush" structure•

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II.

MATERIALS AND METHODS

Five species of urodele amphibians were examined in this study.

These species were Triturus viridescens

viridescens (Rafinesque), Desmognathus fuscus fuscus (Rafinesque), Eurycea bislineata bislineata (Green), Ambystoma mabei (Bishop), and Necturus maculosus maculosus (Rafinesque).

Ambystoma and Necturus were

obtained from zoological dealers.

Triturus. Eurycea,

and Desmognathus were collected by the author in brooks and ponds in Staten Island, New York, through­ out the year at regular intervals.

The eft or land

stage of Triturus was collected in the Bear Mountain region of New York, since this intermediate stage was not found in Staten Island. Preliminary examination showed that developmental patterns in all the named species were similar. However, certain phases of development were observed more clearly in one or the other of the species.

It

was decided to use a given species for the study of that phase which it showed to most advantage.

For

example, Triturus was best suited for the study of early growth and yolk deposition, since larval, eft, and adult stages were available. Similarly, although all species showed the ’’lampbrush" structure, most observations on this

phenomenon were made on the oocyte of Necturus which was larger and showed the structure to best advantage. No attempt was made to verify chromosome counts which have been reported by other authors using male germ cells. The animals were killed by decapitation, and the ovaries were put into various fixatives.

In the case of

the species collected by the author, the materials were fixed either in the field or immediately upon return to the laboratory.

In the case of shipped material,

the animals were killed and ovaries fixed immediately upon receipt.

For the study of larval stages of

Triturus, gravid females and sexually mature males were collected in May and put into a large glass aquarium containing clear pond water and a few leaves Flodea canadensis. Fertilized eggs were deposited and affixed to the submerged leaves.

The eggs were

allowed to develop, and the larvae so obtained were kept for several weeks, then fixed whole, embedded, and sectioned serially. The following fixatives were used in the course of the investigation: BouinTs, San Felice, Carnoy's, Smith1s Fluid, and the Glacial-Acetic mixture recom­ mended by Makino (1934).

All fixatives proved satis­

factory for the early ovarian stages before the depo­ sition of yolk, but only Smiths Fluid provided for

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The whole larvae were

fixed in Bouin's fluid. After embedding in paraffin (52 degrees F.), serial sections were cut at 10, 15, 17, 20, and 25 micra.

It

was found that sections cut at 15 micra were best for the study of the germinal vesicle and the oocyte in general. Larval stages were cut at 12 micra. Each stage studied was stained in Heidenhain^ ironalum hematoxylin and by the Feulgen method.

Representa­

tive sections were also stained by the eiethy1-greenpyronine method, as described by Brachet (1940).

The

Feulgen reaction was used for two reasons, namely, because of its specificity for desoxyribonucleic acid and because it does not cause a color change in yolk. Hematoxylin, on the other hand, stains yolk granules heavily.

The methyl-green-pyronine method was used as

a test for ribonucleic acid.

The sections of whole

larvae were stained with Heidenhai^s iron-alum hematoxylin only. Photographs were taken on 35 mm. Kodak Microfile film with a Leica camera equipped with the Micro-ibso attachmentf and with a Leitz Macca using 4.5 x 6 cm. plates.

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III.

OBSERVATIONS AND RESULTS

Origin of the germ cells There are two methods applicable in the study of the origin of germ cells.

One method is con­

cerned with the larval stage and the earliest ap­ pearance of the sex cells following the formation of organs.

The other method is concerned with the

production of new cells in the adult animal. Larva.

The first appearance of g e m cells in

ontogeny occurs in the genital ridge of the larva. The germ cells are at first indistinguishable from ordinary somatic embryonic cells.

Those cells which

are to become gonia increase in size, undergoing a growth period in which the nucleus increases in size until it occupies almost the entire cell volume.

At

the end of this growth period, the chromatin material of the nucleus resolves itself into threads, and the cell becomes surrounded by smaller, undifferentiated cells of the genital ridge. cells are known as auxocytes.

At this stage, tha germ Figures 1 and 2 show

several such cells in the larvae of Desmognathus and Triturus. The average diameter of the auxocytes in these sections is 30 micra. Adult.

In all the species studied, new germ cells

were seen to arise in cysts formed from cells of the cyst wall epithelium (fig. 3), and less commonly of the ovarian epithelium.

The cells in the cysts are

at first undifferentiated and resemble the oogonia of the genital ridge.

Later developmental relationships

parallel those of the larval condition.

Figures 4, 5,

and 6 show cyst cells in fifferent stages of development When the oogonium has reached maximum size, undiffer­ entiated epithelial cells migrate into the cyst and surround the germ cell (fig. 5).

Figure 4 shows a

telophase stage of the last mitotic division before prophase; in this cyst, all the cells are oogonia and measure about 30 micra in diameter.

Figures 5 and 6

show later development, in which some of the oogonia have become oocytes. The membranes The ovary is a sac containing groups or cysts of oocytes.

The lining of the ovarian sac is a single­

layered epithelium.

Each cyst of oocytes within the

ovary is also surrounded by a single-layered epithelium, the cyst wall epithelium (fig. 7).

Each oocyte is

surrounded by a single-layered epithelium, the follicle (fig. 8). In places where new cysts are to be formed, the

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cyst wall epithelium (and occasionally the ovarian epithelium) thickens until it becomes multi-layered. Some of the cells in this new formation become oogonia and then oocytes, the remaining cells either migrating to form follicles around the new oocytes or moving to the periphery to form a new cyst wall epithelium.

At

an early stage, the cyst looks like a spindle-shaped sac, with its two extremities joining the single-layered cyst wall epithelia of the adjacent areas (fig. 3). Most of the follicular cells of the growing oocyte show chromatin arrangements that suggest mitotic ac­ tivity, but actual mitotic figures are not common.

In

young oocytes, the follicular cells are rounded; in older cells, they are flattened.

The follicle presents

a striking appearance when the preparation is stained by means of the Feulgen reaction (fig. 9).

Structure of the oocyte The yolk-laden oocytes of vertebrates are the largest known forms of cells.

In the moderately telo­

lecithal oocyte of amphibians, the cytoplasm is dis­ tributed throughout the structure and not limited to a blastodisc as is the case in reptiles and birds. Serial sections were made of ovarian cysts of the eft stage of Triturus. A single diplotene oocyte in

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n one of the cysts was studied by measuring the diameter of the germinal vesicle and of the oocyte as they ap­ peared in each cut or section of the series.

Table I

is a record of measurements made with an ocular mi­ crometer of one oocyte cut in serial sections at 15 micra.

The results indicate that the largest diameter

of the oocyte is 315 micra, while that of the germinal vesicle is 195 micra.

In sections 5 and 15 of the

series (Table I), the nucleoli are scattered over the entire cut surface of the germinal vesicle (fig. 7, g.v.-l).

In all other sections of the series in which

the germinal vesicle appears, however, the nucleoli are found only at the periphery of the vesicle (fig. 8)• This distribution suggested a method of counting the number of nucleoli present in the germinal vesicle at this stage.

Focussing with the fine adjustment reveals

that there are two layers of nucleoli in each section cut at 15 micra; the average diameter of the nucleolus is 4 micra.

Since between 40 and 50 nucleoli can be

counted in each section and since nucleoli appear in ten sections of the series (Table I), there are between 400 and 500 nucleoli in the germinal vesicle of the eft oocyte studied. The oocyte is typically ovate.

This fact is deduced

from the measurements recorded in Table I. l

The widest _i

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diameter measured in the serial sections is 240 micra.

This measurement may be taken as one of the

axes of the oocyte, in fact, of the short axis, for the long axis is computed to be 315 micra.

The long

axis is determined by counting the number of sections obtained in the complete series and multiplying this number by 15, which is the thickness of each section in micra.

In many cases, the outline of the oocyte

is very irregular, a condition due to mechanical pres­ sure (fig. 9).

The germinal vesicle is typically

spherical and maintains a central position in the oocyte in the early stages of development.

At the

approach of metaphase-I, the germinal vesicle will migrate to the periphery of the oocyte, preparatory to its dissolution and the resolving of cleavage chromosomes (Makino 1934).

Since metaphase does not

occur until the oocyte is extruded from the ovary, these changes were not studied in the present investi­ gation. By comparing the oocyte-germinal vesicle ratios at different levels of development, it was seen that the over-all size of the oocyte increases faster than that of the germinal vesicle.

The ratios for representative

stages were obtained from the type of measurement illus­ trated in Table I. L

For example, by counting the number _i

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n of times that both oocyte and germinal vesicle appear in the serial sections and multiplying by the thickness of the sections, it is seen that the ratio for the oocyte recorded in Table I is 315/195; this is an oocyte in early diplotene, containing no yolk.

In an

early "lampbrush" stage of the ^riturus oocyte, con­ taining an appreciable amount of yolk, the ratio was found to be 1156/470. the ratio was 1300/300.

In a very late "lampbrush" stage, In the last case, the germinal

vesicle was in an eccentric position, undergoing pe­ ripheral migration (fig* 10).

It is noted that the

germinal vesicle in the late "lampbrush” oocyte is smaller in diameter than the one in the early "lampbrush" oocyte, while the reverse is true of the diameters of the oocytes.

This indicates that the germinal vesicle

loses some of its material during the peripheral migra­ tion, while the oocyte itself continues to increase in size. The cytoplasm and yolk The early oocyte undergoes prodigious increase in size.

The enlargement is caused partly by the elabo­

ration of and deposition of yolk, but a proportionately large amount of cytoplasm to contain the yolk is also elaborated.

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In the earliest oocytes, the cytoplasm

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makes up only a minor part of the cell (figs, 1 and 5), By the time early diplotene is reached, the amount of cytoplasm is considerable (figs, 7 and 8),

The cyto­

plasm is seen as a lightly-staining, homogeneous mate­ rial containing scattered granules.

Even in the late

diplotene and "lampbrush" stages, characterized by large deposits of yolk, the cytoplasm remains a re­ latively clear material, staining faintly with hema­ toxylin and showing a delicate, reticular network (fig. 17) . Except for a few isolated preparations (fig. 11), definite structures in the cytoplasm comparable to the vitelline bodies described by earlier authors (Munson, 1912) were not found in the species studied. In oocytes of the eft stage of Triturus, however, there were observed discrete areas in the cytoplasm which stain noticeably with hematoxylin but not with either the Feulgen reaction or the methyl-green-pyronine mixture.

These areas occur in a circular pattern

near the periphery of the oocyte (figs. 7 and 8).

In

development, the stained vitelline areas elongate, and join adjacent areas to form a continuous ring under­ neath the follicle.

Actual yolk spherules are not

discernible at this stage.

The vitelline areas differ

from the rest of the cytoplasm only in staining a l

deep brown with hematoxylin (fig.

1 2 ).

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When yolk spherules appear, the continuous ring of vitelline areas is no longer visible.

The yolk

is deposited in a circular pattern beneath the follicle, in the same position formerly occupied by the vitelline areas.

Yolk eventually fills up the

whole volume of the oocyte outside of the germinal vesicle (fig. 13).

It can be seen from figure 13

that deposition of yolk spherules progresses from •i

the periphery to the middle of the oocyte. In a few sections of young oocytes of the adult Triturus before the formation of yolk, there are observed in the cytoplasm strongly basophilic bodies (fig. 11).

These structures, found in groups of

five and six, are larger than nucleoli which they resemble in their staining affinities and measure from nine to twelve micra in diameter.

They are

very similar to the definite vitelline bodies de­ scribed by Smith (1912). Yolk digestion was noted in large oocytes of Desmognathus, in the case of specimens that had been kept in a tank at room temperature for several weeks.

The cells of the follicle round out, break

off, and wander into the mass of yolk.

Clear areas

around the wandering cells indicate digestion of yolk (figs. 14, 15, and 16). L

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The nucleoli Oogonia and oocytes of all species studied show one nucleolus.

Certain sections appear to contain

many nucleoli, but these dark-staining areas are chromomeres on the chromosomal thread or bends in the chromosomes that can be resolved by focussing.

The

multiple nucleolus condition characteristic of the later stages is observed for the first time in early diplotene (fig. 18).

In some cases, nucleoli are found in the cyto

plasm otitside of the germinal vesicle (fig. 18) . The extruded nucleoli are normal in shape, and the wall of the germinal vesicle is undamaged.

Nucleoli vary in size

within the same vesicle; the variation may be slight or extreme (fig.17).

Nucleoli are stained heavily with

hematoxylin but only faintly with the methyl-greenpyronine mixture (fig. 19).

They are not stained with

the Feulgen reaction. In some sections, when the iron-alum-hematoxylin preparation is over-destained, the nucleoli appear to consist of several black-staining kernels surrounded by a lighter-staining spheroidal mass (fig. 20).

By focus­

ing, as many as seven or eight spheroidal, heteropycnotic bodies can be seen embedded at different levels of the nucleolus.

The largest of the nucleoli in this

preparation (fig. 20) measured four to five micra in diameter. l

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Explanation of Table TABLE I

Measurements of the diameters of serial sections of one oocyte and its germinal vesicle.

Eft stage of Triturus

viridescens viridescens, Measurements were made with an ocular micrometer on each section of the series.

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Number of section

1 2 3 4 5 6 7

Diameter of oocyte

100 micra 130 130 150 150 160 165

Diameter of germinal vesicle

35 micra 70 80

8 9 10

200 220 220

100 100 120

11 12 13 14 15 16 17 18 19 20 21

230 240 240 230 210 200 200 200 170 150 140

130 130 120 90 80 80 30

TABLE I

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Explanation of Figures All figures are mierophotographs of sections stained with iron hematoxylin, unless otherwise noted.

1.

2.

Triturus viridescens viridescens. 1 cm. larva.

Oogonia and oocytes in the genital

ridge.

Magnification X 800.

Desmognathus fuscus fuscus. 2.5 cm. larva. Oogonia and early oocytes in the genital ridge.

Magnification X 800.

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Fig. 1

Fig. 2 L

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Explanation of Figures

3.

Desmognathus fuscus fuscus. Cyst of oogonia and oocytes developing in a cyst wall epithelium.

4.

Magnification X 800.

Desmognathus fuscus fuscus. Cyst of oogonia and oocytes, showing telophase of the last mitotic division before prophase of meiosis.

Magnification

X 1550.

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Fig. 3

Fig. 4 L

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Explanation of Figures

5.

Desmognathus fuscus fuscus. Portion of a cyst containing cells in different stages of development.

Note undifferentiated epi­

thelial cells surrounding oogonia to form follicles.

6.

Magnification X 1630.

Necturus maculosus maculosus. Portion of a cyst containing cells in different stages of development.

Magnification X 1630.

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Fig. 6 L

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Explanation of Figures

7*

Triturus viridescens viridescens. Eft stage. A cyst of oocytes showing the fullest de­ velopment in the eft stage.

Magnification

X 100. c., cyst wall epithelium;

g.v.-l, an end

section of germinal vesicle showing nucleoli spread over the entire section;

g.v.-2,

middle section of germinal vesicle showing nucleoli around periphery only. 8•

Triturus viridescens viridescens. Eft stage. Portion of a cyst to show vitelline areas. Magnification X 230.

L

26

n

r

i

r

f

Fig. 7

Fig. 8

L_

27

r

~i

Explanation of Figures

9*

Necturus maculosus maculosus. Late oocyte to show Fwulgen-negative germinal vesicle and yolk; the follicle is Feulgenpositive.

Stained by the Feulgen reaction.

Magnification X 130.

10.

Triturus viridescens viridescens. Section of large oocyte to show peripheral migration of the germinal vesicle. X 130.

L

Magnification

28

r

Fig.

L

10

29 r

Explanation of Figures

Triturus viridescens viridescens. Large basophilic bodies in the cytoplasm.

These

bodies are comparable to the definite bodies described clasically as yolk-nuclei. Magnification X 800.

12*

Triturus viridescens viridescens. Vitelline areas in two adjacent oocytes showing the homogeneous nature of the cytoplasm, even in the stained areas.

L_

Magnification X 1000.

-J

30

r

Fig. 11

Fig.

L

12

_l

31 r

Explanation of Figures

13.

Triturus viridescens viridescens. Section of oocyte in the "lamphrush" stage, showing deposition of yolk from periphery almost completed. epithelium.

14.

Note younger oocytes within Magnification X 100.

Desmognathus fuscus fuscus. Degenerative changes in a large oocyte.

(

Cells of the

follicle are seen in a phagocytic role. Magnification X 130.

L_

32

n

Fig. 13

Fig.

L

14

33 r

"i

Explanation of Figures

15*

^esmognathus fuscus fuscus. Portion of the periphery of a yolk-laden oocyte, showing the rounded follicular cells in a phagocytic role.

16.

Magnification X 760.

Desmognathus fuscus fuscus. Portions of three oocytes showing rounded follicular cells in a phagocytic role.

Stained with

the methyl-green-pyronine mixture. Magnification X 800.

34

r

— -------

-

Fig.

L

16

.______ |

-i

35

r

i

Explanation of Figures

I?*

Triturus viridescens viridescens. Section of large oocyte to show germinal vesicle, cytoplasm, and yolk spherules.

Note the definite membrane

surrounding the germinal vesicle.

Magnification

X 800. g.v., germinal vesicle; n., nucleolus; c., cytoplasm; y., yolk.

18*

Necturus maculosus maculosus. Section to show a whole oocyte in early diplotene. Note the nucleoli in the unstained cytoplasm. Magnification X 800.

-j

36

n

r

Fig. 17

Fig.

18

37

Explanation of Figures

Necturus maculosus maculosus. Section of the germinal vesicle in the "lampbrush" stage to show nucleoli and "lampbrush" loops stained by the methyl-green-pyronine mixture. nucleoli.

Note alveolar structure of the Arrows point to dtained loops.

Methyl-green-pyronine stain.

Magnification

X 1550.

20.

Triturus viridescens viridescens. Eft stage. Over-destained nucleoli to show heteropycnotic kernels embedded within a lighterstaining spheroidal mass. X 1650.

L

Magnification

38

39 : 318 - 327, __________________ , 1948.

Gytological observations

on the oogenesis of certain Indian lizards. II.

Structure and function of the yolk

nucleus in lacertilian eggs.

Transactions

of the American Microscopical Society, J37 : 341 - 349. Swanson, C. P., 1942a.

Meiotic coiling in

Tradescantia.

The Botanical Gazette, 103 :

457 - 474. _____________ , 1942b.

Differences in meiotic

coiling between Trillium and Tradescantia. Papers of the Michigan Academy of Science, Arts, and Letters, 28 : 133 - 141. Wilson, E. B., 1925.

The cell in development and

heredity, New York, von Wittich, G. H., 1845.

Observationes quaedam

des Aranearum ex ovo evolutions. Saxonum.

L_

Halis

VITA

Mario Austin Fontanella, son of Ferdinand and Lena Fontanella, was born in New Haven, Connecticut, on October 12, 1915.

His high school studies were

pursued at Fordham Preparatory School, New York City, from which institution he was graduated in June 1933. He attended the University Heights College of New York University, New York City, and received the degree of Bachelor of Science from that insti­ tution in June 1937.

He received the degree of

Master of Arts from Columbia University, New York City, in February 1940. He served in the Army Air Corps from February 1941 until October 1945. In February 1946, he was accepted as a gradu­ ate student in the Graduate School of Arts and Sciences of Fordham University, New York City. In September 1947, he was appointed instructor in biology at Fordham College, New York City.