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Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects
1978
The ultrastructure of cell division in male reproductive branches of Polysiphonia denudata
Cynthia Louise Bosco College of William & Mary - Arts & Sciences
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Recommended Citation Bosco, Cynthia Louise, "The ultrastructure of cell division in male reproductive branches of Polysiphonia denudata" (1978). Dissertations, Theses, and Masters Projects. Paper 1539625018. https://dx.doi.org/doi:10.21220/s2-7acb-nw24
This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. THE ULTRASTRUCTURE OF CELL DIVISION
IN MALE REPRODUCTIVE BRANCHES
OF POLYSIPHONIA DENUDATA
A Thesis
Presented to
The Faculty of the Department of Biology
The College of William and Mary in Virginia
In Partial Fulfillment
Of the Requirements for the Degree of
Master of Arts
by
Cynthia L. Bosco
19T8 APPROVAL SHEET
This thesis is submitted in partial fulfillment of
the requirements for the degree of
Master of Arts
Author
Approved, May 19 72
Robert E. L. Black
i. (m Lawrence L. Wiseman
O Q A Q • ! 0 <3 0 D 0 t TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS...... iv
LIST OF FIGURES...... v
ABSTRACT...... viii
INTRODUCTION...... 2
METHODS AND MATERIALS...... 8
OBSERVATIONS...... 10
DISCUSSION...... 23
KEY TO ABBREVIATIONS...... 32
FIGURES...... 33
BIBLIOGRAPHY...... 58 ACKNOWLWDGEMENTS
The writer would like to thank her committee chairman and mentor,
Dr. Joseph L. Scott, for his encouragement, wise advice and high standards throughout the investigation.
The writer would also like to express sincere appreciation to Dr.
Robert E. L. Black and Dr. Lawrence L. Wiseman for their valuable critical comments on the manuscript. LIST OF FIGURES
Figure Page
1. Polysiphonia denudata (male plant)...... 33
2. Light micrograph of several male reproductive branches...... 33
3. Light micrograph of a single male reproductive branch...... 33
4. Montage of a longitudinal section through a young male reproductive branch...... 34
5. Longitudinal section of the basal cell of a male reproductive branch...... 34
6. Longitudinal section of an interphase cell...... 35
7. High magnification of a chloroplast...... 35
8. Longitudinal section through an interphase central cell...... 35
9. Cross-section of an early prophase cell...... 36
10. Cross-section through a polar ring in a prophase cell...... 36
11. Longitudinal section through an early prophase cell...... 37
12. Longitudinal section of a polar ring during early prophase...... 37
13. Longitudinal section of an early prophase cell showing polar ring migration...... -...... 38
14. Cross-section through a prophase nucleus...... 38
15. Longitudinal section of a mid-prophase cell...... 39
16. High magnification of a polar ring (longitudinal section)...... 40
17. Longitudinal section of a polar ring and zone of exclusion at one spindle pole...... 40
18. Longitudinal section of a late prophase nucleus...... 41
v Figure Page
19. Longitudinal section of late prophase in a mother cell...... 41
20. Oblique section through a late prophase cell..;.*...... 42
21. Longitudinal section through a prometaphase mother cell...... 43
22. Longitudinal section through a prometaphase mother cell...... 43
23. Longitudinal section through a metaphase nucleus...... 44
24. Longitudinal section through a metaphase nucleus...... 44
25. Longitudinal section through a metaphase nucleus...... 45
26. High magnification micrograph of a metaphase spindle pole...... 45
27. Oblique section through a metaphase nucleus...... 46
28. Longitudinal section of a metaphase nucleus...... '.46
29. Longitudinal section of a metaphase nucleus, illustrating the separation of the polar ring into two separate components...... 47
30. Longitudinal section through an early anaphase nucleus...... 47
31. Longitudinal section through a mid-anaphase nucleus...... 48
32. Longitudinal section through a mid-anaphase nucleus, showing the two components of a polar ring at this stage...... 49
33. Longitudinal section through a late anaphase nucleus...... 49
34. High magnification micrograph of a late anaphase polar ring..•...••.49
35. Longitudinal section through a telophase cell...... * ...... 50
36. Longitudinal section through a mid-telophase cell...... 51
37. Longitudinal section through a late telophase cell...... >.52
38. Longitudinal section through a late telophase cell...... •••••••••52
39. Longitudinal section of cytokinesis (anticlinal condition)...... 53
40. Longitudinal section of cytokinesis .53
41. Periclinal cytokinesis (longitudinal section)...... 54
42. High magnification micrograph of the mid-region of a dividing cell..54
43. Pit connection formation at the termination of the cytokinetic process (longitudinal section) ...... ••••••••...... 55
vi Figure Page
44. Longitudinal section of interphase in a spermatangium...... 56
45. Schematic diagram of cell division in Polysiphonia denudata...... 57
vii ABSTRACT
The purpose of this study is to describe the ultrastructure of vegetative cell division in male reproductive branches of the marine red alga, Polysiphonia denudata. Understanding these events may prove significant in phylogenetic interpretations within the red algae and among other groups of organisms.
Because of the numerous polar fenestrations present in an other wise intact nuclear envelope, dividing nuclei of P. denudata must be described as both open and closed respectively. Two cylindrical organ elles, or polar rings, are situated outside the nucleus throughout karyokinesis. Polar ring migration occurs during prophase to establish opposing spindle poles. The organelles remain polarly associated at least until telophase. Metaphase nuclei contain highly condensed chromatin, well developed kinetochores, a perinuclear endoplasmic reticulum sheath surrounding the nucleus, and both chromosomal and continuous microtubules. Anaphase separation occurs by constriction and elongation of the interzonal spindle. Two daughter nuclei with intact nuclear envelopes are established at telophase following nuclear envelope reformation in the area adjacent to the interzonal spindle. Cleavage furrow formation begins as an impingement of the mid-region of the cell and proceeds centripetally. Vacuoles aid in separation of the daughter nuclei until cytokinesis is completed. Cytokinesis is terminated with the formation of a pit connection between the two cells.
This work contrasts with the three other studies of cell division in the Rhodophyta in several distinct ways, and raises new questions that need to be answered, specifically concerning the origin, development and migration of the polar ring.
viii TEE ULTRASTRUCTURE OF CELL DIVISION
IN MALE REPRODUCTIVE BRANCHES
OF POLYSIPHONIA DENUDATA INTRODUCTION
The enigmatic process of cell division represents one of life's singular opportunities for renewal through growth and change. Crucial components which can affect an organism's viability and biological competitiveness are divided in this process. At no other time is a cell's genetic information so vulnerable to alteration. In order for an organism to be successful over time, this mechanism must be reliable, accurate and rapid. Biologists have long been fascinated by this subject and have focused their attention on the biochemical, structural and physiological processes that occur at this time.
The electron microscope has proved to be an invaluable tool for studying cell division. Due to the increased resolution attainable, structures barely visible at the light microscope level can be easily distinguished. Recent publications on eukaryotic karyokinesis and cytokinesis have greatly furthered the understanding of the dynamic structures and mechanics involved (5, 6, l U , 15, 21-23, 29, 30, 33, 36,
^3-^+7, 57) • These and other ultrastructural investigations have shown the process of cell division to be extremely diverse from one group of organisms to another. However, little is known about cell division in both the red algae (division Rhodophyta) and the brown algae. They are undisputedly the last frontier to be explored with the electron microscope for details of nuclear and cytoplasmic behavior.
The Rhodophyta is a very specialized group of predominantly marine, multicellular plants and is distinguished from other groups of
2 3
organisms by four major features. Firstly, they possess both chloro phyll a and d, the latter molecule being unique to their division.
Secondly, centrioles and cilia or flagella are totally lacking in all stages of their development. Thirdly, the red algae store floridean starch as a reserve product. Finally, unlike all other eukaryotic algae, pit connections, or cytoplasmic plugs formed during the last stages of cytokinesis between any two cells (see h 9 , for general review), are present in most forms of red algae.
The Rhodophyta is divided into two classes: The Bangiophyceae and the Florideophyceae. The former represent unicellular algae and simply organized multicellular forms , whereas the latter is a much larger and more advanced class with complex patterns of both vegetative growth and reproductive life histories.
In the Bangiophyceae, reproduction is primarily asexual. How ever, most Florideophyceae possess a Polysiphonia-type of life history
(ll) in which the newly formed zygote develops parasitically upon the female gametophyte. This diploid stage, known as the carposporophyte, releases carpospores which settle, germinate and develop into free- living diploid sporophyte thalli. The sporophytes are morphologically similar to the gametophytes (13) and typically produce four haploid spores by meiosis in a structure known as a tetrasporangium. Liberated tetraspores give rise to male and female dioecious gametophytes which produce non-motile gametes. The female gametes (carpogonia) are retained upon the female plant. Male sperm cells (spermatia) are dependent primarily upon water movements for transfer to the female plant for fertilization. k
Studies involving reproductive cells of the red algae are of
particular interest to biologists. Such investigations may not only
illustrate the various ways in which apical cells differentiate,
providing valuable developmental information, but they also clarify
questions related to phylogeny and life histories. Florideophycean
reproductive cells which have been studied at the ultrastructural
level include differentiating spermatia (2k, 25, kl, k2, 53, 65),
carposporangia (28, 59, 6 0 , 6l, 63) and tetrasporangia (9, 26 , 38-UO,
52). Within the Bangiophyceae, developing monospores (31) have been
investigated. Excluding the tetrasporangia, all reproductive cells
are produced Toy mitosis.
Due to thick cell walls and small nuclei (3-5 JJm) and chromosomes,
light microscopic studies of nuclear cytology has been difficult in the
Rhodophyta; cytological observations appear best resolved at the
ultrastructural level (ll). Classicial studies of spermatial develop ment (.13, 18) as well as mitosis (6*0 and meiosis (2, 6k) at the light microscope level have contributed to the overall knowledge. Isolated
stages of mitosis (7, 37, *+1, *+2), meiosis (27 , 5*0 and cytokinesis
(8, 17, ^9) at the electron microscope level have also been informative.
To date, there exists only one comprehensive account of cell division in the Rhodophyta at the ultrastructural level (32). This work investigated the vegetative divisions that occurred in the
Florideophycean alga, Memb ran opt era platyphylla. McDonald as well as
Peyriere (*+l) in an earlier study, noted the presence of an unusual spindle pole body present near a prophase nucleus. McDonald termed this organelle a "polar ring." 5
Unquestionably, the specific lack of information on cell division in the red algae is attributable to at least the following reasons.
Primarily, it is difficult to capture mitotic stages because of the apparent rapidity of the process. Additionally, it is hard to deter mine the time of day when cell division is occurring. Dixon (ll), sampling the same species from neighboring beaches discovered that location could affect the time of day at which cell division occurred.
The possibility of periodicity as a triggering mechanism has been explored (3, U. H8) in situ, and Scott (pers. comm.) has noticed a rhythm in cultured plants of Polysiphonia when placed in a controlled environment. Until now there have been no simple, repeatable methods for inducing cell division. However, it should be cautioned that culturing plants can present other problems: as a rule, cultured cells of red algae do not fix well for electron microscopy. Neither are plants in their natural environment easily prepared for electron microscopy. The thick cell walls of most red algal cells prove difficult to penetrate with standard EM fixation techniques. This can cause poor results and make interpretations difficult. Finally, the fact that few people are working in the field has made progress slow.
Research involving cell division in the red algae will prove significant for at least several reasons. Notably, these studies will increase the understanding of cytological events that occur in a largely unknown and underinvestigated group of organisms. Secondly, knowledge of the mitotic and meiotic apparatus in various species of red algae will be useful in systematics (ll) within the Rhodophyta.
Exhaustive studies of mitosis and cytokinesis in the green algae
C^5, ^7» 57) have documented the reliability of using certain 6 ultrastructural features of dividing cells for phylogenetic considera tions. This information has resulted in a major revision necessary for the overall classification of the green algae. Current taxonomy of the various orders of red algae are based upon cumbersome differences in pre- and post-fertilization cellular events that occur on the female plants. Studies of cell division in the Rhodophyta may likewise pro duce a more natural system of classification within the group.
Finally, these results will contribute to the interpretation of phylogenetic interrelationships between the red algae and other groups of lower eukaryotes. Biochemically and structurally, the red algae appear to be more closely related to prokaryotic blue-green algae than to any other algal group; they are considered by a majority of biologists to be the most primitive eukaryotic organisms. Alterna tively, certain vegetative and reproductive characteristics of red algae, as well as particular features of meiosis and mitosis, are very similar to some groups of fungi (10, 20, 35 s 50, 5*0* However, due to the paucity of electron microscopic investigations of cell division within the red algae, such phylogenetic relationships based upon comparative ultrastructure are difficult to establish at this time.
Polysiphonia denudata is an exceptional tool for studying cell division at the ultrastructural level in red algae. As a classical red alga, much is already known regarding its morphology and behavior at the light microscope level and current work serves to add to the growing body of knowledge surrounding the genus. Additionally, P_. denudata is a ubiquitous inhabitant of local shallow marine waters during the summer months, malting collections convenient. The plant is 7
easily transportable to the laboratory and will flourish in crude
culture for several weeks. Furthermore, such laboratory acclimated plants fix well for electron microscopy. This study is specifically
concerned with cell division in the reproductive branches of male
plants. Numerous cells are produced upon these compact, easily
identified, specialized branches. Polysiphonia denudata, therefore, yields the fortuitous combination of plants bearing large numbers of
cells which can be easily adapted to the laboratory. METHODS AND MATERIALS
Specimens of Polysiphonia denudata were collected from the York
River, a few hundred yards west of the York River Bridge (George P.
Coleman Memorial Bridge) at Yorktown, Virginia. Sampling was under taken during low tide in the summer months of 1977* Both free-floating and attached forms were gathered and transported immediately to the laboratory.
Healthy plants bearing spermatangial branches were cultured in von Stosch’s culture medium (58) in a 2U°C incubator set for a 12:12
(light:dark) photoperiod. The algal cultures were allowed to acclimate and grow for k days in their artificial environment prior to fixation for electron microscopy. A k day interval was found to be the optimum time for maintaining healthy, growing plants, while allowing a suffi cient period for adjustment. Approximately two hours after exposure to light on the fourth day, the male plants of P. denudata were immediately fixed. Previous work performed in this laboratory on a different species of Polysiphonia has shown this incubation period to be repeatedly successful in producing large numbers of mitotic figures.
Prior to this schedule, there had been no consistent method for cap turing the division process.
All material was fixed in a solution of 2% glutaraldehyde in 0.1 M phosphate buffer (pH 6 .6 ), with 0.15 M sucrose for 2 hours. Following a brief buffer rinse, postfixaticn was performed for 2 hours using 1j
8 9
OsQ^ in the same phosphate buffer mentioned previously. The material was dehydrated at room temperature in 50% acetone before being left
for l6~2k hours at ^+°C in a solution containing 2% uranyl acetate in
J0% acetone. Dehydration was completed by progressing through a series
of increased concentrations of acetone, ending in 100p acetone which had been stored over molecular sieve. Infiltration was accomplished using 2:1, 1:1, ana 1:2 mixtures of acetone and Epon 812 epoxy resin,
fo3.1owed by 3 days of soaking in pure resin. The algal tissue was then flat embedded in plastic petri plates and placed in a 60°C oven
for 3 days.
Thin sections were cut with a Dupont diamond knife mounted on an
LKB Ultratome III. Formvar-coated, one-hole copper grids were used to collect the sections according to the method of Galey and ITilsson (l6), which were then poststained for 60 seconds in lead citrate (62) before examination with a Zeiss EM 9S-2 electron microscope.
Live material was examined with a Zeiss Photomicroscope II and photographed with 35 mm Panatomic X film. OBSERVATIONS
Polysiphonia denudata (Fig. l) is a ubiquitous inhabitant of local
marine waters during the summer months and can be collected in three
different life forms (bionts): male; female; and tetraspcrangial
plants. Male reproductive gametes, or spermatia, are produced on
non-pigmented branches on the male plants. The mature branches are
lanceolate in shape (Figs. 2, 3) from 115 to 120 um in length, .and
average 37 to ho pm in diameter. Male reproductive branches are
initiated when an apical cell gives rise both to a uniseriate tricho- blast which remains sterile and to another branch which develops
further into a specialized polysiphonious branch modified totally for
the production of spermatia. The basal cell of the reproductive branch is distinctly unlike other branch cells since the cytoplasm
characteristically stains considerably lighter (Figs. h, 5), possibly
caused by the smaller number of ribosomes present.
Numerous mitotic divisions are undergone in the production of a
fertile male branch that ultimately forms spermatia. Initiating the process, a dome-shaped apical cell, through a series of longitudinal
divisions, gives rise to a filament of axial cells (Fig. U) known as
central cells. Mature central cells are large, uninucleate and extremely vacuolated. Each central cell divides in a distinct fashion,
cutting off cells alternately to one side and then the other until a ring of pericentral cells surrounding the central filament is formed.
10 11
In the male reproductive branches, four pericentral cells are usually
formed (19), however the vegetative branches characteristically contain
six pericentral cells which helps distinguish P. denudata from other
local species. Within the male reproductive branches, the ring of peri
central cells divides laterally resulting in the eventual formation of
spermatangia. Spermatangia production can occur in at least three
different ways: l) the pericentral cell, in some cases, can act
directly as a spermatangial mother cell (SMC) by producing spermatangia,
or more commonly, 2) the pericentral cell gives rise to either a
spermatangial mother cell which produces spermatangia, or 3) to an
intervening cell which gives rise to a SMC which then produces sperma
tangia. Spermatangial mother cells are elongated, uninucleate cells
capable of producing 2-5 spermatangia by mitosis; however 3 spermatangia
per mother cell is most common. These cells are cut off in a slightly
diagonal fashion from the mother cell and constitute the outermost
layer of cells in a mature reproductive branch. Spermatangia (Fig.
are ellipsoid, possess an apically located nucleus containing visible heterochromatic regions, and enclose 1-2 basal vacuoles which are
filled with fibrous polysaccharide material formed from the Golgi
apparatus and endoplasmic reticulum ( in 2b), All spermatangia remain
connected by small septal plugs to the mother cell until mature. At that time, the cells are released from the male branch. Spermatia are formed by a direct transformation of spermatangia following the
secretion of the basal vacuole and the rupturing of the surrounding wall material (2U, 53).
This study is concerned predominately with the vegetative divisions that occur within pericentral cells and other cells that give rise to 12
spermatangial mother cells. As such, it represents the first compre hensive report of cell division within a reproductive structure in the
red algae. It is assumed that this type of karykinesis and cytokinesis will be representative of the events that occur throughout the differing types of cells found in the male branch. However, further studies on mitosis within the fertile male branches are necessary to prove this
generalization.
Interphase
Typical interphase cells are nearly isodiametrical and possess a
spherical nucleus which occupies a major portion of the cell volume
(Fig. 6). Within the nucleus, dispersed heterochromatin and a clearly distinguishable nucleolus are present. The nuclear envelope (NE)
contains numerous nuclear pores, evenly distributed throughout it.
Ho polarity is established at this time and the nucleus is situated in the approximate center of the cell.
Although cytoplasmic organelles are not affected to a large extent by events occurring during karyokinesis, they are briefly described below for recognition and identification purposes within this study.
Numerous ribosomes predominate throughout the ground plasm (Fig. 6).
Also, small vacuoles can be found which constitute an insignificant fraction of the cell’s total volume at this stage. Chloroplasts are greatly reduced and contain few thylakoids, imparting a colorless, hyaline appearance to the male branch cells when seen with the light microscope. However, phycobilisomes are distinctly present in small numbers along the thylakoids (Fig. 7). Small amounts of endoplasmic reticulum (ER) are scattered near the nucleus, and granules of floridean 13
starch (Fig. 8), the storage product typical of red algae, may he seen lying free in the cytoplasm. Additionally, an unusual association of the Golgi apparatus (GA) and mitochondria has been observed (Fig.
6); mitochondria are typically found near the forming face of the GA, as has been reported in other red algae (in l). This relationship is believed to couple a site of energy production to an area of energy utilization.
Two additional cellular components are worthy of note. Both concentric membrane bodies (Fig. ll) as well as "lip" crystals (Fig. 8) may be found in a cell whether it is dividing or not. The function of concentric lamellae in reproductive male branch cells is unknown, although Kugrens & West (25) suggest they may contribute to the sperma- tial vacuole. Lip crystals, on the other hand, are found only in vegetative cells, which coincidently serves as a valuable orienting device for determining several cell types within the male branches at the ultrastructural level.
Proohase ■ ■ ! & | Ill ■
An early prophase cell can be distinguished from an interphase cell on the basis of increased chromatin condensation as chromosomes begin to form. Polar rings, or spindle pole bodies (15) 5 are first observed at this time, of which there are eventually two per cell.
However, it is not known definitively how, where or at what exact time polar rings first appear in these vegetative cells.
There are no or very few microtubules associated with the polar ring when it is first seen during early prophase (Figs. 9-11)• Instead, a slight halo, or electron-transparent region, surrounds the organelle Ih
caused by the absence of ribosomes from this area. Superficially, the polar ring appears as a squat, hollow cylinder, composed of electron- dense material (Fig. 10) ; the central axis of the cylinder lies per pendicular to the NE (Fig. ll). Fibrous matter is randomly scattered inside the cylinder. McDonald reported a "groove or notch" around the inside edge of a prophase polar ring, and polar rings of P. denudata appear to contain the same morphology (Figs. 16 , 18). The polar rings as found in ]?, denudata are approximately 167 nm in diameter and ^-7-50 nm in height, and are therefore reasonably similar to those reported by McDonald (.160-190 nm x 70 nm). Because of the small size of the polar ring it is often extremely difficult to observe even one polar ring in a single section. Additionally, planes of nuclear division are unpredictable in the cells of the male branches. For these reasons, proper interpretation of polar ring behavior necessitates tedious serial sectioning through numerous cells.
Contrary to McDonald's finding, it can be asserted that not only are polar rings not associated with microtubules, they also do not
"appear" at the division poles during early prophase. Instead, the organelles have been seen in various non-polar positions implying that either one or both polar rings migrate independently to establish opposing spindle poles (Figs. 11, 13). The polar rings are always found in close association with the nuclear envelope during migration.
"Linkers" or small struts may connect each polar ring to the nuclear envelope (Figs. 12, 16); however, attachment can only be speculated at this tine until well resolved, high magnification micrographs are available. It can be stated that the nuclear envelope regions with which the polar rings are closely associated during migration are 15 specialized in comparison to the rest of the nuclear envelope because of the consistent absence of nuclear pores from the area. Microtubules grow randomly (Fig. 1^+) from the "Microtubule Organizing Centers"
(MT0C)(^3) around the polar rings as the spindle pole bodies become further separated (Fig. 13). The proliferation of microtubules causes a distinct differentiation in the cytoplasm surrounding the polar ring structures, contrasting sharply with the rest of the ground plasm.
Uliis area is tei’med the "zone of exclusion" or "clear area" (32) since it is devoid of ribosomes and instead filled with microtubules.
Because of the insufficient number of microtubules present initially in migration, it appears that microtubules are not directly involved in polar ring motility.
By mid-prophase, migration terminates and the spindle pole bodies are approximately 180° apart (Fig. 15), designating polarity within the cell. At each spindle pole the specialized area of the nuclear envelope associated with the polar ring is stretched out into a plateau-shaped region, termed the "nuclear envelope protrusion" (NEP)
(Figs. 15-19)* The NEP is approximately the same diameter and width as a polar ring. Microtubules are numerous around the NEP and polar ring area, subsequently the zone of exclusion greatly enlarges (Fig.
17). Nuclear pores increase in concentration at the spindle poles
(.Figs. 18, 20) while there is an apparent concomitment decrease in- other regions. The change in pore concentration is thought to aid microtubule monomer (tubulin) entry into the nucleus for eventual spindle fiber formation. However, the NEP remains pore-free (Figs.
18 , 19). 16
By late prophase, a perinuclear endoplasmic reticulum (PER) begins to form along the non-polar sides of the nucleus (Figs. 18-20). The role of the PER is unknown; however, it may provide an area for enclosure and channeling of microtubule protein monomers which are needed for inicrotubule production at this time (32). Finally, the nucleus, which has remained spherical up to this point, tends to elongate in a plane perpendicular to the plane of eventual cell division.
Prometaphase
During this transitory stage, the nucleus becomes indented at both poles (Fig. 21). This flattening can only be seen in sections passing directly through the spindle poles. Considerably condensed chromosomes are located throughout the nucleus and kinetochores are often distinguishable, oriented randomly throughout the nucleus. The nuclear envelope is irregular in outline. Discontinuities, or polar fenestrations, begin to form in the nuclear envelope at the poles
(Fig. 22) and numerous microtubules exist in this area. The fenestra tions serve as a possible site for extranuclear microtubules to enter the nucleus.
The nucleolus, which was present during prophase, disx^erses by this time but remains within the nucleoplasm, becoming loosely associated with the chromosomes.
Metaphase
Metaphase commences as fully condensed chromosomes align on the equatorial plate of the nucleus. Highly visible kinetochores are present, located on the poleward side of each chromosome. The nucleus 17
is now somewhat spindle shaped, being extremely flattened at the poles
and bulging at the equator (Figs. 23-25). Fenestrations in the nuclear envelope at the poles increase in number (Fig. 25) and the plateau
shaped IIEP disappears. Perinuclear endoplasmic reticulum surrounds the entire nucleus, distinctly capping the two polar zones of exclusion.
Unlike prophase these zones are now wide and flattened. Additionally, there is much compartmentalization of the zone of exclusion by the
PER (Fig. 2b). The function of this increase in surface area is unknown.
The spindle apparatus of P. denudata consists of at least two different types of microtubules. Most commonly visible are both continuous (pole to pole) microtubules and chromosomal (chromosome to pole) microtubules. The former tubules extend the length of the nucleus from pole to pole (Fig. 27) whereas the latter anchor a given chromosome to its respective pole. Chromosomal microtubules can be seen directly emminating from the kinetcchores. Interestingly enough, the interface between the kinetochore and chromosomal micro tubules appears to be a splayed, semicircular area (Figs. 26, 31).
Other microtubules which arise from either pole but do not extend the full length of the nucleus may also be present intranucularly but it is difficult to establish from existing evidence. Additionally, some microtubules are present extranucularly between the PER and the nuclear envelope (extranuclear microtubules, Fig. 2b). Although most intranuclear microtubules terminate at the nuclear envelope, some tubules may be seen extending through the fenestrations to the PER region (Figs. 30, 3l). This condition lasts at least into mid anaphase. All intranuclear microtub-*116 3 may function in either 18
chromosome separation or in the movement of the poles away from each
other. 'The role of extranuclear microtubules is unknown.
Particles of dispersed nucleolar material persist inside the
nucleus and contrast with the chromosomal material (Fig. 28). Within
the cytoplasm, vacuoles have begun to coalesce and increase in volume.
Unlike McDonald’s observations, polar rings are found to persist
past prophase (Fig. 2b) in dividing cells. However, the polar ring
undergoes a change in configuration by this time, splitting into two
components, apparently breaking apart in the "groove" region. Subse
quently each polar ring appears to separate into two rings which are
free from one another at this stage of division (Fig. 29). The ring
in closest proximity to the nuclear envelope seems to remain associated
with the nuclear envelope whereas the distal ring is held in place, if
at all, by the cementing material of the MTOC which is scattered
throughout the zone of exclusion. The new polar ring configuration
persists as such at least through anaphase and probably until early
interphase, as observed in ]?. harveyi (Scott & Bosco, in prep.).
Anaphase
Anaphase entails separation and polarization of the duplicated
genomic material. Figure 30 illustrates an early anaphase condition
in which chromosomes begin to separate from each other and move
asynchronously toward their respective poles. Movement is initiated by decreasing the chromosome to pole distance. The nucleus has not
undergone an increase in length at this time, retaining its wide,
flat metaphase configuration.
By mid-anaphase, the pole to pole microtubules have elongated
considerably. As a result, the nucleus adopts a "dumbbell" shape, 19
characterized by a large interzonal spindle located between the sister
chromatids. The elongated isthmus region helps insure successful
separation of the two genomes until cytokinesis can occur. Meanwhile,
chromosome to pole microtubules continue to shorten, isolating the
replicated sets even further. At this time the nuclear envelope
retains its fenestrations at the poles and microtubules continue to
extend through the gaps (Fig. 3i). Dispersed nucleolar material still
remains visible throughout this stage and can be seen in varying
degrees of association with the chromosomes and along the interzonal
spindle.
External to the nucleus, the polar rings persist, unchanged since
metaphase with the proximal half remaining in close association with
the nuclear envelope a.nd the distal half lying unattached (Fig. 32).
The PER continues to surround the nucleus on all sides and the zone of
exclusion remains basically -unchanged since metaphase, enduring in a
wide and flat configuration contained by sheaths of PER. Vacuoles
continue to increase from their metaphase size and are frequently
observed in a central position, adjacent to the interzonal spindle.
By late anaphase, the dumbbell nucleus has stretched almost the
entire length of the cell. With elongation there is a subsequent
narrowing of the interzonal spindle, caused possibly by the lack of
new nuclear envelope formation. Kinetochores are in very close
association with the nuclear envelope and microtubules continue to
serve as the anchoring device for the chromosomes. Figure 33
exemplifies a late anaphase condition. The elongated interzonal
spindle lies slightly below this plane of sectioning, as determined
from examination of adjacent sections, and thus appears absent in this micrograph. The proximal half of the polar ring remains closely associated, with the nuclear envelope during late anaphase (Fig. 3*0. The distal half was not observed, but is believed to persist within the zone of exclusion. Also at this stage, the PER disappears from the polar
regions and there is a concurrent reduction in number of the fenestra tions found at the poles.
Telophase
The final stage of karyckinesis , telophase, includes further separation of the polarized chromatids followed by reformation of the two daughter nuclei with intact nuclear envelopes, each with a single reformed nucleolus,
Early telophase can be distinguished by the presence of a dehiscing interzonal spindle. Three distinct products from karyo- kinesis are recognizable at this point. There are two reforming daughter nuclei and one interzonal spindle which appears to break down very rapidly.
Reforming telophase nuclei lie at the extreme ends of the cytoplas
(Fig. 35). Kinetochcres are still visible adjacent to the nuclear evelope and nucleolar material is evident, lying loosely within the nucleus. The PER is still present around the non-polar sides of the nucleus, most heavily concentrated in the areas where nuclear envelope reformation will occur.
With the disappearance of the spindle apparatus there is a simultaneous appearance of coalesced vacuoles in the interzonal region.
These vacuoles possibly function in providing a barrier that separates the newly formed daughter nuclei. Figure 36 most likely represents a 21
mid-telophase stage. Kinetochores are slightly discernable and
reformation of the nuclear envelope has commenced. New nuclear
envelope growth appears to be synthesized de_ novo, and reformation
occurs in close proximity to the dehiscing interzonal spindle region.
By late nelophase, reformation of the nuclear envelope is complete
along with the reappearance of an intact nucleolus (Fig. 37). Micro
tubules are noticeably absent from inside the nuclei and are only
found extranucularly once karykinesis is completed (Fig. 38). Kineto
chores are no longer distinguishable and the zone of exclusion has
disappeared. The PER is no longer adjacent to the nucleus; however,
the area in between the separated nuclei typically contains layers of
endoplasmic reticulum. These stacks of cisternae will remain through
out cytokinesis, possibly assisting in pit connection formation.
Polar rings were not observed during telophase, however, this
does not mean they do not exist at this stage. Because polar rings
are present in interphase spermatangia, the end product of male repro
ductive differentiation (Fig. UU), and because polar rings have been
found in telophase and early interphase nuclei of another species
of Polysiphonia (Scott & Bosco, in prep.), the possibility exists
that polar rings persist through all stages of cell division and are
passed along to each different cell type through the process of cell
division.
Cytokinesis
The division of the cytoplasm into two separate masses can be
initiated as early as prophase (Fig. 20). However, most of the cyto- kinetic process occurs after the two daughter nuclei are reformed and telophase is completed. Cytokinesis begins as small cleavage furrows invaginate the cell surface in the equatorial region. These indentations progress in a centripetal manner, forming an annular ingrowth of the wall (8). In cells dividing anticlinally, the greatest degree of impingement is always seen on the side closest to the periphery of the male branch
(Fig. 39). Periclinal divisions (Fig. bl) characteristically proceed more evenly; the cleavage septa proceed inward at the same rate.
During this time, vacuoles in the mid-region of the dividing cell coalesce (Fig. 37) to gradually form a single, large vacuole (Fig. ^0)
Inward-growing cleavage septa ultimately unite with the central vacuoles.
No microtubules, microfilaments or dictyosomes appear in the area of the forming cleavage furrow (Fig. k 2 ). However, stacks of EE are typically concentrated near the growing septa (Fig. 37). As cytokines progresses, small septal plugs (pit connections) are ultimately formed (Fig. k 3 ). A permanent barrier results once these structures are completed, cutting off all cytoplasmic associations between the two cells. Pit connection formation has been described in the marine alga Pseudogloiophloea (^-9) and the process in Polysiphonia denudata appears to follow very closely to that account. DISCUSSION
Several hundred vegetative male branch cells of Polysiphonia denudata were examined throughout the course of this investigation.
Over half of the cells examined contained prophase nuclei. Roughly
20 individual cells were observed at metaphase, 8 at anaphase and 8 at telophase. Late stages of karyokinesis appear to proceed extremely rapidly, and often the nuclei in these stages contrast less well against the ground plasm, making detection more difficult.
The successful location of abundant mitotic figures in this study is attributable to both the method employed in obtaining material and to the unique alga utilized for examination. Laboratory acclimated plants were found to fix comparably to plants collected in_ situ, but were also manipulatable and minimized adverse collection conditions.
Collecting plants from nature but maintaining them for four days prior to fixation in the laboratory unexpectedly provided healthy, actively growing plants which were accessible at all times and preserved well.
Similarly, finding cells in active stages of mitosis 2 hours after the incubator lights came on of the fourth day was a fortuitous, but repeatable, discovery. Additionally, male plants of P. denudata consti tute an excellent tool for studying cell division at the electron microscope level because the male reproductive branches grow rapidly and contain numerous, small cells. An exceptional section examined with the electron microscope may contain 3 immature reproductive
23 2k
branches, each with 20 or more cells vhich are potentially mitotically active. Such large numbers of cells visible at one time logically increases the probability of viewing dividing cells.
General cytological features of the vegetative male branch cells of Polysiphonia denudata support the observations of recent red algal ultrastructural literature (12). Although Simon-Bichard-Breaud (55,
56) reported the presence of flagella in spermatia of Bonnemaisonia hamifera, the poor quality of her fixation caused difficulty in inter preting her figures and they are widely discounted.
Mitosis in P. denudata concurs with the three previous accounts of cell division in the Rhodophyta, only one of which was reasonably comprehensive, and also reveals additional information. From this study, polar rings were found not to be transient structures. Polar rings were first observed in prophase, and contrary to McDonald’s findings, these organelles were not a first polarly associated, nor were microtubules associated with the organelles at this early SLage.
Seemingly one or both polar rings migrate from their initial position to establish opposing spindle poles. As the polar rings become further separated, microtubules begin to concentrate near the organelles.
Because microtubules are not present both initially near the spindle pole bodies and during the early stages of migration, it appears that microtubules have no dominate role in polar ring movement. Instead, polar ring migration appears to be accomplished by movement of the specialized areas of the nuclear envelope to which the polar ring is associated. As these areas move, the polar ring(s) are pulled along with them. Similar phenomena have been observed in certain fungi as the spindle pole bodies migrate to establish the division poles (15). 25
Once the spindle poles are established in P. denudata prophase nuclei, a previously unreported plateau shaped configuration, the NEP,
develops in the specialized area of the NE. The NEP is believed to be the product of opposing forces at work on the region. At this time the nucleus tends to indent and flatten at the poles , pulling the NE inward. Meanwhile the polar ring, which is closely associated and possibly attached to the specialized membrane area, offers some resis tance to the inward pulling. The "drag" on the NE results in a plateau shaped region precisely the same diameter as the polar ring. It is theorized that eventually, tension on the polar ring becomes so great that the polar ring splits in half, yielding in the area of least resistance, McDonald’s "groove" region. This implies that the half rings are bound less tightly to each other than either the bond connecting the proximal half of the polar ring to the NE or the fastening between the distal half of the polar ring and the MTOC material in which are anchored numerous microtubules. Subsequently, the polar ring appears as two separate rings from prometaphase on with only one ring in close proximity to the NE at each pole. It is proposed, therefore, that during early prophase the polar ring is actually a compact, double-ringed structure, held in close association either by small struts similar to those thought to connect the polar ring to the NE or by some cementing substance. The "groove or notch" that McDonald reported around the inside edge of a prophase polar ring cylinder most likely represents an area of contact between the 2 half rings .
Unlike McDonald’s observations, the polar ring was found to persist past prophase, distinctly enduring as two half-rings at least 26
through anaphase. McDonald additionally erroneously reported the production of daughter nuclei by constriction of the elongated telo phase nucleus, resulting in two end products, the 2 daughter nuclei.
Examination of serial sections in this study revealed daughter nuclei reformation to occur instead by de_ novo synthesis of NE material in close association to the deteriorating interzonal spindle, resulting in 3 end products: 2 daughter nuclei, plus the interzonal spindle.
Although Polysiphonia and Membranoptera belong to the same order
(Ceramiales}, some discrepancies were found upon comparing the two accounts of cell division. Unfortunately, the only two other accounts of mitosis in the red algae to which this study can be compared are not complete. Notably, Peyriere's pioneer work in Griffithsia flosculosa (Ul) located the polar ring for the first time at the ultrastruetural level in a red alga and documented a metaphase nucleus; however, other division stages were entirely lacking. Six years later, Bronchart and Demoulin (T) published a highly incomplete study of mitosis in the Bangiophyceae. Only brief descriptions of prophase and metaphase in Porphyridium purpureum were presented, yet the work was titled an "unusual mitosis." The authors admit to examining few cells (only one metaphase cell was oriented so that both spindle poles could be examined) yet they confidently state that
Porphyridium lacks polar rings, kinetochores, well condensed chromo somes and a well developed PER on the basis of their evidence. From this they conclude that nuclear division is quite distinct from that of Membranoptera■
The importance of serial sectioning in studies such as these cannot be emphasized enough. The discrepancies between McDonald’s 27
work and this one appear to be caused both by examining red algal tissue having a relatively limited potential for active growth and by foregoing a detailed investigation utilizing serial sectioning. What
McDonald interpreted as constriction of the interzonal spindle during anaphase was actually a figure similar to Figure 33. Sectioning further into the tissue, he would have found a completely connected interzonal spindle. Failure to observe early prophase as well as post prophase polar rings must also be attributed to the lack of serial sectioning. This technique is imperative in studies such as these,
o for out of a possible sixteen 800 A-thick, longitudinal sections passing through the central axis of a metaphase nucleus, only three of these sections will contain the polar ring. Similarly, Bronchart and
Demoulin failed to provide convincing evidence of an unusual mitosis because of the low number of cells inspected which lacked serial examination. Kinetochores, polar rings and condensed chromatin may not always be readily apparent in any single section. Likewise,
Peyriere could have added additional insights into the persistence of the polar ring by serial sectioning; however, she was disadvantaged by undertaking the original work in this area.
Cell division in P. denudata is both similar and different to the classical mitosis found in higher plants and animals. Spindle development and composition as well as chromosome movement appears much the same in these organisms. However, unlike vascular plants and higher animals, separation of the genetic material occurs inside a basically intact nucleus. Polar gaps at the spindle poles constitute the only discontinuities in the nuclear envelope throughout division.
Other differences include the presence of unique spindle pole bodies 28
external to the dividing nucleus, PER, dumbbell shaped nuclei during anaphase, and the formation of cytoplasmic plugs at the completion of cytokinesis.
The lack of ultrastructural information on cell division within the red algae makes comparisons to more primitive organisms a bit tenuous and premature. Unquestionably, if Simon-Bichard-Breaud*s work proved correct (the possession of flagella by red algae), serious phylogenetic implications would ensue. Most proposed phylo genetic schemes rely heavily on the lack of flagellation to establish red algal relatedness to other organisms, however, there is tremendous controversy over what this trait implies. Most biologists consider the Rhodophyta to be the most primitive eukaryotic group of organisms and have linked them to the blue-green algae because of the l) absence of flagella, cilia and centricles , 2) similarity of their pigments to those of blue-green algae, and 3) primitive chlorcplast structure where thylakoids are not associated in stacks or grana as in other algae and higher plants. In all probability, this group separated from a primordial cell line that later evolved the centriole/flagellar system passed onto most other groups of living organisms (^T)> as evidenced by the fact that the condition of the nuclear envelope in some green algae (in 32) is similar to the situation found in the
Rhodophyta, while the polar ring shows no homology to existing centrioles as found in the Chlorophyta. However, another theory (see
3^+ for review) has proposed that lack of flagella in the Rhodophyta is actually an indication of evolutionary advance (a trait that has been gained and then lost) since the red algae already possess a well developed spindle which is believed to have come from a sexual flagellate ancestor (heterokont). 29
Such controversies over phylogenetic associations stem from individual preferences as to which criteria taxonomy should he based upon. Biologists have generally utilized cytological and histochemical characteristics in establishing natural classes and orders. Because the mitotic apparatus is a structural feature that would be expected to retain many conservative properties throughout evolution (UU) the use of cell division for phylogenetic interpretations can no longer be dismissed.
Interestingly enough, many similarities have been found between the red algae and some forms of Ascomycete and Basidiomycete fungi.
Lack of flagellated cells , presence of noncentriole-like spindle pole bodies outside a basically intact nuclear envelope during mitosis, occurrence of a uninuclear meiosis (.10, 23, 5*+) , similarity between floridean starch and glycogen as a storage product, and parallelism in reproductive morphology and complex life histories tend to suggest a common thread. Additionally, pit connection formation during cyto kinesis within the Rhodophyta, such as P. denudata, is unique. Fungi, however, also utilize somewhat similar structures, or septa, which are formed between adjacent cells by centripetal invaginations (15).
Nonetheless, the phylogenetic association between red algae and fungi must remain speculative until further studies are performed in other genera of the red algae.
Although the systematics and interrelationships of the red algae may not be clear at this time, further investigations into the micro anatomy of the red algae and various other lower eukaryotes, coupled with biochemical and physiological studies, should prove useful in providing a more accurate phylogenetic scheme. Additional work in the 30
Rhodophyta needs to determine where, how and when the polar ring originates in the apical cell, the first cell to initiate the male reproductive branch. Other studies must trace the development, if any, of the polar ring through the vegetative cellular divisions to the mother cells , terminating in the spermatangia. Work in progress
(Scott & Bosco, in prep.) tends to indicate that the polar ring is present at interphase in apical, central, pericentral and sperma- tangial cells, implying that the polar ring is a persistent organelle through all stages of division. Examination of telophase cells in another species of Polysiphonia (Scott & Bosco, in prep.) reveals adjacent attachment of both of the two separated, half-ring structures to the NE in early interphase nuclei. Therefore, does each half-ring structure synthesize new polar ring material prior to the next pro phase? Do the rings rejoin into a single, compact polar ring structure and replicate a completely new polar ring prior to the next prcphase?
Could one or both polar ring(s) appear de novo?
Our understanding of polar ring migration patterns likewise remains unresolved. Recent findings (Scott & Bosco, in prep.) in vegetative male branch cells suggest that the two polar rings may, in some cases, rotate by movement of the nucleus to establish a single spindle pole from which a single polar ring migrates to establish the opposing pole. In other cases, the two polar rings are already situated at one of the polar regions, thus the nucleus with attached polar rings need not rotate into position prior to the migration of a single polar ring. These migration/rotation patterns, if actually functioning as theorized, could logically account for the distinctive cell symmetry known to exist in red algae, resulting from divisions 31
occurring alternatively to the right and then the left during peri
central cell formation. Preliminary work in apical and spermatangial
cells tends to suggest a different pattern of migration. In these cells,
the polar rings are initially found in the eventual metaphase plate
region. Each polar ring then migrates 90° to establish opposing
poles. However, additional work is needed in each cell type to con
firm such observations.
Furthermore , the mechanism which provides separation of the two
polar ring halves during prometaphase must be elucidated. The presence
of linkers needs to be established conclusively by studying high-
rnagnification micrographs at increased resolution. Possibly a different
type of fixation schedule or the use of electron microscopes with
better resolving power would provide the increased necessary resolution.
Finally, because P. denudata is a dioecious, haplodiplontic plant,
other studies need to determine the similarity of mitosis within
vegetative and reproductive cells throughout the life history of the
organism. According to Scott & Bullock (51)» cell division must be
examined in all vegetative and reproductive entities of a given species before a particular observed mitotic sequence can be considered
characteristic of a given organism. Only then can the ultrastructure
of cell division be used successfully as a comparative tool for
understanding evolutionary relationships among organisms.
It is hoped that this study concerning the fine structure of vegetative cell division in male reproductive branches of the marine
red alga Polysiphonia denudata can benefit those toying with and untangling the phylogenetic string. Key To Abbreviations
ac axical cell ape apical cell be basal cell BrW branch vail c chloroplast Ch chromosome cmb concentric membrane bodies cr lip crystals er endoplasmic reticulum F fenestrations fs floridian starch G Golgi apparatus IS interzonal spindle K kinetochores m mitochondria mtoc microtubule organizing center N nucleus NEP nuclear envelop protrusion No nucleolus nom nucleolar material NP nuclear pore pc pericentral cell PER perinuclear endoplasmic reticulum PR polar ring SMC mother cell sp septal plug tb trichoblast branch V vacuole * zone of exclusion
32 Figure 1: Photograph of a herbarium specimen of Polysiohonia denudata (male plant).
Figure 2: Light micrograph of several specialized male reproductive branches. Arrow points to released spermatia. (X 33^)
Figure 3: Light micrograph of a single male reproductive branch. (X 892) CtNTIM!ILRS Figure *+: Montage of a longitudinal section through a young male reproductive branch illustrating the various types and positions of the cells it contains. Arrows point to septal plugs formed between adjacent cells. (x 83*0
Figure 5: High magnification of the basal cell of the male reproduc tive branch shown in Figure *+. The cytoplasm of a basal cell is always electron transparent in comparison to other reproductive branch cells. (X 18,56*0
THE FOLLOWING FIGURES ARE ELECTRON MICROGRAPHS OF LONGITUDINAL SECTIONS THROUGH EITHER PERICENTRAL CELLS OR INTERVENING CELLS IN THE FORMATION OF SPERMATANGIAL MOTHER CELLS, EXCEPT WHERE NOTED.
Figure 6 Interphase cell. Note the large, spherical nucleus and intact nucleolus. The Golgi apparatus and mitochondria are often found in close association. (X 21,500)
Figure 7 High magnification of a single chloroplast. Thylakoids are net numerous. Arrows point to phycobilosomes located along the thylakoids. (x 2^,000)
Figure 8 Interphase nucleus in a central cell. Lip crystals and floridean starch are present. (X 11,900)
Figure 9 ’ Cross-section through an early prophase cell. The polar ring is in close association with the nuclear envelope. Few or no microtubules are present adjacent to the polar ring at this time. (X 31,500)
Figure 10: High magnification of the polar ring in Figure 9» The large arrow points to the halo surrounding the polar ring formed hy the absence of ribosomes. The small arrow shows fibrous matter scattered inside the polar ring cylinder. (X 86,000) m
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slip © „ 3 . r 4 _.. •* '■ V***? * ' t*i' Vi ' *»?■ :ojr; | # 1 ,• < :© V j * 5 4 . V’ ** ‘ «ss ■” : »{ , 1 ' Hr ■;.
,. ■ -4 - N ■'■\i > ; • v X :f k i'"-.v" r’ v XS ^ f?'f ■ '** * . f ’ y 1 *il» SfcaT^-V ^Si"* * ,-tL* ■Wfc: •« . / Sr Figure 11: Early prophase condition. The polarity of the cell is represented by the arrow. One polar ring is seen in a non-polar position, indicating migration of the organelle. Concentric membrane, bodies are present in the cytoplasm. (X 30,000)
Figure 12: High magnification of the polar ring shown in Figure 11. Faint struts (arrow) may connect the polar ring to the nuclear envelope, A few microtubules are visible near the polar ring at this stage. (X 109*000)
Figure 13: Early prophase. Asterisks indicate the tvo zones of exclusion. Note one polar ring is in a non-polar position and probably migrating to establish opposing spindle poles. (X 16,530)
Figure ih: Cross-section through a prophase nucleus, shoving polar ring. Microtubules are concentrated near the microtubule organizing center material surrounding the polar ring. (X 23,000) .m toe Figure 15: Mid-prophase. A polar ring is located at each spindle pole. At this time, the specialized areas associated with the polar rings along the nuclear envelope stretch out into plateau shaped regions called the nuclear envelope protrusion. The NEP's are formed by inward pulling forces of the nucleus. Nuclear pores are con- sistantly absent from the NEP regions. (X 31,000) ii-i.A* :*M Figure 16: High magnification of a polar ring shoving possible struts connecting polar ring to the nuclear envelope (large arrows) Note the lack of nuclear pores in the nuclear envelope protrusion. The polar ring appears to consist of two separate rings, separated by a thin, electron transparent region (McDonald’s ’’groove or notch”). (X 157,950)
Figure 17: Note the large zone of exclusion that surrounds the polar ring during mid-prophase. Nuclear pores are absent in the nuclear envelope protrusion, but concentrated else where. (x 61,500) ’. > . V ^ **■ PR V ’ ,
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• 'r- . * s W * ■' %A • *. sf-i ■ % > • .;" ’. ® 3? r ■ ■" i» .V*5 :• J1 Figure 18: Late prophase nucleus. Large arrow points to the "groov or notch" in the polar ring. Nuclear pores are concen trated at the poles, but not in the nuclear envelope protrusion, at this time. A perinuclear endoplasmic reticulum has begun to form along the non-polar sides of the nucleus. (X 3T»000)
Figure 19: Late prophase in a mother cell. (X 19,800)
Figure 20: Oblique section through a late prophase cell. Arrows point to the centripetally growing cleavage furrows which have commenced at this time. (X 23 5800) ft Figures 21 and 22: Prometaphase condition in a mother cell. The nucleus is "becoming spindle-shaped, and flattened at both poles. Polar fenestrations begin to form at the spindle poles. (X l6,6UO and X 22,080 res pectively) 'I: Figure 23: Metaphase cell, illustrating the spindle shaped nucleus. Perinuclear endoplasmic reticulum encloses both the nucleus and the zone of exclusion at this time. Kineto- chores are highly distinguishable inside the nucleus, located on the poleward side of each chromosome. The nucleolus has dispersed within the nucleus. (X 1398oU)
Figure 2h: Metaphase nucleus. The polar ring is visible as a single ring structure at one of the poles. Note the intranuclear microtubules and extranuclear microtubules (arrow). (X 36,000)
Figure 25: Metaphase nucleus, showing numerous polar fenestrations in the nuclear envelope and a well developed perinuclear endoplasmic reticulum. (X t0,000)
Figure 26: High magnification of one of the spindle poles of a metaphase nucleus. The zone of exclusion is highly com partmentalized. Within the nucleus, several microtubules appear to emminate directly from the kinetochore {chromo somal microtubules). The interface between the kineto chore and microtubule appears to be a splayed, semicircular area. (X Ul,600)
Figure 27 : Oblique section through a metaphase nucleus showing continuous microtubules. (X 36,000)
Figure 28 : Metaphase nucleus. Note dispersed nucleolar material evident within the nucleus. Vacuoles have begun to coalesce and increase in size. (X 3^+}000)
Figure 29: Metaphase nucleus. Note that the polar ring at one of the spindle poles has separated into two components by this stage, hereafter referred to as the proximal and distal half of a polar ring.
Figure 30: Early anaphase nucleus. Chromosomes have begun to pull away from the equatorial plate. Note that the nucleus has not yet undergone an increase in length, retaining a spindle shaped configuration. Microtubules can be seen extending through the nuclear envelope (large arrow). (X 2^,000)
Figure 31: "Dumbell” shaped anaphase nucleus, showing large inter zonal spindle between the sister chromatids. Microtubules continue to extend through the polar fenestrations. Large arrows point to the splayed interface between chromosomal microtubules and kinetochores. Nucleolar material remains dispersed within the nucleus. (X i+2,500)
Figure 32 : Anaphase nucleus. The polar ring remains unchanged since metaphase, with the proximal half remaining in close association to the nuclear envelope and the distal half lying unattached. Vacuoles are frequently seen in a central position, adjacent to the interzonal spindle. (X 30,000)
Figure : Late anaphase nucleus. Kinetochores are in close associ ation with the nuclear envelope, and the dividing nucleus has stretched almost the entire length of the cell. Peri nuclear endoplasmic reticulum has disappeared from the polar regions. (X ^,393)
Figure 3^ : High magnification of the polar ring in Figure 33. The proximal half of the polar ring continues to remain in close association with the nuclear envelope. 'There are few or no fenestrations in the nuclear envelope at the poles by this time. (X 88,000)
Figure 35• Telophase condition. Reforming telophase nuclei lie at the extreme ends cf the cell. Kinetochores are. visible adjacent to the nuclear envelope. The perinuclear endo plasmic reticulum is most heavily concentrated in the areas vhere nuclear envelope reformation will occur. Vacuoles are concentrating between the reforming nuclei. (X 25,200)
Figure 36 Mid-telophase. Reformation of the nuclear envelope has commenced (arrows). Kinetochores are still discernable. (X 25 ,200) |
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rv & $ ! & Figure 37: Late telophase. The nuclear envelope has reformed around each daughter nucleus. Kinetochores and intranuclear microtubules are no longer distinguishable within the nucleus. Coalescing vacuoles are present between the daughter nuclei, and endoplasmic reticulum is concentrated in the mid-region as well. Arrows mark the cleavage furrows. (X 19,2^0)
Figure 33; Late telophase. An intact nucleolus is visible at this stage. Arrows mark cleavage furrows. (X 15,000)
Figure 39• Cytokinesis. In anticlinal divisions, the greatest degree of impingement by the cleaveage furrows is always seen on the side closest to the periphery of the male reproduc tive branch wall. (X 12,900)
Figure U0: Cytokinesis. Note that a small vacuole in the mid-region of the cell has coalesced (arrow) with the central vacuole. Inward growing cleavage septa (large arrows) ultimately unite with the central vacuoles. (X 17,700) BrW
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' . , ' - ’ .- $ * ' . i it: i " ) ■’ • Figure kl : Periclinal cytokinesis. In this case, the cleavage furrows (arrows) proceed at an even rate find parellel to the branch wall. (X 15,232)
Figure b2 : High magnification of the mid-region of the cell in Figure 40, illustrating the lack of microtubules, microfilaments or Golgi apparatus in the vicinity of the forming cleavage furrows (arrows). (X 20,700) fvtnx Figure 43: Septal plug, or pit connection, formation marks the e of cytokinesis. A permanent barrier results once the structures are complete. (X 10,31^-) •v ' Figure Interphase in a spermatangium. Spermatangia constitute the outermost layer of cells in a mature male reproductive branch. Note the presence of a polar ring in close assoc! ation with the nuclear envelope. (X U2,000) B r W
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*'~L ■ r i-4 V I JL- : Schematic diagram of cell division in Polysiphonia denudata. 1) Interphase - early prophase condition. 2) Early prophase. 3) Late prophase. k) Metaphase nucleus. 5) Anaphase nucleus. 6 ) Telophase, with cytokinesis occurring. per BIBLIOGRAPHY
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Cynthia Louise Bosco
Born in Arlington, Virginia, k September 1953. Graduated from
McLean High School in McLean, Virginia, June 1971. B.S. in Biology
from the College of William and Mary, 1975. Candidate for M.A. at the
College of William and Mary 1975-1973.
63