The ultrastructure of the spermatozoon and spermiogenesis in Cryptocotyle lingua (digenea: Heterophyidae)

lnrernarionnl Journnifor Parasifology, Vol.9, pp.405-419. F’ergamon Press Ltd. 1979. P&led in Greaf Britain.

THE ULTRASTRUCTURE OF THE SPERMATOZOON SPERMIOG~NESIS IN CR YPTOCOTYLE LINGUA (DIGENEA: HETEROPHYIDAE) F. Department

GWENDOLEN

AND

REES

of Zoology, University College of Wales, Aberystwyth,

U.K.

(Received 11 October 1978) Abstract-REEs F. G. 1979. The ultrastructure of the spermatozoon and spermiogenesis in Cryptocotyle lingua (Digenea: Heterophyidae). International Journal .for Parasitology 9: 405-419. During spermiogenesis two lateral flagellar processes and a median process arising from the apex of the zone of differentiation, fuse to form the elongated unipartite spermatozoon. Two axial units, therefore, with the ‘9+ 1’ pattern of microtubules are incorporated into the spermatozoon. The nucleus, in the head region, contains dense lamellar subunits arranged in a spiral in the long axis. These are formed by condensation of the ~hromatin during s~rmiogenesis. The single elongated mit~hondrion, resulting from early fusion of small mitochondria, extends through the head and middle regions of the spermatozoon. Peripheral microtubules, present originally in the zone of differentiation, are arranged in straight dorsal and ventral rows, along the length. fi glycogen particles accumulate in the spermatozoa after they have separated from the residual cytoplasm. Spermatozoa are present in the testes on the second day after infection of the bird host and accumulate in the vesicula seminalis from the third day onwards. INDEX KEY WORDS: spermiogenesis.

Digenea;

Cryptocotyie

lingua

ultrastructure;

unipartite

MATERIALS THIS PAPER is one of CryptocotyZe liizgua,

a series on the ultrastru~ture of at various stages in the life cycle (Rees, 1974,1975a, h, 1977, 1978; Rees &Day, 1976; Day, 1976) and concerns the ultrastructure of the spermatozoon and spermiogenesis. Cable (1931), in a light microscope study, described spermatogenesis in this species, determining the number and size of the chromosomes and their behaviour during somatic and germinal development. Few ultrastructural studies have been made on spermiogenesis in platyhelminths. The most complete, among Digenea, are those of Gresson & Perry (1961) in Fas~io~a heputica, Sate, Oh & Sakado (1967) in ~~~~g~~~~~~ ~~~~ffzff~~j,Burton (1972) in ffuemato/oechas medioplexus and Grant, Harkema & Muse (1976) in Pharyngosfomaides procyonis. Studies involving part of the process, or spermatozoa only, are those of Shapiro, Hershenov & Tulloch (1961), Hershenov, Tulloch & Johnson (1966) and Burton (1960, 1967) all in H. mediop/exus and Morseth (1969) in Dicrocoelium dendriticum. Ultrastructural spermiogenesis

studies have also

of spermatozoa and been made in cestodes

(Rosario, 1964; von Bonsdorff & TelkkZ, 1965; Morseth, 1969; Swiderski, 1968, 1976a, 6; Maamouri & Swiderski, 1975), in monogeneans (Tuzet & Ktari, 1971; Halton & Hardcastle, 1976) and in some turbellarians (Silveira & Porter, 1964).

spermatozoon;

AND METHODS

Adult specimens of C. ~~ng~fawere obtained by infection, of day-old domestic chicks, with metacercariae encysted in the fins of Gob&s minutas previously exposed to cercariae of C. /ingua emerging from Littorina &urea. Each of six chicks was given, by mouth, 50-60 3% to 45day old encysted metacercariae. As the worms become sexually mature in 2-3 days after arrival in the intestine of the host, in laboratory conditions, the birds were killed one on the first day, two on each of the second and third days and one on the fourth day post infection (d.p.i.). Worms recovered from the intestine were washed in either Ringer-Locke solution or 0.75% saline at 40°C. On 3 d.p.i. and 4d.p.i. spermatozoa could be teased from the vesicula seminalis on a slide in Ringer-Locke soIution. Spermatozoa had not completed their development by one d.p.i. and although present in the testes, on the second day, very few had reached the vesicula seminalis. In l- and 2-day old worms, therefore, testes were teased apart. Live sperms were viewed with bright light and phase contrast microscopes. Neutral red was used as an intra vitam stain and some preparations were stained with acetoorcein. Worms intended for sectioning for light microscopy were fixed in Carnoy’s or Lillie’s fixatives. They were cut at 5 pm, in various planes, to include the testes and vesicula seminalis. Histochemical tests for glycogen were made with periodic acid-Sichiff (PAS) and Best’s carmine using the salivary digestion control (Pearse, 1968, 1972). Specimens for electron microscopy were fixed for 2 h at 4”C, in 5 % glutaraldehyde in 0.2 M-cacodyiate buffer at pH 7.2. After rinsing in

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(b)

Is

mt

Id

mt

Cd)

(h

(e)

QOSt.

(I)

FIG. 1 Cryptocotyle lingua. (a)-(k) diagrams of a series of transverse sections througn a spermatozoon from the vesicula seminalis. (a), (b) anterior projection; (c)-(e) head region; (f) middle region; (g)-(j) terminal region; (k) posterior projection: (1) spermatozoa from the vesicula seminalis (left) and from the testis (middle and right). Abbreviations: a.z. attachment zone; ant. anterior; ax. axial unit; gl. p glycogen; 1.d. less dense area of nucleus; Is. lamellar subunits of nucleus; mt. microtubule; n.m. nuclear membrane; p.m. plasma membrane; post. posterior

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buffer overnight they were post-fixed in 1% osmium tetroxide, in buffer, for 4 h at 4”C, stained in 3 % aqueous uranyl acetate for 3 h, dehydrated in ethanol, treated with propylene oxide and embedded in TAAB resin Mix C. Sections were stained with 5 % aqueous uranyl acetate and lead citrate (Reynolds, 1963) and viewed with an AEI 6B electron microscope.

RESULTS The spermatozoon The spermatozoon is elongated, thread-like and tapering posteriorly with a maximum length of 285 pm [Fig. 1 (I)]. Free flagella and undulating membranes are absent. Living spermatozoa removed from the vesicula seminalis undergo slow two dimensional undulatory movements along the whole length. Those teased from the testes, and which may not have completed their development, are slightly shorter and show more rapid shallow undulatory movements of about the posterior sixth of the length [Fig. 1 (I)]. Random movements may also occur in this region which may be thrown into a loop. The anterior extremity, which at this stage is attenuated, may also loop or move randomly. These movements, of both extremities of the spermatozoon, may assist in freeing it from the residual spermatid cytoplasm. When the spermatozoon has completed its development and is moving freely in the lumen of the testis the anterior process shortens to a mere projection, incapable of separate movement. Undulations along the whole length, together with peristaltic contractions of the vasa efferentia, then enable the spermatozoa to move from the testes into the vesicula seminalis. The spermatozoon shows no external division into head, middle and terminal regions but these regions are recognizable, in ultra thin sections, by their contained organelles. A slight flattening throughout the length, but more pronounced in the middle and terminal regions, is apparent in spermatozoa in the vesicula seminalis [Fig. 1 (b)-(j)]. Fawcett (1962) suggested that flattening in spermatozoa should be regarded as dorso-ventral, a terminology which is adopted here. The spermatozoon is bounded by a smooth trilaminate plasma membrane of uniform thickness. Head region. The head region contains the nucleus, one axial unit apically, but later two axial units and the anterior end of the mitochondrion [Figs. I (c)-(e), 3, 5 & 61. The uneven distribution of the axial units will be explained in the description of The elongated nucleus, broad spermiogenesis. anteriorly and tapering posteriorly, commences at the apex of the head region immediately behind the small anterior projection. It is impossible to measure the length of the nucleus, in longitudinal sections, owing to the undulations of the greatly elongated cell. The anterior end of the head region is pyriform

407

in profile in transverse section and measures 0.66 x 0.40 urn [Figs. 1 (c) & 31. The nucleus is very slightly flattened in the same plane as the cell and measures 0.34 x0.32 pm in diameter. It tapers gradually posteriorly. The chromatin of the nucleus is in the form of a large number of closely packed lamellae arranged in a helical pattern relative to the longitudinal axis of the nucleus [Figs. 1 (c)-(e), 3, 5 & 61. Occasionally less dense and less abundant material, which also follows a spiral course, is present in the nucleus [Figs. 1 (c), 3 & 161 and is probably a residual protein as noted, also, by Silveira & Porter (1964) in some turbellarians. The nuclear membrane is usually separated from the condensed chromatin by a narrow clear zone [Figs. 1 (c), 3 & 51. Only one axial unit is present at the anterior end of the head region [Figs. 1 (c), (d), 3 & 51. The second axial unit commences a short distance behind the anterior end, on the opposite side of the nucleus, where the latter begins to decrease in diameter [Figs. 1 (e) & 61. At the distal end of the head region the profile of the spermatozoon, in transverse section, is elliptical measuring 0.68 x 0.39 urn. The nucleus is more or less central and the axial units lateral and in close proximity to the plasma membrane. Each axial unit, measuring 0.20 nrn in diameter, consists of nine doublet tubules arranged in a cylinder around a central complex (‘9+ 1’) [Figs. 1 (a)-(j), 2-71. Nine radial strands or ‘spokes’ join the doublet tubules to the central complex. The two tubules of each doublet are morphologically distinct. Tubule A, in which an electron-dense region partially occludes the lumen, bears a pair of ‘arms’ directed clockwise when viewed antero-posteriorly. Tubule B is simple and slightly longer. The structure is similar to that described by Burton (1967) and by others to be mentioned later. The central complex, 0.06 urn in diameter, is cylindrical consisting of a dense central fibril 0.02 urn in diameter surrounded by two ringlike structures. The outer ring, or central sheath, has a density similar to that of the central fibril while the inner ring, or central matrix, is homogeneous and electron-lucent (Figs. 3, 4 & 12). In longitudinal section the central sheath is seen to be composed of subunits, which are arranged in a double helical pattern (Fig. 30), appearing as dark and light bands at an angle of 45”. It is rarely possible to see both coils of the helix in the same longitudinal section. The subunits of the helix appear to be tubular but, owing to the angle at which they are cut, only two or three of the eighteen components can be seen, in profile, in any one section (Fig. 4). Serial sections are necessary to reveal others, in sequence. The nine radial strands, 0.04 nm in length, pass from the central sheath to tubule A of each of the nine doublets [Figs. 1 (a)-(j), 2-91. They are probably arranged in a helical pattern which may explain why those on opposite sides are sometimes the more

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prominent in transverse section. The radial strands and central fibril have an indistinct granular structure. The mitochondrion is a single elongated body (Fig. II), which begins shortly behind the anterior end of the nucleus, and lies between the nucleus and the single axial unit present in this region [Figs. 1 (d) & 51. On the appearance of the second axial unit, on the opposite side, the nucleus and mitochondrion occupy the central region [Figs. 1 (1) & 61. Sato & Sakoda (1967), in their study of Paragonimus miyazakii, suggested that the mitochondrion should be regarded as being ventral to the nucleus. A single row of 20-22, rarely up to 25, cortical microtubules orientated in straight lines, parallel to the long axis of the spermatozoon, lie immediately below the plasma membrane and are arranged in a dorsal and ventral row, in the central area, between the lateral axial units [Figs. 1 (d), (e), 5 8c 61. The number, dorsally and ventrally, may vary slightly. At the anterior end of the head region, however, where the nucleus is at its maximum diameter and only one axial unit is present the microtubules are arranged in two wedge-shaped groups between the nucleus and the axial unit [Fig. 1 (c)l. They are extremely difficult to see in transverse sections as, owing to the rapid increase in diameter of the spermatozoon, they are usually cut obliquely. The microtubules, which probably serve a cytoskeletal function, so necessary in such a long slender cell, are hollow cylinders with dense walls. In some transverse sections, more particularly in developing stages (Fig. 24), a very short straight connection can be seen between each microtubule and the plasma membrane. Ribosomes, polyribosomes and Golgi bodies are absent from the cytoplasm in the central region, but aggregates of 8 glycogen particles are abundant dorsally and ventrally to the nucleus and mitochondrion [Figs. 1 (d), (e), 5 & 61. In sections of 3 d and 4 d worms spermatozoa, packed in the vesicula seminalis, give

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a positive reaction with Best’s carmine and PAS, which is diastase fast, indicating glycogen. The particles, also, have the morphological appearance of 8 glycogen. In front of the head region the short anterior conical projection contains, apically, the anterior of the two axial units, surrounded by a ring of about 20 peripheral microtubules [Figs. 1 (a) & 2 (a)]. As the diameter of the projection increases the axial unit comes to lie on one side, immediately below the plasma membrane, while the microtubules form a semicircle around the small adjacent area of cytoplasm [Figs. 1 (b) & 2 (b)]. The anterior projection then widens rapidly to become continuous with the anterior end of the head region. The anterior end of the nucleus lies immediately behind the small area of cytoplasm, at the base of the projection, the axial unit continuing along one side of it into the head region [Figs. 1 (b), (c) & 31. At the level of the anterior end of the nucleus the microtubules separate into dorsal and ventral wedge-shaped groups of about 10 each [Fig. 1 (c)l, which later become arranged in a single row dorsally and ventrally [Figs. 1 (d), (e), 5 & 61. Middle region. The middle region begins immediately behind the nucleus and extends throughout the remainder of the length of the mitochondrion. It appears to be the longest of the three regions judging from the relative number of sections of this region which are invariably present. The elliptical profile, in transverse section, measures anteriorly 0.60 x 0.38 urn, slightly less than the head region, as the spermatozoon is tapering gradually [Fig. 1 (f)]. The two axial units are lateral and the mitochondrion central, the cytoplasm around it still containing 8 glycogen particles [Figs. 1 (f) & 71. The peripheral microtubules remain unchanged. Terminal region. The terminal region extends from the end of the mitochondrion to the posterior extremity. The tapering continues so that the axial

FIGS. 2-9. Cryptocotyle lingua: Transverse sections through the spermatozoon. FIG. 2. (a) apical projection showing the single axial unit (ax.) surrounded by peripheral microtubules (mt.), (b) apical projection, proximally, showing microtubules surrounding the cytoplasmic area (cy.). FIG. 3. Anterior

end of head region showing the nucleus (n.) with less dense areas (I.d.), nuclear membrane (n.m.) and one axial unit. FE. 4. The axial unit showing the subunits of the central sheath (t.s.) and the plasma membrane (p.m.). FIG. 5. Head region showing the mitochondrion (m.) and fl glycogen particles (gl.).

FIG. 6. Head region showing two axial units, peripheral microtubules and nucleus with lamellar subunits (1,s.). FIG. 7. Middle region with mitochondrion

(mt.), mitochondrion

(m.)

(m.), microtubules (mt.), b glycogen particles (gl.) and no nucleus. FIG. 8. Terminal region showing decrease in number of peripheral microtubules (mt.). FIG. 9. Terminal region showing one peripheral microtubule dorsally and ventrally (mt.) and attachment zones (a.z.).

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units approach one another, the transverse diameter of the spermatozoon decreasing to 0.30 urn [Figs. 1 (g)-(j), 8 & 91. A longitudinal groove appears, dorsally and ventrally, between the two axial units [Fig. 1 (h) k(j)]. The central cytoplasm still contains g glycogen particles, anteriorly [Figs. 1 (g) & 81. The dorsal and ventral peripheral microtubules decrease gradually in number until only one remains dorsally and ventrally [Figs. 1 (h) & 91. These eventually also disappear [Figs. 1 (j) & 10 (a)]. Two dense plaques, attached to the inner surface of the plasma membrane, are visible dorsally and ventrally between the axial units [Figs. 1 (h) & 91. These are more clearly apparent in sections through developing spermatozoa and will be referred to later. At the posterior tip of the spermatozoon only the posterior axial unit remains [Figs. 1 (k) & 10 (c)l. This extends a short distance beyond its partner and is enclosed by the plasma membrane only, forming a short posterior projection which differs from the anterior projection, in the absence of peripheral microtubules. Irregularities appear in the doublet tubules and central complex at the posterior extremity [Figs. 1 (k) & 10 (c)]. Occasionally a variation of the above arrangement has been observed towards the end of the terminal region. The axial units, instead of being symmetrically arranged, approach one another dorsally. This results in an unusual profile in transverse section [Fig. 10 (b)]. Only one microtubule lies between the axial units dorsally and the remainder in a row ventrally, necessitating a larger area of plasma membrane on this side. The row of microtubules, however, gradually decreases in number posteriorly until only one remains dorsally and ventrally as before.

Spermiogenesis

The thirty-two spermatids of a cluster develop synchronously into spermatozoa. The first stage in spermiogenesis is the beginning of the formation of the zone of differentiation distal to each of the spermatid nuclei. This region bulges out slightly and a row of microtubules appears below the plasma membrane (Fig. 14). The zone of differentiation grows into a conical process, clearly distinguished from the spermatid cytoplasm (Fig. 18), measuring 1.62 urn in length and 0.88 urn in diameter at the base. The base of each sinks back slightly, below the surface, so that it becomes surrounded by a shallow circular groove and collar. Profiles of cisternae of endoplasmic reticulum lie below the plasma membrane of the collar (Fig. 21). The microtubules elongate as the process grows and become arranged in a continuous ring of 52-54 lying immediately below the plasma membrane, forming a cytoskeleton extending from the apex to the base. Meanwhile the spermatid nucleus undergoes changes. At first it is oval in profile 4.80 x 2.50 urn and contains evenly distributed chromatin granules and a dense nucleolus. The nucleus gradually elongates along the axis of the zone of differentiation and the chromatin condenses into lamellae, which are first visible near the centre of the nucleus, giving it a longitudinally striated appearance in longitudinal section (Fig. 13) and a honeycomb appearance in transverse section (Fig. 14). The central lamellae seem to be folded into scrolls around areas of lesser density, the honeycomb appearance in transverse section being due to the folded lamellae coming into contact with one another. The lamellae eventually spread throughout the nucleus (Figs. 15 & 16) but

FIGS. 10-16. Cryptocotyle lingua: sections through the spermatozoon and early stages of spermiogenesis. FIG. 10 (a) Transverse section through the end of the terminal region showing two axial units (ax.), attachment zones (az.) and the absence of microtubules. (b) Terminal region showing an irregular arrangement of a large number of microtubules, ventrally (mt.). Section slightly oblique. (c) Terminal projection showing irregularities in the doublet tubules (d.t.) and central complex (cc.) of the single axial unit. Microtubules absent. 11. Longitudinal section showing mitochondrion (m.), with cristae almost longitudinal, surrounded by f3glycogen particles. A doublet tubule (d.t.) and central complex (c.c.) of an axial unit also shown. (Slight blemish across mitochondrion.) FIG. 12. Longitudinal section through the axial unit showing a doublet tubule (dt.), central sheath (c.s.), central filament (c.p.) and radial strands (rs.). FIG. 13. Spermatid nucleus in an early stage of condensation of the chromatin, into lamellar subunits (I.s.). FIG. 14. Formation of the zone of differentiation (z.d.). Nucleus, in transverse section, shows honeycomb pattern of lamellae (I.s.). Mitochondria (m.) present. Microtubules (mt.) below the plasma membrane. FIG. 15. Spermatid nucleus, showing spiral twisting of lamellar subunits (I.s.). Ribosomes (r.) occur in the cytophore (ct.). FIG. 16. Spermatid nucleus in later stage showing dense fibrillar subunits (I.s.), less dense spirally arranged material (1.d.) and nucleolus (nu.).

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the honeycomb order is not perfect, especially near the circumference (Fig. 15). The nucleus continues to elongate, the diameter decreasing to 0.45 urn. It then begins to twist in a spiral, the lamellae following the spiral of the nucleus (Figs. 15 & 16). The nucleolus breaks up into two or three very dense bodies which continue to be recognizable for a time. There is some evidence, though admittedly not clear, of a row of microtubules in close association with the distal end of the nuclear membrane in the early stages of condensation of the chromatin (Fig. 17), but not apparent later. The nucleus now lies with one extremity in the proximal region of the zone of differentiation and the remainder in the cytophore (Fig. IS). While the nuclear changes are progressing two flagellar processes begin to grow out, laterally, from the zone of differentiation (Fig. 23). Each wilI consist, eventually, of an axial unit surrounded closely by an extension of the plasma membrane (Figs. 17, 18, 22 & 30). The axial units arise, each from a basal body (Fig. 22 & 30), one on each side of the central body to be referred to later. At first the basal bodies and flagellar processes are at right angles to the central body, now in its early stage of formation (Fig. 23). but as development proceeds their orientation changes and they rotate gradually, through an angle of 90”, about the central body (Fig. 25). Eventually they come to lie parallel to the longitudinal axis of the developing spermatozoon and one on each side of, though not in the same plane as, the median cytoplasmic process which develops, simultaneously, at the apex of the zone of differentiation (Figs. 20, 22 & 30). The basal bodies are composed of nine sets of triplet tubules (Fig. 26). The dense central complex of the axial unit arises at the distal end of the basal body. The tapering striated rootlets, associated with the basal bodies, extend along each side of the nucleus into the cytophore below the zone of differentiation, anchoring the basal bodies and the

FIGS. 17-22.

Cryptocotyle

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developing axial units (Figs. 20 & 30). The rootlets show a major transverse periodicity of 0.06 urn. Lesser transverse striations occur half way between these and further finer striations between these again. The two basal bodies and consequently the two rootlets are not precisely level, one being embedded slightly distally to the other, in the zone of differentiation (Fig. 30). Early in spermiogenesis a structure consisting of a number of dense elements or layers, of equal length, measuring 0.34 urn in length x0.20 urn in breadth appears between the basal bodies. This is referred to as the central body, its origin and function being unknown (Figs. 25, 26 & 30). It consists of nine layers of dense material arranged parallel to the long axis of the spermatid, the densest elements being the ones on each side of the less dense central element (Figs. 25 & 30). The outermost element on each side is somewhat indistinct. The cytoplasm of the cytophore (Figs. 14 & 19) polyribosomes, large contains free ribosomes, mitochondria with few cristae near the nucleus and a few profiles of granular endoplasmic reticulum. Golgi elements and crystalline bodies have only very occasionally been observed. The cytoplasm of the zone of differentiation contains, at first, a few ribosomes and polyribosomes but later these are restricted to the cytophore. Immediately below the plasma membrane in the zone of differentiation the cytoplasm, in the vicinity of the microtubules, is electron-dense (Fig. 21). The cytoplasm of the median process contains no inclusions except for the 20-2.5 peripheral microtubules (Fig. 22). With the appearance of the median and lateral flagellar processes the nucleus passes through the zone of differentiation, between and slightly dorsally to the basal bodies, and on into the median cytoplasmic process (Figs. 20, 24, 25 & 30). During its progress the advancing extremity becomes narrower. At the same time mitochondria which have accumulated near the nucleus (Fig. 14) fuse end to end, to

sections through the testes showing stages in spermiogenesis.

FIG. 17. Zone of differentiation,

with spermatid nucleus in an early stage of chromatin condensation, showing what appear to be microtubules near the nucleus (mt.). Median processes (m.p.), flagellar process (f.p.) and peripheral microtubules present. FIG. 18. Two zones of differentiation (z.d.) developing synchronously. A groove (gr.) and collar surrounds the base of each, separating them from the cytophore (ct.). FIG. 19. The mitochondrion (m.), resulting from the fusion of several, entering the zone of differentiation (z.d.), ventral to the nucleus (n.) and rootlet (rt.) of an axial nuit. Groove (gr.) still apparent. (Details of structures. other than the mirochondrion, shown elsewhere.) FIG. 20. Two Ragellar processes (f.p.) arising from the apex of the zone of differentiation (z.d.). Flagellar rootlet (rt.) showing transverse periodicity. Nucleus (n.). FIG. 21. Detail of the margins of the groove, at the base of the zone of differentiation, showing cisternae of endoplasmic reticulum (e.r.) and dense cytoplasm (d.c.) below the plasma membrane (p.m.). FIG. 22. A later stage in spermiogenesis, showing the median cytoplasmic process (m.p.) and one of the two flagellar processes (f.p.) containing an axial unit (ax.) arising from a basal body (b.b.).

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form a long narrow cylindrical mitochondrion which also passes into the zone of differentiation ventrally to the nucleus (Figs. 19, 24 & 26). Eventually the nucleus comes to rest in the proximal end of the median cytoplasmic process which will become the head region of the fully developed spermatozoon. The mitochondrion, however, moves further, coming to lie mainly distally to the nucleus in what will become the middle region of the spermatozoon. The mitochondrion becomes very much stretched during the process, and the cristae consequently markedly oblique (Figs. 11 & 19). Meanwhile the median cytoplasmic process and the lateral flagellar processes fuse along their lengths, to form the unipartite shaft of the spermatozoon. Fusion begins proximally and is apparently accomplished fairly rapidly. It takes place along the strip of plasma membrane, between the paired attachment zones, along the length of the median cytoplasmic process on each side (Figs. 27-30). The plasma membranes break down on contact and the three processes are incorporated into one shaft (Figs. 28 & 29). The profile of the shaft, in transverse section, is at first rounded but later takes on the typical outline of the three regions of the fully developed spermatozoon (Figs. l-9). Of the 52-54 peripheral microtubules, in the zone of differentiation (Figs. 24 & 26), only those in the centre, dorsally and ventrally, totalling 20-22 or rarely up to 25, continue into the median cytoplasmic process, gradually decreasing in number towards the posterior extremity. Those which are lateral cease near the origin of the lateral flagellar processes. Normally the number of microtubules, dorsally and ventrally, in the fully developed spermatozoon is equal or nearly so; but occasionally, as already mentioned, when the lines of fusion of the median

FIGS. 23-29.

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and lateral processes curve dorsally the number of microtubules is greater ventrally (Figs. 27 & 28). The 32 spermatozoa of a cluster are eventually liberated into the testis lumen leaving a residual mass of cytoplasm, the original cytophore. The basal bodies, rootlets and central body have never been seen in the liberated spermatozoon. It is assumed, therefore, that they remain in the residual cytoplasm and presumably degenerate. The residual cytoplasm contains free ribosomes, a few degenerating mitochondria and occasional whorls of membranes which may have a lysosomal function. Separation of the spermatozoon takes place at the point of origin of the three processes, at the apex of the zone of differentiation. As it separates a small portion of cytoplasm, surrounded by plasma membrane, is drawn out into a filamentous process which is eventually torn away from the residual cytoplasm. At the base of the process is the extremity of the anterior axial unit surrounded by a ring of microtubules [Figs. 1 (a) & 2 (a)]. The posterior axial unit begins a little further back in the head region [Figs. I (e) & 61. This arrangement is presumably due to one basal body and consequently one axial unit being at all times slightly in advance of the other in the zone of differentiation. The more distal becomes the posterior axial unit, the more proximal the anterior axial unit of the free spermatozoon. After

a short time in the testis lumen, the spermatozoon completes its development. It increases slightly in length, the anterior process shortens to a mere projection, the nuclear lamellae become more compact and g glycogen particles accumulate in the cytoplasm. The spermatozoon is then ready to pass out of the testis into the vesicula seminalis.

through

the testes showing

stages

in spermiogenesis.

FIG. 23. An early stage in the formation of the zone of differentiation, showing the flagellar processes (f.p.) arising laterally and at right angles to the central body (c.b.), which latter is in an early stage of development and consequently not clear. Nuclear (n.) chromatin not yet condensed. Peripheral microtubules present (m.c.). FIG. 24. Transverse section through the zone of differentiation (z.d.) showing the mitochondrion ventral to the nucleus (n.), the two rootlets (r.t.) of the axial units and peripheral microtubules attached to the plasma membrane. FIG. 25. Slightly advance

oblique longitudinal section showing of the other (b.b.), the mitochondrion

FIG. 26. Slightly

oblique transverse central body

the central body (c.b.), one basal body (m.) and peripheral microtubules (mt.).

section showing the triplet tubules (c.b.), nucleus (n.) and mitochondrion

(t.t.) of the basal (m.).

body,

FIG. 27. Transverse section of the median cytoplasmic process (m.p.), with paired attachment (a.z.) and the two adjacent flagellar processes (f.p.) shortly before fusion. FIG. 28. The three

processes

fused

(m.) (mt.)

to form the unipartite rounded profile.

spermatozoon,

middle

region.

in the

zones Note

the

FIG. 29. Transverse section of the head region of the spermatozoon shortly after fusion. Profile rounded. Two axial units (ax.), mitochondrion (m.) and attachment zones (a.z.) present.

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FIG. 30. Cryptocotyle tion of a late stage

lingua. Diagrammatic reconstrucin spermiogenesis, showing the parallel median cytoplasmic (m.p.) and the lateral flagellar (f.p.) processes arising from the apex of the zone of differentiation (z.d.), the base of which is marked by a groove (gr.). The central sheath (c.s.) of the axial unit (ax.) shows a double helical arrangement of subunits (d.h.). The basal bodies (b.b.) lie one slightly in advance of the other, with the central body (c.b.) between. The rootlets (r.) of the basal bodies extend into the cytophore. The nucleus (n.) and mitochondrion (m.) are entering the zone of differentiation. Doublet tubules (d.t.), radial strands (r.s.), microtubules (mt.) and attachment zones (a.z.) also shown.

DISCUSSION The elongated thread-like unipartite spermatozoon, incorporating two axial units, is characteristic of those of Digenea so far described (Gresson & Perry, 1961; Herschenov et ul., 1966; Burton, 1960, 1967, 1972; Sato & Sakoda, 1967; Tulloch & Herschenov, 1967; Grant c’t al., 1976). The same arrangement exists in two monogeneans (Tuzet & Ktari, 1971; Halton & Hardcastle, 1967) and in some pseudophyllidean, tetraphyllidean and trypanorhynchan cestodes (von Bonsdorff & TelkkZ,

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1965; Maamouri & Swiderski, 1975; Swiderski, 1974, 19766) while in cyclophyllidean and caryophyllidean cestodes only one axail unit is incorporated into the spermatozoon (Rosario, 1964; Lumsden, 1965; Swiderski, 1968, 1970, 19761~; Morseth, 1969; Featherston, 1971). Some turbellarians also have thread-like spermatazoa (Klima, 196 I ; Hendelberg, 1965, 1969), while others possess two long free flagella (Silveira & Porter, 1964). The sequence of events in spermiogenesis, in C. lingua, resembles that described by Maamouri & Swiderski (1975) for two tetraphyllidean cestodes and which they listed under five headings. The elongation of the nucleus in the longitudinal axis of the spermatid and the condensation of the chromatin material into electron-dense lamellae, coiled in a spiral, have been noted in other digeneans (Gresson & Perry, 1961; Burton, 1972; Grant et al., 1976) and also in other classes of platyhelminths (Silveira & Porter, 1964; Halton & Hardcastle, 1976). Rudzinska & Porter (19.55) were the first to record a honeycomb structure in the arrangement of the nuclear lamellae, seen in transverse section, when studying the macronucleus of Tokophrya infusionurn. Later Yasazumi & Ishida (1957) and Kessel (1967) noted a hexagonal pattern, in the nuclei, in spermiogenesis of the grasshopper as did Gibbons & Bradfield ( 1957) in Locusta migratoria. Kessel (1966) described microtubules, arranged in groups, lying in longitudinal furrows along the nuclear membrane in spermiogenesis of a dragonfly, and suggested that as they are no longer present in the mature spermatozoon, they play a role in the elongation of the nucleus. Kessel (1967) later described a helical arrangement of microtubules around the elongating spermatid nucleus of the grasshopper and associated these, also, with nuclear elongation. There seems, however, to be no proof of this and Fawcett, Anderson & Phillips (1971), in a review of factors influencing the shape of the sperm head in various animal groups, are of the opinion that microtubules are not responsible for shaping the nucleus, but that the shape is largely determined by intrinsic forces of a different nature. They quote a number of references in support of this, as does Phillips (1974). It is not known what may be the function of the microtubules, associated briefly with the spermatid nucleus, in C. lingua. The present study, however, is not aimed to resolve this issue. As noted by Burton (1972) in Haematoloechus medioplexus the flagellar processes in C. lingua arise, from the zone of differentiation, shortly before the median process. Their origin seems to be associated with the central body consisting of elongated electron-dense elements. A similar structure, in other platyhelminths, has been named the middle filament by Gresson & Perry (1961), a centriole by Silveira & Porter (1964), a centriole-like body by Burton (1972) and Maamouri & Swiderski (1975), a condensation centre by Hitchin & Butler (1973) and a centriole-

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like body or centre body by Halton & Hardcastle (1976). Grant et al. (1976) referred to it as the microtubule organizing centre, using the terminology of Pickett-Heaps (1969, 1971) for a somewhat similar body in dividing plant cells, and suggested, as did Burton (1972) and Hitchin & Butler (1973), that it is involved in the assembly of peripheral microtubules of the axial unit. This, however, is presumably the function of the basal bodies which agree with the definition of a centriole as a short, cylindrical organelle, with the characteristic nine triplet tubules and which is essential for the initial assembly of the subunits that form the nine doublet tubules. The centre body has, apparently, some close association with the basal bodies but its precise origin and function are not known. It may possibly be associated with the central complex which has no template in the basal body. Rotation of the flagellar processes, so that they lie alongside the median cytoplasmic process, has been observed by Burton (1972) and Grant et al. (1976). The median process was named ‘the middle snook’ by Hendelberg (1962) in a light microscope study of the spermatozoa of Fasciola hepatica and Dicrocoelium dendriticum. This term was adopted by von Bonsdorff & TelkkB: (1965), in describing spermatozoa of Diphyllobothrium latum, but does not seem to be generally used. The ‘9-t 1’ pattern of microtubules in the axial units of platyhelminths was first reported by Shapiro et a/. (1961) and with the exception of Schistosoma martsoni where the pattern is ‘9fO’ (Kitajima, Paraense & Correa, 1976) has been found to be usual in spermatozoa of platyhelminths so far examined (Burton, 1960, 1967, 1972; Rosario, 1964; von Bonsdorff & Telkkg, 196.5; Hershenov et al., 1966; Sato & Sakoda, 1967; Swiderski, 1968, 1976~7, b; Morseth, 1969; Grant et a/., 1976; Halton & Hardcastle, 1976). A helical pattern of subunits in the central sheath has been noted by Silveira & Porter (1964), Swiderski (1968) and Sato & Sakoda (1967). A double helical pattern of eighteen subunits, as in C. lingua, was mentioned by Burton (1967) and Henley, Costello, Thomas & Newton (1969). In C. lingua the nucleus remains at the proximal end of the spermatozoon. Burton (1972) found that in Haematoloechus medioplexus it migrates to the distal end as did Hendelberg (1962) in, FascioIa hepatica and Dicrocoelium dendriticum. Swiderski (1968) mentioned an unfixed localization of the nucleus in spermatozoa of Moniezia expansa as did von Bonsdorff & Telkkg (1965) in Diphyllobothrium latum. In Echinococcus granulosus Morseth (1969) and Taenia hydatigena (Featherston, 1971) the nucleus is wound around the single axial unit. Burton (1972) noted that numerous mitochondria accompanied the nucleus into the median cytoplasmis process where they fused end to end to form a long mitochondrion. In C. lingua fusion of the mitochondria takes place at the proximal end of the

of differentiation and before entry into the median process. Mitochondria have been described in spermatozoa of other digeneans (Grant et al., 1976; Sato & Sakoda, 1967) and in turbellarians (Hendelberg, 1965), but have not been found in spermatozoa of cestodes (Rosario, 1964; von Bonsdorff & Telkkg, 1965; Morseth, 1969; Featherston, 1971; Swiderski, 19760, 6). Almost all animal spermatozoa are dependent on mitochondria for energy necessary for movement, so that cestodes represent an unusual exception. Reserves of glycogen are common in spermatozoa. Axman (1947) found that in several digeneans including C. lingua developing spermatozoa were free of glycogen but the mature sperm gave a diffuse reaction to tests for glycogen. Some other records are those of Anderson & Personne (1970)? Silveira (1973), von Bonsdorff & Telkkg (I 965) and Swiderski (19760, b). Glycogen in spermatozoa represents a store of food material, which is used as an energy source during the relatively long life and for active movement over a relatively long distance. Two free flagella occur in some triclad spermatozoa but, as Franz& (1956) suggested, these spermatozoa may have evolved from the primitive type, within the Metazoa, as doubling of the flagellum occurs in other groups. A single short free flagellum was described by Kitajima et a/. (l976), for the spermatozoon of Schistosoma mansoni, but they consider this to be a rudimentary type ‘probably related to the low activity required for fertilization’. Free flagella were probably the original condition in Turbellaria, but in some of these and in Digenea, Monogenea and Cestoda, the flagella have become incorporated into the extremely long unipartite spermatozoon. As spermatozoa in Cyclophyllidea and Caryophyllidea possess only one axial unit, whereas those of Tetraphyllidea, Trypanoryncha and Psueophyllidea have two, as do Digenea (except S. mansoni) and Monogenea, further studies of spermatozoa might contribute to the understanding of phylogenetic relationships within the platyhelminths. zone

Acknowledgements-l am most grateful to The Royal Society, London, for a grant to support this investigation. I am also greatly indebted to Mr. P. C. Lloyd for valuable assistance with electron microscopy and also to Mr. D. R. Williams. Professor B. M. Jones has generously provided departmental facilities.

REFERENCES ANDERSON W. A. & PERSONNE P.

1970. The localization of glycogen in the spermatozoa of various invertebrate and vertebrate species. Journul of Cell Biology 44: 29-51. AXMAN M. C. 1947. Morphological studies on glycogen deposition in schistosomes and other flukes. Journal of Morphology 80: 321-334.

418

F.

GWENDOLEN REES

BURTONP. R. 1960. Gametogenesis and fertilization in the frog lung fluke Haematoloechus medioplexus Stafford (Trematoda: Plagiorchiidae). Journal oj Morphology

107: 93-122.

BURTONP. R. 1967. Fine structure of the unique central region of the axial unit of lung-fluke spermatozoa. Journal of Ultrastructure

Research

19: 166-172.

BURTONP. R. 1972. Fine structure of the reproductive system of a frog lung fluke. III. The spermatozoon and its differentiation. Journal of Parasitology 58: 68-83. CABLE R. M. 1931. Studies on the germ-cell cycle of Cryptocotyle lingua (Creplin). 1. Gametogenesis in the adult. Quarterly Journal of Microscopical Science 74: 563-589. DAY M. F. 1976. Changes in the epidermis of Cryptocotyle lingua (Creplin) during metamorphosis of the cercaria

to the metacercaria in the second intermediate (fish) host. Parasitology 73: XXIV. FAWCET~D. W. 1962. Sperm tail structure in relation to the mechanism of movement. In Spermatozoon motility (Edited by BISHOPD. W.). American Association for the Advancement of Science, Washington, DC, No. 72. FAWCETTD. W., ANDERSONW. A. & PHILLIPS D. M. 1971. Morphogenetic factors influencing the shape of the sperm head. Developmental Biology 26: 230-251. FEATHERSTON D. W. 1971. Tuenia hydatigena. III. Light and electron microscope study of spermatogenesis. Zeitschrif fiir Parasitenkunde 37: 148-168. FRANZ&NA. 1956. On spermiogenesis, morphology

spermatozoon, invertebrates.

and

biology

of fertilization

of the among

Zoologiska bidrag fr& Uppsala 31: 354482. GIBBONSJ. R. & BRADFIELDJ. R. G. 1957. The fine

structure of nuclei during sperm maturation in the locust. Journal of Biophysical and Biochemical Cytology 3: 133-140.

GRANT W. C., HARKEMAR. & MUSE K. E. 1976. Ultrastructure of Pharyngosfomoides procyonis Harkema 1942 (Diplostomatidae). I. Observations on the male reproductive system. Journal of Parasitology 62: 3949. GRESSON R. A. R. & PERRY M. M. 1961. Electron microscope studies of spermioteleosis in Fasciola henatica L. Exoerimental Cell Research 22: l-8. HA&ON D. W. & HARDCASTLE A. 1976. Spermatogenesis in a monogenean, Diclidophora merlangi. International Journal for Parusitology

6: 43-53.

HENDELBERC~ J. 1962. Paired flagella and nucleus migration in the spermiogenesis of Dicrocoelium and Fasciola (Digenea, Trematoda). Zoologiska bidrag fr&n Uppsala 35: 569-588.

HENDELBEKG J. 1965. On different types of spermatozoa in Polycladida, Turbellaria. Arkiv fiir zoologi, Uppsala 18: 267-304.

HENDELBERGJ. 1969. On the development of different tvoes of spermatozoa from spermatids with two flagella in-the Tuibellaria with remarks on the ultrastructure of flagella. Zoolorriska bidrag fr&t Uppsala 38: l-50. HENLEYC., COSTELLOD. ~,-THoM~~ M. B. & NEWTON W. D. 1969. The ‘9+ 1’ pattern of microtubules in soermatozoa of Mesostoma (Platyhelminthes, Turbellaria). Proceedings of the National Academy of Sciences of the United States

of America

64: 849-856.

HERSHENOV B. R. G., TULLOCHG. S. & JOHNSONA. D. 1966. The fine structure of trematode sperm-tails. Transnctions 85: 480483.

of the American

Microscopical

Society

I.J.P. VOL. 9. 1979

HITCHIN E. T. & BUTLER K. R. 1973. Ultrastructural studies of the commensal suctorian Choanophryra infundibufifera Hartog. II. Tentacle morphogenesis. Zeitschrift .ftir Zellforschung Anatomie 144: 59-73.

und

mikroskopische

KITAJIMAE. W., PARAENSEW. L. & CORREAL. R. 1976. The fine structure of Schistosoma mansoni sperm (Trematoda: Digenea). Journal qf Parasitology 62: 215-221.

KERNEL R. G. 1966. The association between microtubules and nuclei during spermiogenesis in the dragonfly. Journal of Ultrastructure

Research

16: 293-304.

KESEL R. G. 1967. An electron microscope study of spermiogenesis in the grasshopper with particular reference to the development of microtubular systems during differentiation. Journal of Ultrastructure Research

18: 677-694.

KLIMAJ. 1961. Elektronenmikroskopische Studien iiber die Feinstruktur der Tricladen (Turbellaria). Protoplasma 54: 101-162.

LUMSDENR. D. 1965. Microtubules in the peripheral cytoplasm of cestode spermatozoa. Journal of Purusitology 51: 929-93 1. MAAMOURI F. M. & SWIDERSKIZ. 1975. &ude en microscopic Clectronique de la spermatogtntse de deux cestodes Acanthobothriumfilicolle benedenii Loennberg, 1889 et Onchobothrium uncinatum (Rud., 1819) (Tetraphyllidea, Onchobothriidae). Zeitschrift ftir Parasitenkunde

47

: 269-28

1.

MORSETHD. J. 1969. Spermtail finestructure of Echinococcus granulosus and Dicrocoelium dendriticum. Experimental Parasitology

24: 47-53.

PEARSEA. G. E. 1968, 1972. Histochemistry, Theoretical and Applied, 3rd ed., Vol. 1, 1968; Vol. 2, 1972. Churchill, London. PHILLIPSD. M. 1974. Spermiogenesis. Academic Press, London. PICKETT-HEAPSJ. 1969. The evolution of the mitotic apparatus: an attempt at comparative ultrastructural cytology in dividing plant cells. Cytobios 1: 257-280. PICKETT-HEAPSJ. 1971. The anatomy of the centriole: fact or fallacy. Cytobios 3: 205-214. REEKF. G. 1974. The ultrastructure of the body wall and associated structure of the cercaria of Cryptocotyle lingua (Creplin) from Littorina littorea (L.). Zeitschrift ftir Parasitenkunde 44: 239-265. REEK F. G. 1975~. Studies on the pigmented

and unpigmented photoreceptors of the cercaria of Cryptocotyle linaua (Creolin) from Littorina littorea (L.). Proceedings of-the Xoyal society, London Series B 188: 121-138. REF.SF. G. 19756. The arrangement and ultrastructure of the musculature, nerves and epidermis, in the tail of the cercaria of Cryptocotyle lingua (Creplin) from Littorina littorea (L.). Proceedings of the Royal Society, London Series B 190: 165-186. REESF. G. 1977. The development of the tail and the excretory system in the cercaria of Cryptocotyle lingua (Creplin) (Digenea: Heterophyidae) from Littorina littorea CL.). Proceedings of the Royal Society, London Series B‘lk: 425-452: REFS F. G. 1978. The ultrastructure, development and mode of operation of the ventrogenital complex of Cryptocotyle lingua (Creplin) (Digenea: Heterophyidae). Proceedings of the Royal Society, London Series B 200: 245-267.

I.J.P. VOL.9. 1979

Spermiogenesis

in Cryptocotyle lingua

REESF. G. & DAY M. F. 1976. The origin and develop-

1810) (Cyclophyllidea,

ment of the epidermis and associated structures in the cercaria of Cryptocotyyle fingua (Creplin) (Digenea: Heterophyidae) from Littorina littorea (L.). Proceedings of the Royal Society, London Series B 192: 299-321. REYNOLDSE. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy.

Poloniae 18: 475486.

Journal of Cell Biology 17 : 207-2 12.

ROSARIO B. 1964. An electron microscope study of spermatogenesis in cestodes. Journal of UItrastructure Research 11: 412-427. RUDZINSKAM. A. &PORTER K. R. 1955. Observations on the fine structure of the nucleus of Tokophrya infusionum. Journal of Biophysical and Biochemical

Cytology

1: 421-428. SATO M., OH M. & SAKODAK. 1967. Electron microscopic study of spermatogenesis in the lung fluke (Paragonimus miyazakii). Zeitschrift fur Zellforschung und mikroskopische Anatomie 77: 232-243.

SHAPIROJ. E., HERSHENOV B. R. & TULLOCHG. S. 1961. The tine structure of Haematoloechus spermatozoan tail. Journal of Biophysical and Biochemical Cytology 9: 211-217.

SILVEIRAM. 1973. lntra-axonemal glycogen in ‘9fl’ flagella of flatworms. Journal of Ultrastructure Research 44: 253-264.

SILVEIRAM. & PORTERK. R. 1964. The spermatozoids of flatworms and their microtubular systems. Protoplasma 59: 240-265.

SWIDERSKIZ. 1968. The fine structure of the spermatozoon of sheep tapeworm, Moniezia expansa (Rud.,

419

Anoplocephalidae).

Zoologica

SWIDERSKIZ. 1970. An electron microscope study of spermatogenesis in cyclophyllidean cestodes with emphasis on the comparison of fine structure of mature spermatozoa. Journal of Parasitology 56: 337. SWIDER~KI Z. 1974. Comparative fine structure of spermatozoa in some tetraphyllidean and trypanorhynthen cestodes. Parasitology 69: III-IV. SWIDERSKIZ. 1976a. Fine structure of the spermatozoon of Glaridacris catustomi (Cestoidea, Caryophyllidea). Proceedings of the 6th European Microscopy, Jerusalem. 307-308.

Congress of Electron

SWIDERSKIZ. 19766. Fine structure of the spermatozoon of Lacistorhynchus tenuis (Cestoda, Trypanorhyncha). Proceedings of the 6th European Congress of Electron Microscopy, Jerusalem. 309-3 10.

TULLOCHG. S. & HERSHENOVB. R. 1967. Fine structure of platyhelminth sperm tails. Nature 213: 299-300. TUZET 0. & KTARI M. H. 1971. La spermatogenese et la structure du spermatozoide de Microcotyle mormyri Lorenz, 1878 (Monogenea). Compte rendu hebdomadaire des seances de l’dcademie des sciences 212: 2702-2705. VONBONSDORFF C. H. & TELKKAA. 1965. The spermatozoon flagella in Diphyllobothrium latum (fish tapeworm). Zeitschrift fur Zellforschung und mikroskopische Anatomie 66: 643-648.

YAWZIJMI G. & ISHIDA H. 1957. Spermatogenesis in animals as revealed by electron microscopy. II. Submicroscopic structure of developing spermatid nuclei of a grasshopper. Journal of Biophysical and Biochemical Cytology 3: 663-668.