TISSUE & CELL 1975 7 (3) 525-534 Puhli.shed h.v Longmrtn Group Ltd. Printed in Great Bfitnitr

JEAN ROSS

CYLINDRICAL ORGANELLES ASSOCIATED WITH MICROTUBULES DURING SPERM ELONGATION IN A MEALYBUG

ABSTRACT. Spermiogenesis in the mealybug Pwutkmrcos ohscrwus involves the elongation of a cytoplssmic papilla from the surface of a spermatid. Longitudinally oriented fibers and microtubules, with diameters of 250 8, and a non-9+2 arrange- ment, are associated with this process. Fibers alone are found in early stages of papilla emergence and in the basal region of the elongated papilla. They appear to be con- tinuous with the microtubules in the more distal region of an elongated papilla. Disk- shaped, circular, and rectangular profiles are associated with the basal ends of the libers. Probably the fibers are microtubular ‘anlagen’ and the associated structures may be microtubular ‘organizing centers’.

introduction

THE spermatozoa of the mealybug Pseudo- COCCUY ohscurus are motile, yet they lack the 9 + 2 arrangement of microtubules usually associated with motile cilia and flagella (Ross and Robison, 1969). Instead, each sperma- tozoon contains a highly ordered array of 56 microtubules which extends throughout most of its length. Furthermore, these spermatozoa contain no basal bodies or centrioles (Hughes- Schrader, 1946, 1948; Ross and Robison, 1969) which are usually associated with the 9+2 axoneme in cilia and flagella and from which microtubules commonly emanate in many cell types (see Porter, 1966; deHarven, 1968, for review). Therefore, the early stages of spermiogenesis were examined in search of a microtubule organizing center (centriole substitute) which may be associated with the microtubules during their formation in the development of these unusual spermatozoa (see preliminary report, Ross, 1970).

From the Department of Biology, University of Virginia, Charlottesville, Virginia, and the Depart- ment of Biology, Yale University, New Haven, Corm. Present address: Department of Biology, C~niversity of Chicago, 1103 E. 57th Street, Chicago, Illinois 60637.

Received 5 December 1974.

Materials and Methods

A culture of the mealybug Pseudococcus

obscwrrs Essig (Homoptera, Coccoidea) was maintained on well-sprouted potato tubers (Solanum tuberosum) in cloth-covered jars and kept in the dark at room temperature.

Testes from third-instar males were dis- sected and fixed for 23 hr in ice cold 0.25 M glutaraldehyde and 0.17 M sucrose in 0.05 M cacodylate buffer at pH 7.4. The tissue then was washed in cold 0.07 M cacodylate buffer with 0.35 M sucrose, post- fixed for l-2 hr on ice in 1 .Oo/:, osmium tetroxide in sodium phosphate buffer (Mil- lonig, 1962) at pH 7.4, dehydrated at room temperature in an ethanol series and em- bedded in Epon 8 12. Sections were cut on an MT-2 Sorvall Porter-Blum microtome, stain- ed in ethanolic uranyl acetate followed by lead citrate and observed with a Philips 200 electron microscope.

Results

Mature spermatozoan .rftwfwe

The spermatozoa of P. ohsrurus are Ma-

mentous structures which lack morphological divisions into head, midpiece, and tail (Ross and Robinson, 1969). Throughout its length,

525

ROSS

have no central lumen are associated with the region of papilla emergence; they lie between the nucleus and the cell membrane. The extending nucleus and the fibers have similar contours and form a cone extending from the cell. The region of the nucleus which remains within the cell body is surrounded by cyto- plasm which is relatively clear and free of organelles except for structures which appear as either solid disks or hollow circles in sections and are 350450 8, in diameter and sometimes are surrounded by halos (Fig. 1).

Late papilla development. In a later stage of spermiogenesis each spermatozoon, or papil- la, consists of a highly elongated portion of the spermatid nucleus surrounded by a system of longitudinal structures which vary in their internal detail when transected at different levels along the papilla. In the basal region of the papilla, which lies within the cell mass of the spermatid, 28-30 dense fibers are arranged in a single row around the nuclear membrane (Figs. 4, 5). The majority of the fibers are approximately 250 8, in diameter, though some are smaller. The fibers lie within a distinguishable layer of cyto- plasm which is delimited by an intracellular membrane (Figs. 4, 5). In the papilla proper, beyond the point of emergence, there is an increase in the number of fibers so that approximately 56 fibers lie as a single row around the nucleus (Fig. 3). Some of the fibers contain small, electron-lucent centers which are smaller than those of the microtubules in the mature spermatozoon.

At a presumably later stage of develop-

each spermatozoon consists of a micro- tubular array surrounding a central core of chromatin. The microtubules are oriented parallel to each other and to the long axis of the spermatozoon. Throughout most of the length of the spermatozoon there are 56 microtubules arranged as 23 concentric rings. In one end of the spermatozoon there are dense fibers, 200-250 A in diameter, which appear to be continuous with the micro- tubules (Ross and Robison, 1969).

Spermiogenesis

The development of the spermatozoa is synchronous and the testes of a third instar insect are filled with binucleate spermatids which are grouped into cysts (Hughes- Schrader, 1948; Nur, 1962; Ross, 1969). Spermiogenesis involves the elongation of two cytoplasmic papillae from the wall of each spermatid (Figs. 1, 2, 3); each papilla represents a prospective spermatozoon. The papillae continue to grow out and around their respective cysts and during this out- growth nuclear material moves from the cell into the papillae (Nur, 1962). This type of spermiogenesis is typical in coccid insects (Schrader, 1921, 1923 ; Hughes-Schrader, 1946, 1955; Nur, 1962) and has been describ- ed ultrastructurally for Steatococcus tubercu- Iatus (Moses and Wilson, 1970).

Early papilla development. In an early stage of the outgrowth of a papilla, the nucleus is positioned close the cell membrane and partially extends into the papilla (Fig. 1). Dense fibers which are 250 A in diameter and

Fig. I. An early stage of papilla emergence. The nucleus (N) lies adjacent to the cell membrane of the spermatid and a portion of it extends as a cone out of the cell. Dense fibers (F) lie between the nucleus and the cell membrane; the fibers are shown in transection in the inset. The nucleus is surrounded by cytoplasm which is relatively clear except for structures which appear as solid disks or hollow circles surrounded by halos (arrows). x 30,000. Inset x 35,000.

Fig. 2. A spermatid out of which two spermatozoan papillae elongate. Only the basal portion (BP) and the region of emergence (E) of each papilla are in the plane of the section. Dense fibers (F) extend into each papilla. The proximal ends of the fibers lie in clear cytoplasm (C) which also contains small, dense disk-like structures (arrows). x 21,000.

528

ment, the dense fibers in the basal region approximate a concentric arrangement which closely resembles that of the fibers in one end of the mature spermatozoon and the nuclear material is no longer present in this region. The number of fibers varies from about 30 to 50, and probably is dependent on the level of transection. The fibers are still surrounded by clear cytoplasm (Fig. 6).

The basal region of the dense fibers in this later stage of development appears to be associated with structures which exhibit various profiles in sections (Figs. 3, 7, 8, 9). In some sections these structures appear to be approximately rectangular (ca. 450 by 700 A) with an electron-lucent center (Fig. 3). In other cases, they appear to be either circular or disk-like with a diameter of 450-500 A (Figs. 2, 7, 8, 9). Occasionally, a single fiber

ROSS

appears to abut directly upon one of these structures (Figs. 3, 7, 9). In favorable longi- tudinal sections, where the structures have a rectangular profile, the end of a fiber appears to contact the surface of the central region of one of these structures at a plane normal to its long axis (Fig. 3). Some of the circular and disk-shaped profiles also contact the fibers but many do not and lie free in the clear cyto- plasm surrounding the fibers (Figs. 2,7,8,9).

In the extended portion of a papilla, the longitudinal structures are microtubular in form with diameters of about 250 A and a central lumen (Fig. 6). There are usually 55 to 57 microtubules and they are arranged in three concentric rows in this area. Even more distally in the spermatozoon the micro- tubules have larger central lumens and are arranged in 2+ concentric rows (Fig. 10) as

Fig. 3. Longitudinal and cross sections of portions of spermatozoa papillae (P) which have elongated out of the spermatid cytoplasm (S). The dense fibers (F) surround the thread-like chromatin (Ch). The proximal end of one fiber appears to abut on a rectangular structure (RS). This structure is shown in (A); it has a dense periphery and a less-dense central area. Another papilla (long arrow) is transected in its proximal region and has 56 fibers (F) which lie in a single row in the peripheral cytoplasm. Some fibers are shown in enlargement (B) and have small electron-lucent centers (short arrow). x 25,000; A and B, x 54,000.

Fig. 4. Oblique section of the basal region of a spermatozoan papilla. This region lies within the cytoplasm of the larger cell mass of the spermatid, but is separated from it, partially, by an intracellular membrane (IM). Dense fibers (F) surround chromatin (Ch). x 33,000.

Fig. 5. Transection through the basal region of a papilla as illustrated in Fig. 4. Twenty-eight dense fibers (F) lie in a single row around the nucleus in a distinguishable layer of cytoplasm. Chromatin (Ch), nuclear membrane (NM), intracellular membrane (I M). x 42,000.

Fig. 6. Transection through the basal region of one papilla (BP) and a more distal region of another (DR). The basal region lies within the cell; the dense fibers approxi- mate a concentric arrangement and are surrounded by clear cytoplasm. The more distal portion of the papilla (DR) is extended beyond the cell mass and consists of 55 micro- tubules arranged as three concentric rows and surrounded by cytoplasm. x 42,000.

Figs. 7-9. Oblique sections through the basal region of papillae. The proximal ends of the fibers are associated with clear cytoplasm and with circular and disk-shaped structures (arrows).

Fig. 7. Fibers appear to abut directly on the disk-like structures. x 38,000.

Fig. 8. Several disk-like structures (single-stemmed arrow) and circular structures (double-stemmed arrow) are in the clear cytoplasm. x 43,000.

Fig. 9. Each of two dense rods is in close association with a circular structure (arrow). x 69,000.

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MICROTUBULES DURING SPERM ELONGATION

papillae. The tubules have small lumens surround the chromatin (Ch). x 80.000.

Fig. IO. Transection throueh the distal region of relatw3v mature soermatozoar dre arranged in 2! coIxentr/c row. and -1

found in the mature spermatozoon (Ross and Robison, 1969). Eventually, the papilla

dissociates from the cell body and is engulfed into a sperm bundle with I5 other sperma-

tozoa, as described by Ross (1971).

Discussion

During the elongation of a papilla from the surface of a spermatid, dense fibers and microtubules are prominent but are not in a 9t2 arrangement. In early stages, dense fibers are present in the area of papilla emergence and follow the contour of the papilla. In later stages of elongation, each spermatozoon contains dense fibers in the basal region of the papilla and microtubules in the extended portion. A fiber and a tubule may represent a continuous, longitudinal structure in which the central area changes, spatially, from electron dense to lucent. This suggestion is based on the similar size,

number, and disposition of the fibers and the microtubules, and on the variability of the

central areas. The smallest and least consis- tent electron-lucent centers are associated

with the fibers in the basal papilla while the larger ones are consistently in the more distal,

longitudinal structures, or microtubules. A similar continuity has been noted between dense fibers and microtubules in one end of thematurespermatozoon (Ross and Robison, 1969). The dense fibers in the immature spermatozoon are probably equivalent IO those in one end of the mature spermatozoon since they are the same in number and appearance.

The basal regions of the dense fibers, in turn, are associated with unusual structures which display various profiles in section, The circular, disk-shaped and rectangular pro- files, which are seen in later stages of papilla elongation, can all be explained by an organelle with the shape of a hollow cylinder

ROSS

centers frequently occur as small dense bodies in the cytoplasm (de The, 1964; Szollosi, 1964; Robbins et al., 1968; Bowers and Korn, 1968; Gibbins et al., 1969; Rattner and Brinkley, 1972) or areas of dense amorphous material (Tilney, 1971a; Bouck and Brown, 1973). Various other sites have been described (see Tilney, 1971b, for review). It has been suggested that these various sites associated with the ends of microtubules may initiate or orient micro- tubule assembly (Porter, 1966; InouC and Sato, 1967; Tilney, 1968; Gibbins et al., 1969; Brown and Bouck, 1973) but this has not been demonstrated unequivocally.

It has also been suggested that nucleation centers, in the form of disk-like structures, are required for the assembly of micro- tubules in vitro (Borisy and Olmsted, 1972). Similar ring- and spiral-like structures have been seen by other investigators in associa- tion with the in vitro polymerization and depolymerization of microtubules (Granett et al., 1973; Kirschner et al., 1974) but their role in microtubule assembly is undetermined.

The sites for initiation of microtubule assembly, both in vivo and in vitro, have not been well defined even though the ends of microtubules often are associated with various structures. The cylindrical organelles as well as the dense fibers, which are associ- ated with the microtubules in the elongating spermatozoa of P. obscurus may be another form of microtubule ‘organizing center’. Furthermore, the microtubules in the mature spermatozoon assume a very ordered arrange- ment which must be controlled by some factor, possibly the ‘organizing centers’.

with closed ends, 450-500 A in diameter and 700 8, long, which has been sectioned in dif- ferent planes. Thus, the hollow rectangular profile (ca. 450 by 700 A) would represent a longitudinal section through the cylinder, while the circular and disk-like profiles (ca. 450 A in diameter) would represent tran- sections through the mid- and end-regions, respectively, of the cylinder. It appears that a single fiber terminates basally on a single cylindrical organelle, in the central region of the organelle and in a plane normal to its long axis. Since only circular and disk-like profiles are observed in the basal region during early stages of spermiogenesis, perhaps these are precursors of the cylindrical organelles, but they may represent an entirely different structure. Unlike the fibers and the micro- tubules, the associated organelles are no longer present in the mature spermatozoon (Ross and Robison, 1969).

The cytoplasm at the base of the papilla is interesting in that it contains no organelles except for the fibers and the organelles just discussed. Moses and Wilson (1970) have also noticed distinct cytoplasm at the base of the papillae in Steutococcus tuberculutus. Possibly, this area is specialized for the pro- duction of microtubules and the associated structures serve as ‘organizing’ or ‘nucleating’ centers for formation of the dense fibers which are continuous with the microtubules; this suggests that the dense fibers represent microtubular anlagen. This hypothesis re- quires that the microtubules in the papillae be assembled at their basal end. This, in fact, has been indicated to be the case in the papillae of the related coccid, Steutococcus tuberculatus (Moses et al., 1968; Moses and Erickson, 1973). A microtubule ‘anlagen’ has also been described in the papillae of S. tuberculatus (Moses et al., 1968; Moses and Wilson, 1970); this is a fuzzy structure in the proximal region that later takes on the charac- teristic appearance of a microtubule. The microtubule anlagen in these two related insects may be analogous but the cylindrical organelles have been seen only in the papillae of P. obscurus.

There are numerous reports demonstrating the association of ends of microtubules with various loci other than basal bodies. These

Acknowledgements

I am grateful to Dr Gerald Robison Jr for his help in this study and his advise during the preparation of the manuscript. The work was supported by grants from the National Science Foundation (GB 5850) and the United States Public Health Service (FR- 07094-01 and FR-07094-02) to Dr W. Gerald Robison Jr and by United States Public Health Service predoctoral training grant (5 Ti HD-29) and Postdoctoral Fellowship (HD-54208) to the author.

MICROTUBULES DURING SPERM ELONGATION

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