ultrastructural changes in major organelles during spermatial differentiation inbangia (rhodophyta)

Protoplasma 102, 253--279 (1980) PSOTOPLASMA �9 by Springer-Verlag 1980

Ultrastructural Changes in Major Organelles During Spermatial Differentiation in Bangia (Rhodophyta)

KATHLE~N COLE * and R. G. SHEATH 1

Department of Botany, University of British Columbia, Vancouver

Received August 8, 1979 Accepted November 30, 1979

Summary The major organelles within the cells of male Bangia atropurpurea (Roth) C. Ag. filaments undergo a series of ultrastructural transformations during the production of spermatia. Initially, thylakoids within the large axial chloroplast develop a reticulate pattern commencing at the central pyrenoid region. Subsequent changes involve loss of lobes and diminution of volume through division; chloroplasts in final stages contain a few dilated, distorted thylakoids and many plastoglobuli. During differentiation the large nucleolus disappears from the nucleus and four masses of chromatin aggregate near the nuclear envelope. Furrows originating from the nuclear envelope form double membranes around each of the chromatin masses and most of the nucleoplasm is eliminated. Several types of fibrillar vesicles are formed during the process and large floridean starch reserves are utilized. Multilamellar bodies and microbody-like structures occur within the cells during certain phases of spermatiogenesis.

Keywords: Bangia; Differentiation; Organelles; Rhodophyta; Spermatiogenesis; Uitrastruc- rural transformation.

1. I n t r o d u c t i o n

The small , n o n - m o t i l e ma le gametes , spe rma t i a , p r o v i d e a d i s t inc t ive f ea tu re

of the red algae. The re are a n u m b e r o f pub l i shed repor t s on the u l t r a s t ruc tu r e o f spermat iogenes i s in the Rhodophyta (PEYRI~RE 1971, 1974, SIMON-

BICHARD-BI~AUD 1971, 1972 a, b, KUGRENS a n d WEST 1972, SCOTT and

DIXON 1973, KOCR~NS 1974, P~EL a n d DUCKETT 1975, YOUNG 1977, HAWKES

I978) . H o w e v e r , mos t o f these dea l m a i n l y w i t h deta i l s r e l a t ed to the fo r - m a t i o n of spe rma t i a l vesicles and the cond i t i on of the re leased spe rma t i a

*Correspondence and Reprints; Department of Botany, University of British Columbia, Vancouver, B. C., Canada, V6T 1W5.

1 Present address: Department of Botany, University of Rhode Island, Kingston, RI 02881, U.S.A.

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0033-183X/80 /0102 /0253 /$ 05.40

254 KATHLEEN COLe and R. G. SHEATH

rather than the complete process. With the exception of HAWKES' (1978) paper on Porphyra gardneri, a member of the Bangiophyceae, these published data have been restricted to species within the Florideophyceae. There are only two other known accounts of spermatiogenesis in the Bangiophyceae, both unpublished (McBRIDE 1972, MCDONALD 1972 a). In Porphyra and Bangia, two genera in the Bangiophyceae, packets of 32-128 spermatia are produced from vegetative cells of the gametophytic thallus by a series of cell divisions (CoLe 1972, CoNwav et al. 1975). Male filaments which occur in dioecious populations of the marine form of Bang& atropurpurea are particularly suitable for a complete cytological study of spermatia production. The various stages of the process can be followed in sequence distally along the filament from the deeply-pigmented more proximal vegetative cells through an area of lighter pigmentation to the colourless tip where mature spermatia are released. A detailed ultrastructural study of spermatiogenesis in Bang& atropurpurea was conducted using material collected each year for four years. It was observed that major organelles within the ceils undergo a sequence of transformations during this process, some of which had not been recorded previously for the Rhodophyta. Details of these changes are discussed in this paper.

2. Mate r i a l s and Methods

Diacious Bang& atropurpurea (Roth) C. Ag. was collected from rocks in the upper part of the littoral zone near Point No Point, Vancouver Island, B.C., Canada (48~ 123~ The marine form of the taxon previously had been named Bang& fuscopurpurea (Dillw.) Lyngb. However, from the results of adaptation experiments subjecting the algae to different degrees of salinity, GEESlNK (1973) concluded that the fresh water and marine forms of

Figs. 1, 2, 22, 23, 34, light micrographs; Figs. 3-21, 24-33, 35-40, electron micrographs

Fig. 1. Three easily distinguishable types of filaments occurring in populations of the marine form of Bangia atropurpurea; female (iv), male (M), and asexual (A). •

Fig. 2. Maturing tip of a male filament showing the gradual loss of pigmentation during raaturation of the spermatia. X 30

Figs. 3-5. Vegetative ceils of the male filament Fig. 3. Typical vegetative ceil showing the large lobed chloroplast with central pyrenoid (P) and a number of plastoglobuli; large nucleus (N), adjacent to the chloroplast, containing heterochromatic bodies; a number of floridean starch granules (S); several mitochondria (M); thick ceil wall (W). • Fig. 4. Interphase nucleus (N) with a prominent nucleolus (NI) and several chromatin bodies. The nuclear envelope has a number of pores (double arrows). Note the mitochondrion (M) containing tubular cristae adjacent to the nucleus, as well as endoplasmic reticulum (arrow) near the plasma membrane. X 10,000

Fig. 5. Portions of three adjoining cells showing the cell wall (W) and endoplasmic reticulum (arrows) in the cytoplasm near the plasma membrane. C chloroplast. • 11,000

Organelle Transformations During Bangia Spermatiogenesis 255

Figs. 1-5

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256 KATHL~rN COL~ and R. G. SIqEATH

Bangia are conspecific, the name of the fresh water form [Bangia atropurpurea (Roth) C. Ag.] having priority. However, it is interesting that sexual filaments are restricted to the marine fornl. Male filaments of Bangia were selected from the collected material prior to or immediately following fixation. These were easily distinguished from the female filaments because they are shorter and thinner, and possess long, colourless or yellowish tips (Figs. 1 and 2). Fila- ments were fixed for electrorl microscopy in the field with 5~ glutaraldehyde in phosphate buffer at pI-I 7.2, postfixed in 1% osmium tetroxide, dehydrated with a graded ethanol series and embedded in Spurr's epoxy resin. Fixation was carried out at 4 ~ Thin sections were stained with uranyl acetate (DAwEs 1971) and iead citrate (R~xNoI.Ds 1963) and examined in a Zeiss EM-10 electron microscope. For light microscopy, male filaments were fixed in 3 : 1 ethanol-acetic acid solution and stained for &romosome studies using WITTMMqN'S (1965) aceto-iron-haematoxylin-chloral hydrate technique.

3. Results

Commencing with a vegetative cell of the male filament of Bangia, the production of spermatia may be described in three sequential stages, each of which has distinctive ultrastructural features. Initially, the vegetative cell divides to form a packet of 16 ceils which will be defined as the spermatangia. This is followed by a period when major differentiation occurs within the cells and two more divisions per cell occur. As a result, each spermatangium produces four spermatia. In the final stage, the walls around the spermatia dissolve, the outer wall around the packet breaks and approximately 64 mature spermatia are released (CoL~ 1972).

3.1. The Vegetative Cell The vegetative cell has an average diameter of 12.5 ~tm and possesses typical ultrastructurat features of the Bangiaceae (Fig. 3). The large stellate chloroplast with a central pyrenoid averages 10 ~m in diameter and occupies most of the cell. It contains single, parallel thylakoids which are evenly distributed;

Figs. 6-10. Formation of cell packets

Fig. 6. A packet of cells formed from the vegetative cell during the division phase. Note the association of the nucleus (N) and chloroplast (C). The cytoplasm is filled with starch reserves (S) and vesicles. • 5,000

Fig. 7. One cell in a packet showing the reduced size of the chloroplast with its large pyrenoid (P). The envelope of the chloroplast is beginning to separate from that of the nucleus (N), and starch granules and vesicles are located between them at one side. X 10,000

Fig. 8. Chloroplast (C) containing extensive reticulation of the thylakoids, commencing in the pyrenoid (P). The adjacent nucleus (N) contains several heterochromatic bodies. Floridean starch granules (S) are still evident. Many vesicles have fused with the plasma membrane causing a highly irregular outiine of the protoplast. • 10,000

Figs. 9 and 10. Two microbody-like structures (arrows) in the cytoplasm just outside the nucleus (N). In Fig. 9 they are situated within an invagination of the nuclear envelope. Fig. 9, )<30,000; Fig. 10, •


Figs. 6-10

258 KATHLEEN COL~ and R. G. SHEATH

some thytakoids penetrate the pyrenoid and there is no peripheral thylakoid. A few plastoglobuli are scattered through the stroma. The large, slightly-lobed interphase nucleus, 5-6 ~m in diameter, is situated adjacent to the chloroplast; the outer membranes of these two organelles are very closely associated (Fig. 3). There is a large number of evenly-spaced pores in the nuclear envelope (Fig. 4). The nucleoplasm is finely-granular and contains a number of small heterochromatic bodies and one to three nucleoli. A few small dictyosomes are scattered through the cytoplasm; mitochondria with characteristic tubular cristae are often situated close to the dictyosomes. A small number of fibrous filled vesicles are produced from the dictyosomes and endoplasmic reticulum is located near the plasma membrane (Fig. 5). Some floridean starch granules and a few multilamellar bodies are distributed through the cytoplasm.

3.2. Formation of Cell Packets

During the formation of packets of cells by division of the vegetative cell, the cells are reduced in size by partitioning to approximately 6-8 ~m in diameter, the chloroplast and nucleus become proportionately smaller, and many vesicles and floridean starch granules are produced, filling much of the cytoplasm (Fig. 6). Initially, the thylakoid distribution within the chloroplast becomes disrupted and the membranes develop a reticulate pattern, commencing in the pyrenoid (Fig. 8). The reticulation is less obvious toward the end of this division phase when the chloroplast has been reduced to approximately half the volume of the cell and has fewer prominent lobes. At this stage the pyrenoid is no longer present. The nucleus becomes more lobed remaining close to the chloroplast (Fig. 7). The inner membrane of the nuclear envelope forms short invaginations and the outer one appears to form short extensions resembling the endoplasmic reticulum. Microbody-like structures (usually in pairs) are often located in the cytoplasm near the nuclear envelope (Figs. 9 and 10). They are approximately 170 nm in diameter and have a

Figs. 11 and/2. Formation of cell packets (continued)

Fig. 11. A dictyosome (D) in the peripheral cytoplasm forming fibrillar filled vesicles which appear to be fusing and moving to the plasma membrane. Fibrillar material (large arrow) is present outside the plasma membrane. A large mitochondrion (M) is situated near the forming face of the dictyosome. Note the endoplasmic reticulum membrane system parallel to the plasma membrane (small arrow) and also the presence of large starch granules (S) and fibrous vesicles within the cytoplasm. X24,000 Fig. 12. Large vesicles containing amorphous material fusing with the plasma membrane (single arrow). Note the dilation of the rough endoplasmic reticulum (double arrows) which is distributed throughout the cytoplasm. The chloroplast (C) has reticulate thylakoids and the cytoplasm is rich in ribosomes. • 12,000


Figs. 11 and 12

260 KATHLEEN COLE and R. G. SHEAT~t

homogeneous, dense granular matrix. Toward the end of the division period, the outer membrane of the chloroplast becomes separated from that of the nucleus and starch grains, vesicles and other cytoplasmic inclusions are located between them. A number of dictyosomes are present in the cytoplasm, often with the forming face adjacent to mitochondria (Fig. 11). The dictyosomes produce small fibrous filled vesicles which fuse with each other and then move to the plasma membrane (Fig. 11). The plasma membrane develops an irregular outline as many small and large vesicles fuse with it. Endoplasmic reticulum is abundant in the cytoplasm, particularly near the plasma membrane (Figs. 11 and 12). It is often oriented at right angles to the plasma membrane during the initial part of this stage. Dilation of the rough endoplasmic reticulum occurs and granular material is present within the cisternae (Fig. 12). Several parallel layers of rough endoplasmic reticulum are present in areas near the plasma membrane where cytokinesis originates (Figs. 13 and 14). The plasma membrane invaginates and a double-membraned septum forms (Fig. 15). Cell wall material is deposited centripetally as furrowing progresses (Figs. 13 and 14). During the division stage, multiiamellar bodies become more abundant in the cytoplasm, some producing vesicles (Fig. 16). Small multilamellar bodies also appear in a few chloroplasts (Fig. 16).

3.3. Major Differentiation Following the formation of spermatangial packets in the division phase there is a marked differentiation of cellular structures including the appearance of spermatial vesicles, and two more divisions per spermatangium occur (Fig. 17). The chloroplast continues to diminish in size in proportion to the other cellular organelles and becomes more discoid in shape (Fig. 18). With further degeneration it eventually contains swollen thylakoids with numerous plastoglobuli in the stroma (Figs. 19 and 20). The cells contain a number of relatively large mitochondria, often near the chloroplast (Figs. 18 and 20).

Figs. 13-16. Formation of cell packets (continued) Fig. 13. The furrowing process in cytokinesis (arrows). Cell wall material is being deposited centripetally in the invaginations of the plasma membrane. • 8,000 Fig. 14. Several layers of rough endoplasmic reticulum (small arrow) near the invaginated plasma membrane during cytokinesis. The endoplasmic reticulum is dilated in several areas. Wall material (W) is being deposited centripetally (large arrow). Note the large multilamellar body (MB) in the cytoplasm just outside the nucleus (N). X 55,000 Fig. 15. The plasma membrane invaginating during cytokinesis. A double membraned septum (arrows) appears to be associated with it. • 50,000 Fig. 16. A large multilamellar body (MB) adjacent to the chloroplast (C). Note the vesicles forming from this body (small arrows). There are several smaller multilamellar bodies (large arrow) within the chloroplast and associated with the thylakoids. • 45,000


Figs. 13-16

262 KATHLEEN COLe and R. G. SHEATH

The large nucleoli disappear from the interphase nucleus as scattered masses of chromatin aggregate close to the envelope (Figs. 18 and 21). There is a re- markable activity of the nuclear envelope as the nucleus becomes smaller during this stage of spermatial development. The perinuclear space swells and the nucleoplasm appears to be eliminated from the nucleus as furrows originating from the nuclear envelope form double membranes around each of the four chromatin masses (Figs. 18 and 25-27). These four masses of chromatin each measure 1 ~m in diameter and appear to represent the four haematoxylin- stained chromosomes of the same size visible in the light microsocpe (Figs. 21-23). Microtubules, 20 nm in diameter, were observed within the nuclear envelope in a few sections; they appeared to be associated with chromatin, although no kinetochores were evident (Fig. 24). Microbody-like structures occur in the cytoplasm near the nucleus and the plasma membrane; they range in size from 150-300 nm (Fig. 28). No dictyosomes are evident at this stage; however, the endoplasmic reticulum is particularly active (Figs. 28 and 30). Vesicles are produced by inflated endoplasmic reticulum near the plasma membrane. A single spermatial vesicle is formed in each cell by fusion of fibrous filled vesicles (Fig. 29). It continues to enlarge as more vesicles fuse with it, until it eventually occupies approximately half the volume of the cell (Fig. 17). Numbers of large tubules, 40-50 nm in diameter, are present in the cytoplasm, particularly near the plasma membrane in lobed areas of the cell (Figs. 32 and 33). Multilamellar bodies are still evident, usually associated with spermatial vesicles or areas of cytokinesis (Figs. 28 and 29). Small multilamellar bodies are eliminated from the cytoplasm into the wall by exocytosis (Fig. 31). Cyto- kinesis is initiated as a double-membraned septum forms across the cell (Fig. 28). Furrows develop as cell wall material is deposited centripetally

Figs. 17-20. Major differentiation

Fig. 17. Packet of spermatangia showing the large spermatiai vesicles (SV) being formed. In the lower right cell the vesicle has already released its fibrous contents (arrow). The cells have small nuclei (N) containing condensed chromatin with little nucleoplasm. Many small vesicles fill the cytoplasm. Little or no floridean starch is evident. • 15,000

Fig. 18. Differentiating ceil showing several mitochondria (M), a small discoid chloroplast (C) and a number of vesicles. The nuclear envelope is inflated in certain areas (arrows) and is invaginating, cutting off nucleoplasm. Some chromatin is aggregated close to the nuclear envelope. •

Fig. 19. The considerably reduced discoid chloroplast in a differentiating cell. Note that there are numerous piastoglobuli in the stroma between the parallel thylakoids and there is no pyrenoid. • Fig. 20. Two adjacent portions of a sectioned chloroplast (C). The chloroplast appears to be degenerating further as indicated by the dilation of the thylakoids. Note the mitochondria (M) close to the chloroplast, and the microbody-like structure in the cytoplasm (arrow). X 45,000


Figs. 17-20

264 KaTHL~EN COL~ and R. G. SHEAT~t

between the two septal membranes (Fig. 28). The large floridean starch reserves are utilized during this stage so that there are very few remaining in the cytoplasm as the developing spermatia reach the final maturation stage.

3.4. Spermatial Release

As the spermatial differentiation process is completed, the spermatial vesicte releases its contents and disappears. The wall around the cells within the packet dissolves and the outer wall breaks, allowing the spermatia to escape (Figs. 34 and 40). Remnants of material from the spermatial vesicles can be seen between the cells which are being released (Fig. 35). At the time of release spermatia are irregular in outline, approximately 4-5 gm in diameter, and small osmiophilic globules are associated with the plasma membrane (Fig. 35); there is no cell covering. The cytoplasm is rich in ribosomes but contains only a small amount of endoplasmic retlculum and no floridean starch or dictyosomes. There are usually one or two mitochondria present showing signs of degeneration (Fig. 36). The chloroplast is irregularly-discoid in shape, averaging 2 um in diameter (Fig. 36). It contains a few swollen, distorted thylakoids, a number of large plastoglobuli and some DNA-like fibrils. A few double-membraned vesicles, which may be plastid initials, are formed from the chloroplast envelope by a blebbing process (Fig. 37). These vesicles measure about 430 nm and have a finely-granular homogeneous matrix. The nucleus is approximately 2 ~tm in diameter and contains a large, lobed chromatic body, a small amount of nucleoplasm and no nucleoli (Fig. 38). The nuclear envelope lacks pores and the swollen perinuclear space contains a fibrous material. Vesicles may be produced by evagination of the outer membrane of the envelope (Fig. 38). Within the cytoplasm of the mature spermatium there are many vesicles containing electron dense material (Fig. 35). Microtubules similar in diameter to those observed within the nuclear envelope during the previous phase were observed traversing the cytoplasm in sections of one spermatium. They were very close to chromatin masses (Fig. 39).

Figs. 21-23. Major differentiation (continued)

Fig. 21. Four chromatin masses (Ch) within the nucleus. Note the nuclear membrane invaginating at four points (arrows). There are several fibrous filled vesicles in the cytoplasm. X45,000

Fig. 22. Four haematoxylin-stained chromosomes equivalent to the four chromatin masses in Fig. 21. X2,500

Fig. 23. Haematoxylin-stained chromatin material in differentiating cells. The cells in the packet on the right have irregularly-shaped masses of chromatin which may represent the structures formed by the fusion of the four chromosomes shown in the upper cell of the packet on the left and in Fig. 22. •


Figs. 21-23

266 KATHLEEN COL~ and R. G. SHEATH

4. D i s c u s s i o n

The major features of spermatiogenesis reported to date (including this paper) for a few of the rhodophytan algae in the classes Bangiophyceae and Florideo- phyceae are notably similar, particularly regarding the differentiation which occurs during the maturation of the spermatium. This is interesting because, while the spermatia of the two classes are similar, the morphological events which lead to their production differ. Spermatiogenesis in the Bangiaceae within the Bangiophyceae occurs by division and differentiation of vegetative cells of the thallus producing 8-32 spermatangia, each of which then produces four spermatia. Spermatiogenesis in the Florideophyceae usually occurs in cells of specialized spermatangial branches of the thallus which produce a number of spermatangial mother cells, each of which forms two or three spermatangia; one spermatium differentiates within each spermatan- glum. Several prominent features of spermatiogenesis are concerned with changes in the main cellular organelles. In previous reports on other Rhodophyta (e.g., KUCRENS and WEST 1972, KUORENS 1974, HAWKES 1978), it has been pointed out that, during the process in most species, the size of the chloroplast relative to the size of the cell is reduced significantly and the chloroplast becomes less complex, appearing to degenerate or dedifferentiate and even disappear; the nucleus also becomes smaller and, toward the conclusion of the process, contains a large mass of condensed chromatin, little nucleoplasm and no nucleoli; and one or more fibrous filled vesicles produced by the dictyosomes and/or the endoplasmic reticulum. These phenomena were also observed in Bangia in the present study, but in considerably greater detail than previously reported.

4.1. The Chloroplast

The chloroplast is a dynamic organelle which changes its shape and structure in response to varying conditions including light intensity and specialization of the cell in which it is located. In the current study, the chloroplast in the undifferentiated cells of male filaments of Bangia is typical of the Bangiaceae (e.g., COLE and CONWAY 1975, LIN et al. 1977). The sequence of changes during spermatiogenesis involves a modification of the large,


Fig. 24. Microtubules (arrows) within the nucleus associated with chromatin (Ch). • 55,000

Fig. 25. Nucleoplasm (large arrow) eliminated from the nucleus. The nuclear membrane is invaginating (small arrow) between two chromatin masses (Ch). X 35,000

Fig. 26. Swollen nuclear envelope (arrows) cutting off nucleoplasm from the chromatin (Ch). X 30,000

Fig. 27. Two chromatin masses (Ch) and a small amount of nucleoplasm enclosed by enve- lopes following the activity described in Figs. 25 and 26. X 37,000


Figs. 24-27

268 KATHLEEN COLE and R. G. SHEATH

lobed structure containing parallel, single, evenly-spaced thylakoids, and a central pyrenoid into a small discoid form with a few distorted, dilated thylakoids and many plastoglobuli, and no pyrenoid. The single discoid chloroplast within the mature spermatium of Bang& is similar to that in another member of the Bangiaceae, Porphyra gardneri (HAWKERS 1978). The internal changes which occur during the modification of the chloroplast resemble those which are usually associated with aging, degeneration or transformation into chromoplasts (e.g., LICHTL~ 1973). The mature spermatia of most of the Florideophyceae studied to date differ from those in the Bang&ceae because they either lack chloroplasts or have one or two proplastids which usually degenerate (e.g., KUGRENS and WEST 1972, SCOTT and DIXON 1973, YOUNG 1977). This disparity between the two red algal groups may be explained by recognizing the difference related to a) chloroplasts in the vegetative cells prior to differentiation, and b) the series of morphological events involved in the production of spermatia from these vegetative cells, which was mentioned at the beginning of the Discussion. In the Bangiaceae, all divisions in spermatiogenesis occur within the wall of the vegetative cell. The single large axial chloroplast within each cell divides and the products are contributed to daughter cells, first the spermatangia and then the spermatia. On the other hand, in the Florideophyceae, all divisions in spermatiogenesis do not take place within the wall of the vegetative cell; the pattern of cell division is polarized (DIXON 1973). In addition, the several small discoid chloroplasts within each cell in the reproductive structures do not always divide or produce proplastids (KuGRENS 1974), SO that the number of plastids present in each successive cell generation is reduced. In his studies or spermatiogenesis in Janczewskia gardneri (Florideophyceae), KIJCl~ENS (1974) also observed that there appeared to be a polarization within the cytoplasm of some spermatangial mother cells. The small reduced chloroplasts concentrated in the basal part of the cell so that they were not included in the spermatangial cells which were cut off from the upper part of the mother cell. The released spermatia of Bang& do not survive for more than one or two days if unattached to the wall of the carpogonium (COLE, personal observa-

Figs. 28 and 29. Major differentiation (continued)

Fig. 28. Cytokinesis in a differentiating cell. Wall material (W) is being deposited centripetally. A double-membraned septum has formed across the cell (small arrows). There are a number of multilamellar bodies (large arrow) near the septum. The cytoplasm contains large mitochondria (M) and a number of microbody-like structures (double arrow), usually near the plasma membrane. Note the large nucleus (N) with swollen membrane system and chromatin masses. • 45,000

Fig. 29. Small fibrous filled vesicles fusing to form a large spermatial vesicle (SV). A multilamellar body (MB) is adjacent to the spermatial vesicle. • 50,000


Figs. 28 and 29

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270 ~[ATHLEEN COLE and R. G. SHEATH

tion), and undergo rapid degeneration once they do become attached. Con- sequently, they require very little storage material. Mature spermatia in the two bangiacean genera studied ultrastructurally (this paper and HAWKES 1978) have no floridean starch but do have single, reduced chloroplasts which contain a number of plastoglobuli. These lipids could serve as a form of carbon storage available for the few metabolic processes required in these specialized cells. It is interesting to note that the mature spermatia of most of the Florideophyceae studied lack chloroplasts, but many of these have some floridean starch remaining in the cytoplasm (e.g., KUCI~ENS and W~sT 1972, K~rORENS 1974, PE:Cl~I~e,~ 1974). There are no known reports on the longevity of florideophycean spermatia. The reticulation of the thylakoids which commences in the pyrenoid during the initial stages of chloroplast transformation in spermatiogenesis in Bangia is unique. SH~AT~ et al. (1977, 1979) demonstrated that the internal membrane system of pyrenoid-containing chloroplasts in the Rhodophyta is particularly responsive to light conditions. They noted that during the early stages of dark incubation of cells of Porphyridium purpureurn (Banglophyceae) and the chantransia stage of Batrachosperrnum moniliforme (Florideophyceae) some reticulation of the thylakoids occurred. The reticulation pattern of thylakoids visible in the early stages of chloroplast transformation during sperrnatio- genesis in Bangia is similar to that in the above studies on the Rhodophyta except that the thylakoidal reticulation in Bangia chloroplasts commences within the pyrenoid and ultimately extends to the remainder of the chloroplast. It is not likely that this reticulation phenomenon in Bangia is caused by low light conditions because it occurs only in cells of the thallus which are differentiating into spermatia and most of these are at the surface of the filament. However, it may represent the initial stages of preparation of the thylakoids for the differentiation process during which the chloroplast differentiates into a small discoid form lacking a pyrenoid.

4.2. The Nucleus The major nuclear changes which occur during the maturation stages in spermatia of Bangia and most Rhodophyta studied to date are similar in some ways to those which occur in the formation of sperm in other organisms: the


Fig. 30. Inflated rough endoplasmic reticulum (arrows) containing a granular material oriented at right angles to the plasma membrane. • 70,000

Fig. 31. A small multilamellar body being eliminated from the cytoplasm into the wall (W) by exocytosis. X 70,000

Fig. 32. Large cytoplasmic tubules (arrow) near the plasma membrane in a lobed area of the cell. The cytoplasm also contains many vesicles. • 25,000

Fig. 33. Longitudinal and cross sections of the large cytoplasmic tubules. • 50,000


Figs. 30-33

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272 KATttLEEN COLE and R. G. SHEATH

chromatin condenses and much or most of the nucleoplasm is eliminated as the nucleus becomes smaller (e.g., Scow, and DIxoN 1973, PEXRIgRe 1974). The nucleus usually lacks a nucleolus in these specialized cells. The spermatial nucleus in many red algae is particularly interesting at maturity because it contains condensed chromatin bodies which stain readily with any of the standard organic dyes used in chromosomal studies and are visible in the light microscope. They appear to be the chromosomes in a prolonged late prophase stage of mitosis. They were first noted in spermatia of Polysiphonia violacea (Florideophyceae) by YAMANOUCHI (1906) who also observed that the nuclei were in prophase at the moment the spermatium united with the trichogyne of the carpogonium. GRuBB (1925) commented on the definite, constant number of chromatin granules which stained in the released spermatia of several other Florideo- phyceae, as did ISHIKAWA (1921) for Porphyra tenera (Bangiophyceae). The spermatia are formed from the gametophytic thallus by mitosis, consequently, these constant numbers are the haploid chromosome numbers for the species. In Bangia atropurpurea (marine form) the haploid number of four chromosomes has been recorded in spermatia (Cote 1972, as B. fuscopurpurea), as well as in vegetative cells of male and female filaments (COLE, personal observations). In the current ultrastructural study on Bangia it was observed that when the furrows which are formed from the nuclear envelope cut off the nucleoplasm they form double membranes around four spherical chromatin masses, each measuring about 1 gm in diameter. These bodies correspond to the four

Figs. 34-39. Spermatial release

Fig. 34. Mass of spermatia being released from the tip of a male filament. • 90

Fig. 35. Several irregularly-shaped spermatia separating as the intercellular walls break down. Note some fibrillar material from the spermatial vesicle between the cells (arrow). Several of the cell structures can be seen at this magnification: a discoid chloroplast, a nucleus with dense chromatin, and a number of small vesicles. Many small osmiophilic globules are associated with the plasma membrane. • 4,000

Fig. 36. Irregularly-shaped discoid chloroplast (C) containing relatively few thylakoids, many osmiophilic globules and DNA-like fibrils (single arrow) in the stroma. Note what appears to be a degenerating mitochondrion (double arrow) within the spermatium. X 25,000

Fig. 37. Bleb forming (arrow) by evagination of the chloroplast envelope. The bleb contains a granular matrix. The contents of the vesicle beside the bleb are similar; this structure may be a plastid initial. •

Fig. 38. Nucleus of the spermatium showing condensed chromatin (Ch), small amount of nucleoplasm and swollen nuclear envelope (small arrows). The perinuclear space contains some fibrillar material (large arrow). The cytoplasm is filled with ribosomes, some of which are associated with the outer nuclear membrane. X 30,000

Fig. 39. Microtubules (arrows) running through the cytoplasm beside a chromatin mass. X55,000


Figs. 34-39


chromosomes visible in mature spermatia when they have been stained and viewed in the light microscope. However, these individual double membrane- bound bodies are not visible in released, mature spermatia of Bangia at this point in the differentiation process; a single nucleus of 2 ~tm diameter containing condensed chromatin irregular in outline and very little nucleoplasm is present, similar to that also reported in other red algae. Considering the dynamic qualities of the cellular membrane system, it is likely that the four individual bodies fuse forming a single, large, irregularly-shaped chromatin body bounded by a double membrane, and containing little nucleoplasm. There is only one other known report on the ultrastructure of chromatin condensation into distinct spheroidal bodies in the spermatia of the red algae. PEEL and DUCKETT (1975) noted that several electron dense bodies, probably equivalent to the chromosomes, appeared in the nucleus of Corallina officinalis (Florideophyceae) during maturation. However, this phase in spermatiogenesis of Corallina differs from that in Bangia; the furrowing process has not been observed, there is no reduction in the amount of nucleoplasm, and each of the chromatin masses is not enclosed by an envelope at any time during differentiation. As was mentioned earlier, the presence of condensed chromosomes in released spermatia of most Rhodophyta studied to date has led to the assumption that these cells are in a prolonged stage of karyokinesis (GI~UBB 1925, DREW 1951, MAGNE 1952). In her ultrastructural studies on released spermatia of Rhodo- mela confervoides (Florideophyceae), PEYRI~RE (1974) noted: concentrations of nuclear pores in polar regions of the nuclear envelope; cytoplasmic microtubules passing through polar fenestrae and seeming to attach to the dense chromatin within the envelope; highly lobed chromatin; and no individual chromosomes or kinetochores. In the current study on maturing spermatia in Bangia, microtubules were observed in the cytoplasm close to chromatin in a few sections. These are the only two published reports of microtubules within released spermatia of the red algae. This is not surprising because microtubules are particularly sensitive to fixation procedures and are easily destroyed. In addition, the microtubules may be present for only a very brief period during the life of the spermatium. The presence of microtubules as well as dense chromatin strongly suggests that spermatia are in a mitotic prophase at least at some period prior to fertilization. However, more data are required to support this. Whether or not it represents a prophase con- dition, the condensed chromatin present in the nuclei of rhodophytan sper- maria ensures that the genetic material is transferred as concisely as possible during fertilization. The relatively large cytoplasmic tubules near the plasma membrane in maturing spermatia of Bangia are similar in appearance and position to those reported by K~CRENS and WEST (1973) in the maturing carpospores of Levringiella gardneri (Florideophyceae). They probably are not concerned


with wall deposition because they occur at the end of the period of maximum cell wall formation. However, they may serve as cytoskeletal elements similar to the cytoplasmic microtubules in the Chrysophyte Ochromonas danica (BoucK and BROWN 1973).

Fig. 40. Spermatial release (continued)

Fig. 4D. Fibrillar contents (single arrow) remaining within the wall remnants following release of spermatia through the breaks in the thick outer wall (double arrows). X 2,000

4.3. The Endomembrane System

The endomembrane system is active throughout spermatiogenesis in Bangia; single membrane-bound vesicles form from the dictyosomes, endoplasmic reticulum, nuclear envelope and multilamellar bodies. Some of the vesicles which usually contain fibrillar material, fuse with the plasma membrane, releasing material into the cell wall, while others fuse with each other to form large spermatial vesicles. During the early stages both dictyosomes and endoplasmic reticulum are evident. The dictyosomes are primarily responsible for producing vesicles containing wall material as observed in other red algae (e.g., W~T~RB~ and W~sT 1977), and the rough endoplasmic reticulum produces some which remain within the cytoplasm. In cytokinesis during spermatiogenesis in Bangia, several layers of

276 KA'r~LEEN Cote and R. G. SHEa'rH

endoplasmic reticulum are located near the invaginating plasma membrane in areas where furrowing originates. Additional layers of endoplasmic reticulum are arranged in line with the furrow as a double membraned septum forms. Furrows develop as cell wall material is deposited centripetally btween the two septal membranes. Both nuclear and plastid division precede this cytokinesis. The process of cytokinesis which follows karyokinesis in the Rhodophyta is relatively simple and has been observed in only a few species. In ultrastructural studies of dividing cells of Porphyridium (Bangiophyceae) (GANTT and CONTI 1965) and Mernbranoptera platyphylla (McDoNALD 1972 b), it was shown that there is a centripetal furrowing of wall material as the plasma membrane constricts the cellular contents producing two equal daughter cells. In the case of Porphyridiurn, the constriction causes the division of the large central chloroplast. No septum was reported for either Porphyridium or Mernbranoptera. Microtubules have not been observed in the cytoplasm related to the furrowing process in any studies to date. As the period of cell division concludes, the dictyosomes disappear and there is an increased activity of the endoplasmic reticulum. At this stage, small vesicles fuse to form the large vesicles containing a fibrous polysaccharide (e.g., PEYRIkI~e 1971, SIMoN-BmHAI~D-BI~kAUD 1972). These larger vesicles, which were termed spermatial vesicles by KimReNS and WEST (1972), are characteristic of the rhodophytan spermatia; they appear prior to the final maturation stage of spermatiogenesis, and extrude their contents at this final stage (e.g., SCOTT and DIxoN 1973, KtrGReNS 1974). Remnants of material from the spermatial vesicles are visible within the spermatangia between the spermatia of Bangia as they are being released, similar to the report by SCOTT and DIxoN (1973) in the spermatangia of Ptilota densa (Floridiophyceae). It has been suggested that the hydration of the polysaccharides forces the spermatium from the spermatangium. KtmRens and WEST (1973) also proposed that the contents of the spermatial vesicles may serve as an adhesive coating for the spermatium, allowing it to stick to the trichogyne. PEEL and DtrCKETT (1975) noted that the spermatial coat of Corallina of]icinalis contains sulphated polysaccharides. As it is being released, the mature spermatium of Bangia contains very little endoplasmic reticulum and no dictyosomes. However, it does have many small cytoplasmic vesicles. Granular material is visible in the swollen perinuclear space of these specialized cells and the outer membrane forms out- foldings. This is the only report of electron-dense material in the perinuclear space in the Rhodophyta, although there have been a few for other algae. For example, mastigonemes are formed within this space in the antherozoids of the brown algae Fucus and Ascophyllum (BoucK 1969). Small vesicles occur near the plasma membrane in the mature, released spermatia of Bangia, ScoTT and DIXON (1973) noted fusion of small vesicles with the plasma membrane in the mature spermatia of Ptilota densa. These vesicles may be contributing


substances which form part or all of the spermatial coating, but further studies are required to confirm this. Multilamellar or concentric bodies are common to many cells. They are present in vegetative cells of Bangia and increase in size and number in the cytoplasm and chloroplast during the division phase of spermatiogenesis. In the differentiation phase, they are also associated with enlarging spermatial vesicles and areas of cytokinesis. The multilamellar bodies are eliminated at the end of the differentiation period when spermatial vesicles have reached their maximum size. K~GR~NS and WEST (1972) recorded a similar occurrence in spermatia of Erythrocystis saccata (Florideophyceae). Early studies by GANTT and CONTI (1965) on Porphyridiurn cruenturn and by BROWN and W~IR (1970) on Batrachospermum moniliforrne showed that the number of concentric bodies in the cytoplasm and chloroplast increased in older cells. From these sparse data available for the Rhodophyta it is difficult to ascertain the role of the multilamellar bodies. They may represent excess membrane material which is used for septal and vesicle formation in dividing and differentiating cells. When no longer required, as in older cells or cells growing under less than optimum conditions, they may eventually be eliminated either by exocytosis, as seen in this study in mature spermatia of Bangia, or by autolysis, as postulated by BROWN and W~IR (1970) and GU~I~IN-DUMARTI~AIT et al. (1973). Microbodies in the cells of higher plants are usually at sites of metabolic functions and are often termed peroxisomes and glyoxysomes because of their enzymatic activities (ToLB~RT 1971). Consequently, it is interesting that bodies similar in size and structure to these microbodies have been observed in the cytoplasm near the nuclear envelope and plasma membrane during spermatiogenesis in Bangia, as well as near the mitotic spindle poles of Porphyridium (Bangiophyceae) (BRONCHART and DEMOULIN 1977, SCHORN- STEIN and SCOTT 1978) and adjacent to the nuclear envelope in prophase I of Palmaria palmata (FIorideophyceae) (PuESCHEL 1979). OAKLEY and DODGE (1974) demonstrated the presence of peroxidase and absence of catalase in the microbodies within the cells of Porphyridium, while I)UESCHEL'S (1979) tests for Palmaria were negative for both peroxidase and catalase. Of course, the microbodies are localized in different parts of the cytoplasm within the cells of these two algae and are probably responsible for different enzyme activities. The cytochemistry of the microbody-like structures in Bangia is being studied currently. No suggestions regarding the functions of these bodies in red algal cells can be presented at this time.

Acknowledgements The authors are grateful to the N.S.E.R.C. for financial support, Operating Grant 0645 to K.C. and Postdoctoral Fellowship to R.G.S. They also appreciate the technical assistance provided by Mr. L. V~TO and Mrs. L. MORNIN, as well as access to the beach at Point No Point provided by Miss E. PACI~HAM.


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